Advertisement

Face-time with TAR: Portraits of an HIV-1 RNA with diverse modes of effector recognition relevant for drug discovery

  • Sai Shashank Chavali
    Affiliations
    Department of Biochemistry and Biophysics, Center for RNA Biology, and Center for AIDS Research, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
    Search for articles by this author
  • Rachel Bonn-Breach
    Affiliations
    Department of Biochemistry and Biophysics, Center for RNA Biology, and Center for AIDS Research, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
    Search for articles by this author
  • Joseph E. Wedekind
    Correspondence
    To whom correspondence should be addressed: Dept. Biochemistry and Biophysics and Center for RNA Biology, University of Rochester Medical Center, 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 585-273- 4516; Fax: 585-275-6007
    Affiliations
    Department of Biochemistry and Biophysics, Center for RNA Biology, and Center for AIDS Research, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
    Search for articles by this author
Open AccessPublished:May 12, 2019DOI:https://doi.org/10.1074/jbc.REV119.006860
      Small molecules and short peptides that potently and selectively bind RNA are rare, making the molecular structures of these complexes highly exceptional. Accordingly, several recent investigations have provided unprecedented structural insights into how peptides and proteins recognize the HIV-1 transactivation response (TAR) element, a 59-nucleotide-long, noncoding RNA segment in the 5′ long terminal repeat region of viral transcripts. Here, we offer an integrated perspective on these advances by describing earlier progress on TAR binding to small molecules, and by drawing parallels to recent successes in the identification of compounds that target the hepatitis C virus internal ribosome entry site (IRES) and the flavin-mononucleotide riboswitch. We relate this work to recent progress that pinpoints specific determinants of TAR recognition by: (i) viral Tat proteins, (ii) an innovative lab-evolved TAR-binding protein, and (iii) an ultrahigh-affinity cyclic peptide. New structural details are used to model the TAR–Tat–super-elongation complex (SEC) that is essential for efficient viral transcription and represents a focal point for antiviral drug design. A key prediction is that the Tat transactivation domain makes modest contacts with the TAR apical loop, whereas its arginine-rich motif spans the entire length of the TAR major groove. This expansive interface has significant implications for drug discovery and design, and it further suggests that future lab-evolved proteins could be deployed to discover steric restriction points that block Tat-mediated recruitment of the host SEC to HIV-1 TAR.

      Introduction

      Noncoding (nc)RNAs exhibit remarkable architectural diversity that contributes to function in multiple gene-regulatory settings (
      • Cech T.R.
      • Steitz J.A.
      The noncoding RNA revolution–trashing old rules to forge new ones.
      ). Although the human proteome is derived from only a small fraction of the genome (0.05%), the preponderance of the DNA blueprint is transcribed into ncRNA (
      • Pennisi E.
      Genomics. ENCODE project writes eulogy for junk DNA.
      ,
      • Djebali S.
      • Davis C.A.
      • Merkel A.
      • Dobin A.
      • Lassmann T.
      • Mortazavi A.
      • Tanzer A.
      • Lagarde J.
      • Lin W.
      • Schlesinger F.
      • Xue C.
      • Marinov G.K.
      • Khatun J.
      • Williams B.A.
      • Zaleski C.
      • et al.
      Landscape of transcription in human cells.
      ). As a result, ncRNA transcripts provide important opportunities to intervene in a range of biological processes and diseases (
      • Matsui M.
      • Corey D.R.
      Non-coding RNAs as drug targets.
      ,
      • Li Z.
      • Rana T.M.
      Therapeutic targeting of microRNAs: current status and future challenges.
      • Disney M.D.
      • Dwyer B.G.
      • Childs-Disney J.L.
      Drugging the RNA World.
      ). Such pursuits are especially meaningful in light of the fact that only 3.5–10% of the proteome is likely to be druggable (
      • Warner K.D.
      • Hajdin C.E.
      • Weeks K.M.
      Principles for targeting RNA with drug-like small molecules.
      ,
      • Hopkins A.L.
      • Groom C.R.
      The druggable genome.
      ). Substantial evidence demonstrates that a handful of ncRNAs adopt elegant three-dimensional folds with distinct topologies and recurrent architectural motifs (
      • Battle D.J.
      • Doudna J.A.
      Specificity of RNA–RNA helix recognition.
      • Chan C.W.
      • Chetnani B.
      • Mondragón A.
      Structure and function of the T-loop structural motif in noncoding RNAs.
      ,
      • Hendrix D.K.
      • Brenner S.E.
      • Holbrook S.R.
      RNA structural motifs: building blocks of a modular biomolecule.
      ,
      • Lilley D.M.
      The K-turn motif in riboswitches and other RNA species.
      ,
      • Nissen P.
      • Ippolito J.A.
      • Ban N.
      • Moore P.B.
      • Steitz T.A.
      RNA tertiary interactions in the large ribosomal subunit: the A-minor motif.
      • Peselis A.
      • Serganov A.
      Structure and function of pseudoknots involved in gene expression control.
      ), including cavities and deep grooves predisposed to ligand binding (
      • Warner K.D.
      • Hajdin C.E.
      • Weeks K.M.
      Principles for targeting RNA with drug-like small molecules.
      ,
      • Liberman J.A.
      • Wedekind J.E.
      Base ionization and ligand binding: how small ribozymes and riboswitches gain a foothold in a protein world.
      ,
      • Hermann T.
      • Westhof E.
      Rational drug design and high-throughput techniques for RNA targets.
      ). These properties are suited for shape-specific recognition of small molecules or peptides and provide a basis to manipulate conformation or dynamics to alter downstream function. Several notable achievements accentuate such efforts, including the identification of inhibitors that target the following: cancer-associated miR-21; CUG repeats of myotonic dystrophy; riboswitches in pathogenic bacteria; and exon splicing in spinal muscular atrophy (
      • Shi Z.
      • Zhang J.
      • Qian X.
      • Han L.
      • Zhang K.
      • Chen L.
      • Liu J.
      • Ren Y.
      • Yang M.
      • Zhang A.
      • Pu P.
      • Kang C.
      AC1MMYR2, an inhibitor of dicer-mediated biogenesis of Oncomir miR-21, reverses epithelial–mesenchymal transition and suppresses tumor growth and progression.
      • Warf M.B.
      • Nakamori M.
      • Matthys C.M.
      • Thornton C.A.
      • Berglund J.A.
      Pentamidine reverses the splicing defects associated with myotonic dystrophy.
      ,
      • Blount K.F.
      • Megyola C.
      • Plummer M.
      • Osterman D.
      • O'Connell T.
      • Aristoff P.
      • Quinn C.
      • Chrusciel R.A.
      • Poel T.J.
      • Schostarez H.J.
      • Stewart C.A.
      • Walker D.P.
      • Wuts P.G.
      • Breaker R.R.
      Novel riboswitch-binding flavin analog that protects mice against Clostridium difficile infection without inhibiting cecal flora.
      ,
      • Blount K.F.
      • Wang J.X.
      • Lim J.
      • Sudarsan N.
      • Breaker R.R.
      Antibacterial lysine analogs that target lysine riboswitches.
      ,
      • Howe J.A.
      • Wang H.
      • Fischmann T.O.
      • Balibar C.J.
      • Xiao L.
      • Galgoci A.M.
      • Malinverni J.C.
      • Mayhood T.
      • Villafania A.
      • Nahvi A.
      • Murgolo N.
      • Barbieri C.M.
      • Mann P.A.
      • Carr D.
      • Xia E.
      • et al.
      Selective small-molecule inhibition of an RNA structural element.
      ,
      • Howe J.A.
      • Xiao L.
      • Fischmann T.O.
      • Wang H.
      • Tang H.
      • Villafania A.
      • Zhang R.
      • Barbieri C.M.
      • Roemer T.
      Atomic resolution mechanistic studies of ribocil: a highly selective unnatural ligand mimic of the E. coli FMN riboswitch.
      ,
      • Lee E.R.
      • Blount K.F.
      • Breaker R.R.
      Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression.
      ,
      • Sudarsan N.
      • Cohen-Chalamish S.
      • Nakamura S.
      • Emilsson G.M.
      • Breaker R.R.
      Thiamine pyrophosphate riboswitches are targets for the antimicrobial compound pyrithiamine.
      ,
      • Vicens Q.
      • Mondragón E.
      • Reyes F.E.
      • Coish P.
      • Aristoff P.
      • Berman J.
      • Kaur H.
      • Kells K.W.
      • Wickens P.
      • Wilson J.
      • Gadwood R.C.
      • Schostarez H.J.
      • Suto R.K.
      • Blount K.F.
      • Batey R.T.
      Structure–activity relationship of flavin analogues that target the flavin mononucleotide riboswitch.
      ,
      • Wang H.
      • Mann P.A.
      • Xiao L.
      • Gill C.
      • Galgoci A.M.
      • Howe J.A.
      • Villafania A.
      • Barbieri C.M.
      • Malinverni J.C.
      • Sher X.
      • Mayhood T.
      • McCurry M.D.
      • Murgolo N.
      • Flattery A.
      • Mack M.
      • Roemer T.
      Dual-targeting small-molecule inhibitors of the Staphylococcus aureus FMN riboswitch disrupt riboflavin homeostasis in an infectious setting.
      ,
      • Palacino J.
      • Swalley S.E.
      • Song C.
      • Cheung A.K.
      • Shu L.
      • Zhang X.
      • Van Hoosear M.
      • Shin Y.
      • Chin D.N.
      • Keller C.G.
      • Beibel M.
      • Renaud N.A.
      • Smith T.M.
      • Salcius M.
      • Shi X.
      • et al.
      SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice.
      • Dibrov S.M.
      • Ding K.
      • Brunn N.D.
      • Parker M.A.
      • Bergdahl B.M.
      • Wyles D.L.
      • Hermann T.
      Structure of a hepatitis C virus RNA domain in complex with a translation inhibitor reveals a binding mode reminiscent of riboswitches.
      ). These successes underscore the feasibility of sequence-specific targeting of RNAs to create research tools or as a means to treat human disease. Accordingly, delineating principles of molecular recognition represents a cornerstone for therapeutic design, especially as part of a combination-drug strategy to circumvent drug resistance by pathogens that undergo multiple genomic mutations per generation (
      • Wang H.
      • Mann P.A.
      • Xiao L.
      • Gill C.
      • Galgoci A.M.
      • Howe J.A.
      • Villafania A.
      • Barbieri C.M.
      • Malinverni J.C.
      • Sher X.
      • Mayhood T.
      • McCurry M.D.
      • Murgolo N.
      • Flattery A.
      • Mack M.
      • Roemer T.
      Dual-targeting small-molecule inhibitors of the Staphylococcus aureus FMN riboswitch disrupt riboflavin homeostasis in an infectious setting.
      ,
      • Ghosh A.K.
      • Anderson D.D.
      • Weber I.T.
      • Mitsuya H.
      Enhancing protein backbone binding–a fruitful concept for combating drug-resistant HIV.
      ).
      New mandates in HIV eradication and cure research (https://grants.nih.gov/grants/guide/notice-files/NOT-OD-15–137.html) (
      • Dieffenbach C.W.
      • Fauci A.S.
      Thirty years of HIV and AIDS: future challenges and opportunities.
      ,
      • Dahabieh M.S.
      • Battivelli E.
      • Verdin E.
      Understanding HIV latency: the road to an HIV cure.
      ) have led to a resurgence in efforts to target the transactivation response (TAR)
      The abbreviations used are: TAR
      transactivation response
      SEC
      super-elongation complex
      ARM
      arginine-rich motif
      IRES
      internal ribosome entry site
      PDB
      Protein Data Bank
      HCV
      hepatitis C virus
      ASM
      arginine sandwich motif
      BIV
      bovine immunodeficiency virus
      SPR
      surface plasmon resonance
      ITC
      isothermal titration calorimetry
      TBP
      TAR-binding protein.
      element. This 59-nucleotide RNA is located in the 5′-LTR of all viral transcripts and features a conserved hairpin that harbors an apical loop and pyrimidine-rich bulge that are each indispensable for transactivation (
      • Wei P.
      • Garber M.E.
      • Fang S.M.
      • Fischer W.H.
      • Jones K.A.
      A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.
      • Dingwall C.
      • Ernberg I.
      • Gait M.J.
      • Green S.M.
      • Heaphy S.
      • Karn J.
      • Lowe A.D.
      • Singh M.
      • Skinner M.A.
      HIV-1 tat protein stimulates transcription by binding to a U-rich bulge in the stem of the TAR RNA structure.
      ,
      • Dingwall C.
      • Ernberg I.
      • Gait M.J.
      • Green S.M.
      • Heaphy S.
      • Karn J.
      • Lowe A.D.
      • Singh M.
      • Skinner M.A.
      • Valerio R.
      Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro.
      ,
      • Jakobovits A.
      • Smith D.H.
      • Jakobovits E.B.
      • Capon D.J.
      A discrete element 3′ of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator.
      ,
      • Weeks K.M.
      • Ampe C.
      • Schultz S.C.
      • Steitz T.A.
      • Crothers D.M.
      Fragments of the HIV-1 Tat protein specifically bind TAR RNA.
      ,
      • Cordingley M.G.
      • LaFemina R.L.
      • Callahan P.L.
      • Condra J.H.
      • Sardana V.V.
      • Graham D.J.
      • Nguyen T.M.
      • LeGrow K.
      • Gotlib L.
      • Schlabach A.J.
      • et al.
      Sequence-specific interaction of Tat protein and Tat peptides with the transactivation-responsive sequence element of human immunodeficiency virus type 1 in vitro.
      ,
      • Delling U.
      • Reid L.S.
      • Barnett R.W.
      • Ma M.Y.
      • Climie S.
      • Sumner-Smith M.
      • Sonenberg N.
      Conserved nucleotides in the TAR RNA stem of human immunodeficiency virus type 1 are critical for Tat binding and trans activation: model for TAR RNA tertiary structure.
      • Roy S.
      • Delling U.
      • Chen C.H.
      • Rosen C.A.
      • Sonenberg N.
      A bulge structure in HIV-1 TAR RNA is required for Tat binding and Tat-mediated trans-activation.
      ). TAR interacts with the viral Tat protein, which recruits the host pTEFb complex away from inactivating HEXIM–7SK RNA complexes (Fig. 1A) (
      • Barboric M.
      • Yik J.H.
      • Czudnochowski N.
      • Yang Z.
      • Chen R.
      • Contreras X.
      • Geyer M.
      • Matija Peterlin B.
      • Zhou Q.
      Tat competes with HEXIM1 to increase the active pool of P-TEFb for HIV-1 transcription.
      • D’Orso I.
      • Jang G.M.
      • Pastuszak A.W.
      • Faust T.B.
      • Quezada E.
      • Booth D.S.
      • Frankel A.D.
      Transition step during assembly of HIV Tat:P–TEFb transcription complexes and transfer to TAR RNA.
      ,
      • He N.
      • Liu M.
      • Hsu J.
      • Xue Y.
      • Chou S.
      • Burlingame A.
      • Krogan N.J.
      • Alber T.
      • Zhou Q.
      HIV-1 Tat and host AFF4 recruit two transcription elongation factors into a bifunctional complex for coordinated activation of HIV-1 transcription.
      ,
      • McNamara R.P.
      • McCann J.L.
      • Gudipaty S.A.
      • D'Orso I.
      Transcription factors mediate the enzymatic disassembly of promoter-bound 7SK snRNP to locally recruit P-TEFb for transcription elongation.
      ,
      • Schulte A.
      • Czudnochowski N.
      • Barboric M.
      • Schönichen A.
      • Blazek D.
      • Peterlin B.M.
      • Geyer M.
      Identification of a cyclin T-binding domain in Hexim1 and biochemical analysis of its binding competition with HIV-1 Tat.
      ,
      • Sedore S.C.
      • Byers S.A.
      • Biglione S.
      • Price J.P.
      • Maury W.J.
      • Price D.H.
      Manipulation of P-TEFb control machinery by HIV: recruitment of P-TEFb from the large form by Tat and binding of HEXIM1 to TAR.
      • Sobhian B.
      • Laguette N.
      • Yatim A.
      • Nakamura M.
      • Levy Y.
      • Kiernan R.
      • Benkirane M.
      HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP.
      ). When localized to TAR, host kinase CDK9 within a super-elongation complex (SEC) phosphorylates RNA polymerase II, releasing it from a paused state to produce full-length viral mRNA (
      • Kim Y.K.
      • Bourgeois C.F.
      • Isel C.
      • Churcher M.J.
      • Karn J.
      Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation.
      ,
      • Isel C.
      • Karn J.
      Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation.
      • Peterlin B.M.
      • Price D.H.
      Controlling the elongation phase of transcription with P-TEFb.
      ). Structural and biochemical analysis of the Tat–pTEFb complex revealed Tat-specific conformational changes (
      • Tahirov T.H.
      • Babayeva N.D.
      • Varzavand K.
      • Cooper J.J.
      • Sedore S.C.
      • Price D.H.
      Crystal structure of HIV-1 Tat complexed with human P-TEFb.
      ,
      • Gu J.
      • Babayeva N.D.
      • Suwa Y.
      • Baranovskiy A.G.
      • Price D.H.
      • Tahirov T.H.
      Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4.
      ). Because of the essential role of Tat in securing pTEFb for processive viral transcription, efforts are focused on the development of inhibitors that block this key host–virus protein interaction (
      • Mousseau G.
      • Clementz M.A.
      • Bakeman W.N.
      • Nagarsheth N.
      • Cameron M.
      • Shi J.
      • Baran P.
      • Fromentin R.
      • Chomont N.
      • Valente S.T.
      An analog of the natural steroidal alkaloid cortistatin A potently suppresses Tat-dependent HIV transcription.
      ,
      • Wan Z.
      • Chen X.
      Triptolide inhibits human immunodeficiency virus type 1 replication by promoting proteasomal degradation of Tat protein.
      • Narayan V.
      • Ravindra K.C.
      • Chiaro C.
      • Cary D.
      • Aggarwal B.B.
      • Henderson A.J.
      • Prabhu K.S.
      Celastrol inhibits Tat-mediated human immunodeficiency virus (HIV) transcription and replication.
      ). Sustained inhibition of Tat could lock HIV into a state of deep latency and represents one strategy to produce a functional cure (
      • Mousseau G.
      • Valente S.
      Strategies to block HIV transcription: focus on small molecule Tat inhibitors.
      ,
      • Kessing C.F.
      • Nixon C.C.
      • Li C.
      • Tsai P.
      • Takata H.
      • Mousseau G.
      • Ho P.T.
      • Honeycutt J.B.
      • Fallahi M.
      • Trautmann L.
      • Garcia J.V.
      • Valente S.T.
      In vivo suppression of HIV rebound by Didehydro-Cortistatin A, a “Block-and-Lock” strategy for HIV-1 treatment.
      ).
      Figure thumbnail gr1
      Figure 1HIV-1 TAR role in transcription, sequence conservation, and secondary structure. A, cartoon diagram of an inactive pTEFb complex comprising CDK9 and CycT1 in the context of HEXIM protein bound to 7SK ncRNA in the host. The arrow indicates that addition of the HIV-1 regulatory protein Tat competes with HEXIM, removing pTEFb from 7SK, which is then escorted by Tat to the TAR RNA element of HIV-1 (
      • Peterlin B.M.
      • Brogie J.E.
      • Price D.H.
      7SK snRNA: a noncoding RNA that plays a major role in regulating eukaryotic transcription.
      ). TAR is essential for transcription and is depicted as a stem loop interrupted by a central bulge that comprises nucleotides 18–44 of the viral transcript (
      • Muesing M.A.
      • Smith D.H.
      • Capon D.J.
      Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein.
      ,
      • Feng S.
      • Holland E.C.
      HIV-1 tat trans-activation requires the loop sequence within tar.
      ). Tat interacts directly with TAR and promotes formation of a host SEC comprising pTEFb, scaffold proteins such as AFF4, and other factors (
      • He N.
      • Liu M.
      • Hsu J.
      • Xue Y.
      • Chou S.
      • Burlingame A.
      • Krogan N.J.
      • Alber T.
      • Zhou Q.
      HIV-1 Tat and host AFF4 recruit two transcription elongation factors into a bifunctional complex for coordinated activation of HIV-1 transcription.
      ,
      • Sobhian B.
      • Laguette N.
      • Yatim A.
      • Nakamura M.
      • Levy Y.
      • Kiernan R.
      • Benkirane M.
      HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP.
      ,
      • Gu J.
      • Babayeva N.D.
      • Suwa Y.
      • Baranovskiy A.G.
      • Price D.H.
      • Tahirov T.H.
      Crystal structure of HIV-1 Tat complexed with human P-TEFb and AFF4.
      ,
      • Schulze-Gahmen U.
      • Echeverria I.
      • Stjepanovic G.
      • Bai Y.
      • Lu H.
      • Schneidman-Duhovny D.
      • Doudna J.A.
      • Zhou Q.
      • Sali A.
      • Hurley J.H.
      Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex.
      ,
      • Schulze-Gahmen U.
      • Lu H.
      • Zhou Q.
      • Alber T.
      AFF4 binding to Tat–P-TEFb indirectly stimulates TAR recognition of super-elongation complexes at the HIV promoter.
      ,
      • Smith E.
      • Lin C.
      • Shilatifard A.
      The super-elongation complex (SEC) and MLL in development and disease.
      ). CDK9 phosphorylates host RNA polymerase II in its CTD, which releases pausing and stimulates synthesis of full-length viral transcripts (
      • Wei P.
      • Garber M.E.
      • Fang S.M.
      • Fischer W.H.
      • Jones K.A.
      A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.
      ,
      • Zhu Y.
      • Pe'ery T.
      • Peng J.
      • Ramanathan Y.
      • Marshall N.
      • Marshall T.
      • Amendt B.
      • Mathews M.B.
      • Price D.H.
      Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro.
      ,
      • Mancebo H.S.
      • Lee G.
      • Flygare J.
      • Tomassini J.
      • Luu P.
      • Zhu Y.
      • Peng J.
      • Blau C.
      • Hazuda D.
      • Price D.
      • Flores O.
      P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro.
      • Yang X.
      • Gold M.O.
      • Tang D.N.
      • Lewis D.E.
      • Aguilar-Cordova E.
      • Rice A.P.
      • Herrmann C.H.
      TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines.
      ). B, Web-logo showing the sequence conservation of HIV-1 TAR based on circulating forms of the virus compiled as described (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ); blue represents the greatest conservation, and red indicates the poor conservation. Elements of the secondary structure including helical stems s1a and s1b are labeled. C, secondary structures of various TAR RNAs. The canonical Cyt30–Gua34 pair of HIV-1 TAR is supported by chemical modification, NMR, sequence conservation, and CycT1-binding requirements (
      • Dethoff E.A.
      • Hansen A.L.
      • Musselman C.
      • Watt E.D.
      • Andricioaei I.
      • Al-Hashimi H.M.
      Characterizing complex dynamics in the transactivation response element apical loop and motional correlations with the bulge by NMR, molecular dynamics, and mutagenesis.
      ,
      • Dethoff E.A.
      • Petzold K.
      • Chugh J.
      • Casiano-Negroni A.
      • Al-Hashimi H.M.
      Visualizing transient low-populated structures of RNA.
      ,
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ,
      • Kulinski T.
      • Olejniczak M.
      • Huthoff H.
      • Bielecki L.
      • Pachulska-Wieczorek K.
      • Das A.T.
      • Berkhout B.
      • Adamiak R.W.
      The apical loop of the HIV-1 TAR RNA hairpin is stabilized by a cross-loop base pair.
      ,
      • Richter S.
      • Cao H.
      • Rana T.M.
      Specific HIV-1 TAR RNA loop sequence and functional groups are required for human cyclin T1-Tat–TAR ternary complex formation.
      ). A key difference between HIV-1 and HIV-2 TAR is deletion of Cyt24 in the central bulge (

      Foley, B., Leitner, T., Apetrei, C., Hahn, B., Mizrachi, I., Mullins, J., Rambaut, A., Wolinsky, S., and Korber, B., (2018) HIV Sequence Compendium, pp. LA-UR-18–25673, Los Alamos National Laboratory, Los Alamos, NM

      ). Details of the BIV TAR secondary structure were derived from Refs.
      • Puglisi J.D.
      • Chen L.
      • Blanchard S.
      • Frankel A.D.
      Solution structure of a bovine immunodeficiency virus Tat–TAR peptide–RNA complex.
      ,
      • Ye X.
      • Kumar R.A.
      • Patel D.J.
      Molecular recognition in the bovine immunodeficiency virus Tat peptide–TAR RNA complex.
      ,
      • Chen L.
      • Frankel A.D.
      An RNA-binding peptide from bovine immunodeficiency virus Tat protein recognizes an unusual RNA structure.
      . CTD, C-terminal domain.
      Targeting TAR RNA represents a fundamentally different antiviral approach (
      • Mousseau G.
      • Mediouni S.
      • Valente S.T.
      Targeting HIV transcription: the quest for a functional cure.
      ). TAR is one of the most conserved RNA sequences in the viral genome (Fig. 1B). In addition to SEC binding, TAR functions as a pre-miRNA whose Dicer cleavage products block host–cell apoptosis, prolonging the viral life span in infected cells (
      • Klase Z.
      • Kale P.
      • Winograd R.
      • Gupta M.V.
      • Heydarian M.
      • Berro R.
      • McCaffrey T.
      • Kashanchi F.
      HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR.
      ,
      • Klase Z.
      • Winograd R.
      • Davis J.
      • Carpio L.
      • Hildreth R.
      • Heydarian M.
      • Fu S.
      • McCaffrey T.
      • Meiri E.
      • Ayash-Rashkovsky M.
      • Gilad S.
      • Bentwich Z.
      • Kashanchi F.
      HIV-1 TAR miRNA protects against apoptosis by altering cellular gene expression.
      • Ouellet D.L.
      • Plante I.
      • Landry P.
      • Barat C.
      • Janelle M.E.
      • Flamand L.
      • Tremblay M.J.
      • Provost P.
      Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element.
      ). For these reasons, TAR is a high-value drug target whose inhibition could potentially disrupt viral transcription in chronic as well as latent infections. However, no such inhibitors are clinically available, and TAR has resisted the development of therapeutics, despite success in the identification of compounds that target the RNA with specificity and affinity (
      • Davidson A.
      • Patora-Komisarska K.
      • Robinson J.A.
      • Varani G.
      Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein.
      ,
      • Shortridge M.D.
      • Wille P.T.
      • Jones A.N.
      • Davidson A.
      • Bogdanovic J.
      • Arts E.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.
      • Lind K.E.
      • Du Z.
      • Fujinaga K.
      • Peterlin B.M.
      • James T.L.
      Structure-based computational database screening, in vitro assay, and NMR assessment of compounds that target TAR RNA.
      ).
      As with other RNA molecules, TAR is dynamic and adopts multiple conformations (
      • Dethoff E.A.
      • Hansen A.L.
      • Musselman C.
      • Watt E.D.
      • Andricioaei I.
      • Al-Hashimi H.M.
      Characterizing complex dynamics in the transactivation response element apical loop and motional correlations with the bulge by NMR, molecular dynamics, and mutagenesis.
      ,
      • Dethoff E.A.
      • Petzold K.
      • Chugh J.
      • Casiano-Negroni A.
      • Al-Hashimi H.M.
      Visualizing transient low-populated structures of RNA.
      • Borkar A.N.
      • Bardaro Jr., M.F.
      • Camilloni C.
      • Aprile F.A.
      • Varani G.
      • Vendruscolo M.
      Structure of a low-population binding intermediate in protein–RNA recognition.
      ), undermining efforts to obtain high-resolution crystal structures (
      • Schulze-Gahmen U.
      • Echeverria I.
      • Stjepanovic G.
      • Bai Y.
      • Lu H.
      • Schneidman-Duhovny D.
      • Doudna J.A.
      • Zhou Q.
      • Sali A.
      • Hurley J.H.
      Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex.
      ). NMR has bridged many gaps in our understanding of TAR with nearly 20 distinct structures determined of the isolated (apo) RNA and in complex with peptides or small molecules (
      • Ouellet D.L.
      • Plante I.
      • Landry P.
      • Barat C.
      • Janelle M.E.
      • Flamand L.
      • Tremblay M.J.
      • Provost P.
      Identification of functional microRNAs released through asymmetrical processing of HIV-1 TAR element.
      ,
      • Davidson A.
      • Patora-Komisarska K.
      • Robinson J.A.
      • Varani G.
      Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein.
      ,
      • Borkar A.N.
      • Bardaro Jr., M.F.
      • Camilloni C.
      • Aprile F.A.
      • Varani G.
      • Vendruscolo M.
      Structure of a low-population binding intermediate in protein–RNA recognition.
      • Schulze-Gahmen U.
      • Echeverria I.
      • Stjepanovic G.
      • Bai Y.
      • Lu H.
      • Schneidman-Duhovny D.
      • Doudna J.A.
      • Zhou Q.
      • Sali A.
      • Hurley J.H.
      Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex.
      ,
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ,
      • Davidson A.
      • Leeper T.C.
      • Athanassiou Z.
      • Patora-Komisarska K.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein.
      ,
      • Merriman D.K.
      • Xue Y.
      • Yang S.
      • Kimsey I.J.
      • Shakya A.
      • Clay M.
      • Al-Hashimi H.M.
      Shortening the HIV-1 TAR RNA bulge by a single nucleotide preserves motional modes over a broad range of time scales.
      ,
      • Greenbaum N.L.
      How Tat targets TAR: structure of the BIV peptide–RNA complex.
      ,
      • Puglisi J.D.
      • Chen L.
      • Blanchard S.
      • Frankel A.D.
      Solution structure of a bovine immunodeficiency virus Tat–TAR peptide–RNA complex.
      ,
      • Schulze-Gahmen U.
      • Hurley J.H.
      Structural mechanism for HIV-1 TAR loop recognition by Tat and the super-elongation complex.
      ,
      • Ye X.
      • Kumar R.A.
      • Patel D.J.
      Molecular recognition in the bovine immunodeficiency virus Tat peptide–TAR RNA complex.
      ,
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      ,
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ,
      • Muesing M.A.
      • Smith D.H.
      • Capon D.J.
      Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein.
      ,
      • Puglisi J.D.
      • Tan R.
      • Calnan B.J.
      • Frankel A.D.
      • Williamson J.R.
      Conformation of the TAR RNA-arginine complex by NMR spectroscopy.
      ,
      • Aboul-ela F.
      • Karn J.
      • Varani G.
      Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge.

      Saenger, W., (1984) Principles of Nucleic Acid Structure, pp. 1–556, Springer-Verlag, New York

      ). Even so, TAR has been historically challenging for NMR due to significant line broadening of resonances, which has hindered the acquisition of restraints needed to generate high-quality models (
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ,
      • Davidson A.
      • Leeper T.C.
      • Athanassiou Z.
      • Patora-Komisarska K.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein.
      • Merriman D.K.
      • Xue Y.
      • Yang S.
      • Kimsey I.J.
      • Shakya A.
      • Clay M.
      • Al-Hashimi H.M.
      Shortening the HIV-1 TAR RNA bulge by a single nucleotide preserves motional modes over a broad range of time scales.
      ). For this reason, many efforts have focused on closely related HIV-2 or BIV TAR (Fig. 1C), which are structurally better defined (
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ,
      • Greenbaum N.L.
      How Tat targets TAR: structure of the BIV peptide–RNA complex.
      ,
      • Puglisi J.D.
      • Chen L.
      • Blanchard S.
      • Frankel A.D.
      Solution structure of a bovine immunodeficiency virus Tat–TAR peptide–RNA complex.
      ). Additional structural improvements have been attained through engineered RNA constructs to promote crystal contacts or by exploiting structurally well-characterized proteins, such as U1A, as a starting platform for lab-based evolution and structural studies. These developments have led to a series of new structures including: exciting TAR–Tat and TAR–Tat–SEC complexes (
      • Schulze-Gahmen U.
      • Hurley J.H.
      Structural mechanism for HIV-1 TAR loop recognition by Tat and the super-elongation complex.
      ,
      • Ye X.
      • Kumar R.A.
      • Patel D.J.
      Molecular recognition in the bovine immunodeficiency virus Tat peptide–TAR RNA complex.
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      ), a co-crystal structure of TAR bound to a lab-evolved protein from the Wedekind lab (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ), and an ultrahigh-affinity cyclic peptide bound to TAR (
      • Shortridge M.D.
      • Wille P.T.
      • Jones A.N.
      • Davidson A.
      • Bogdanovic J.
      • Arts E.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.
      ). Here, we put these novel discoveries into perspective by considering prior characterization of TAR apo- and bound-state conformations. We then consider molecular recognition by representative small molecules, which are then contrasted with recent high-quality ncRNA–inhibitor complexes. A major take-home message is that peptide-mediated TAR recognition utilizes some common molecular-recognition principles, such as the arginine-sandwich motif (ASM)—a primary determinant of affinity and specificity observed in both natural TAR–Tat complexes, as well as TAR binding by a lab-evolved protein. In contrast, no consistent rules of recognition could be discerned for existing TAR–small molecule complexes, despite the use of common guanidinium groups. As the reader will see, new TAR–peptide and TAR–protein complexes offer the most cogent details to address challenges and opportunities associated with effective TAR targeting. In this respect, the best days of RNA drug discovery appear to lie ahead.

      TAR adopts two major conformations that depend on ligand binding

      The discovery of TAR–Tat-mediated gene regulation in HIV-1 (
      • Muesing M.A.
      • Smith D.H.
      • Capon D.J.
      Regulation of mRNA accumulation by a human immunodeficiency virus trans-activator protein.
      ) started a race to elucidate the underlying molecular determinants that give rise to this unique viral RNA–protein interaction. Major steps were made by NMR analyses of TAR in complex with the arginine analogue argininamide and in a ligand-free (apo) state. This work revealed TAR’s overall hairpin architecture as well as substantial backbone rearrangements at the central bulge resulting from ligand binding (
      • Puglisi J.D.
      • Tan R.
      • Calnan B.J.
      • Frankel A.D.
      • Williamson J.R.
      Conformation of the TAR RNA-arginine complex by NMR spectroscopy.
      ,
      • Aboul-ela F.
      • Karn J.
      • Varani G.
      Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge.
      ). Indeed, when specific effectors interact with the major groove, the RNA adopts a slightly bent (∼165°) helical axis formed by coaxial stacking of stem 1a and stem 1b (s1a and s1b) (Fig. 2A), wherein the bases of the central bulge jut outward. This conformation exhibits a high degree of concave surface suited to ligand binding (Fig. 2, A and B). The TAR major groove is characterized by a narrow width (3.9 ± 0.5 Å) and substantial depth (10.3 ± 0.3 Å) reminiscent of an ideal A-form duplex (i.e. 2.7 Å wide by 13.5 Å deep (

      Saenger, W., (1984) Principles of Nucleic Acid Structure, pp. 1–556, Springer-Verlag, New York

      )). In contrast, the minor-groove width (9.9 ± 0.6 Å) and depth (1.0 ± 0.6 Å) are substantially wider and shallower than a typical A-form helix (5.7 Å wide by 7.5 Å deep (

      Saenger, W., (1984) Principles of Nucleic Acid Structure, pp. 1–556, Springer-Verlag, New York

      )). A hallmark of the ligand-bound conformation is that Uri23 interacts with the Hoogsteen edge of a nearby adenine to form a Uri23·Ade27–Uri38 base triple (Fig. 2C)— a feature observed in most peptide- and protein-bound TAR structures (
      • Davidson A.
      • Leeper T.C.
      • Athanassiou Z.
      • Patora-Komisarska K.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein.
      ,
      • Ye X.
      • Kumar R.A.
      • Patel D.J.
      Molecular recognition in the bovine immunodeficiency virus Tat peptide–TAR RNA complex.
      ,
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ,
      • Puglisi J.D.
      • Tan R.
      • Calnan B.J.
      • Frankel A.D.
      • Williamson J.R.
      Conformation of the TAR RNA-arginine complex by NMR spectroscopy.
      ,
      • Leeper T.C.
      • Athanassiou Z.
      • Dias R.L.
      • Robinson J.A.
      • Varani G.
      TAR RNA recognition by a cyclic peptidomimetic of Tat protein.
      ). Cyt24 and Uri25 extrude from the helical core with bases pointing into solvent. Molecular dynamics simulations of TAR in complex with a lab-evolved protein revealed that this long-range triple is preserved over 16 μs but disintegrates rapidly when the protein is omitted from the simulation (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ).
      Figure thumbnail gr2
      Figure 2Overview of TAR conformational differences in the ligand-bound and ligand-free states. A, surface map of concave and convex features for the bound-state of HIV-2 TAR (PDB entry 6mce) (
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      ). The helical axis (purple line) deviates subtly from linearity with an angle of 165°. B, view of A rotated +45°; convex features map mainly to the apical loop and bulge. C, ribbon model passing through the phosphate backbone showing the Uri23·Ade27–Uri38 major-groove base triple—a hallmark long-range interaction characteristic of the ligand-bound state. The flanking UCU bulge is depicted. Coordinates were derived from the HIV-1 TAR–TBP6.7 complex (PDB entry 6cmn) (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ). D, surface map of concave and convex features for apo-state HIV-1 TAR (PDB entry 1anr) (
      • Aboul-ela F.
      • Karn J.
      • Varani G.
      Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge.
      ). The helical axis bends substantially with an overall angle of 121°. The structure is characterized by more convex surfaces compared with the bound state. E, view of D rotated +45° to emphasize the helical bend. F, ribbon model revealing the Ade27–Uri38 duplex but not the major groove triple. Bases of the flanking UCU bulge penetrate the core contributing to the bend. The helical axis, angle, major-groove width, and depth were calculated by Curves+ (
      • Blanchet C.
      • Pasi M.
      • Zakrzewska K.
      • Lavery R.
      CURVES+ web server for analyzing and visualizing the helical, backbone and groove parameters of nucleic acid structures.
      ); when applicable, parameters were computed as the average of the NMR ensemble. Concave and convex properties for each nucleotide of the lowest-energy NMR structures were calculated by Cx (
      • Pintar A.
      • Carugo O.
      • Pongor S.
      CX, an algorithm that identifies protruding atoms in proteins.
      ) and displayed on a Curves+ output file as a heat-map surface using PyMOL (Schrödinger, LLC). Here and elsewhere, perceived hydrogen bonds and related interactions are depicted as broken lines.
      In the absence of interacting ligands, the TAR helical axis is bent more acutely to 121° (Fig. 2D). The major groove is extraordinarily wide (13.1 ± 4.2 Å) and shallow (4.4 ± 3.2 Å) compared with an A-form helix. These features are accompanied by a relative reduction of concave surface in the major groove (Fig. 2, D and E versus A and B), a property that is less conducive to binding by small molecules or peptides (
      • Pintar A.
      • Carugo O.
      • Pongor S.
      CX, an algorithm that identifies protruding atoms in proteins.
      ). Because Uri23 and Cyt24 reside inside the duplex without base pairing to the adjacent strand, the s1a–s1b coaxial stack is wedged apart yielding an underpacked core (Fig. 2, D and F) (
      • Aboul-ela F.
      • Karn J.
      • Varani G.
      Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge.
      ). Whereas Uri23 and Cyt24 adopt stacked and inclined base orientations relative to underlying base pairs, Uri25 loops out of the duplex. These features prohibit formation of the hallmark base triple, leaving only the canonical Ade27–Uri38 pair (Fig. 2F). As a result, the central bulge exhibits significantly more conformational flexibility in the apo-state compared with the ligand-bound state, as observed by NMR analysis and molecular dynamics simulations (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ,
      • Aboul-ela F.
      • Karn J.
      • Varani G.
      Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge.
      ).
      Overall, the propensity of TAR to adopt two major bulge conformations is well-suited to ligand binding. Solution studies showed that the major groove is narrow and deep in the presence of ligand (
      • Puglisi J.D.
      • Chen L.
      • Blanchard S.
      • Frankel A.D.
      Solution structure of a bovine immunodeficiency virus Tat–TAR peptide–RNA complex.
      ,
      • Puglisi J.D.
      • Tan R.
      • Calnan B.J.
      • Frankel A.D.
      • Williamson J.R.
      Conformation of the TAR RNA-arginine complex by NMR spectroscopy.
      ,
      • Puglisi J.D.
      • Chen L.
      • Frankel A.D.
      • Williamson J.R.
      Role of RNA structure in arginine recognition of TAR RNA.
      ). As such, the RNA is capable of folding around a ligand—such as the unstructured peptide of Tat—giving rise to a complementary interface with a substantial buried surface area (
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      ). Understanding the details of such RNA–peptide interactions provides insight into the basis for affinity and specificity, while revealing stereochemical features that are unique to the respective apo- and ligand-bound states. Such information is of high value for the design of novel antivirals that target the HIV TAR element.

      Targeting TAR with small molecules

      During the past 2 decades, multiple labs have worked to identify small molecules that bind HIV-1 TAR (
      • Lind K.E.
      • Du Z.
      • Fujinaga K.
      • Peterlin B.M.
      • James T.L.
      Structure-based computational database screening, in vitro assay, and NMR assessment of compounds that target TAR RNA.
      ,
      • Crawford D.W.
      • Blakeley B.D.
      • Chen P.H.
      • Sherpa C.
      • Le Grice S.F.
      • Laird-Offringa I.A.
      • McNaughton B.R.
      An evolved RNA recognition Motif that suppresses HIV-1 Tat/TAR-dependent transcription.
      • Stelzer A.C.
      • Frank A.T.
      • Kratz J.D.
      • Swanson M.D.
      • Gonzalez-Hernandez M.J.
      • Lee J.
      • Andricioaei I.
      • Markovitz D.M.
      • Al-Hashimi H.M.
      Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble.
      ,
      • Patwardhan N.N.
      • Ganser L.R.
      • Kapral G.J.
      • Eubanks C.S.
      • Lee J.
      • Sathyamoorthy B.
      • Al-Hashimi H.M.
      • Hargrove A.E.
      Amiloride as a new RNA-binding scaffold with activity against HIV-1 TAR.
      ,
      • Lalonde M.S.
      • Lobritz M.A.
      • Ratcliff A.
      • Chamanian M.
      • Athanassiou Z.
      • Tyagi M.
      • Wong J.
      • Robinson J.A.
      • Karn J.
      • Varani G.
      • Arts E.J.
      Inhibition of both HIV-1 reverse transcription and gene expression by a cyclic peptide that binds the Tat-transactivating response element (TAR) RNA.
      ,
      • Richter S.
      • Parolin C.
      • Gatto B.
      • Del Vecchio C.
      • Brocca-Cofano E.
      • Fravolini A.
      • Palù G.
      • Palumbo M.
      Inhibition of human immunodeficiency virus type 1 tat-trans-activation-responsive region interaction by an antiviral quinolone derivative.
      ,
      • Mei H.Y.
      • Mack D.P.
      • Galan A.A.
      • Halim N.S.
      • Heldsinger A.
      • Loo J.A.
      • Moreland D.W.
      • Sannes-Lowery K.A.
      • Sharmeen L.
      • Truong H.N.
      • Czarnik A.W.
      Discovery of selective, small-molecule inhibitors of RNA complexes–I. The Tat protein/TAR RNA complexes required for HIV-1 transcription.
      • Abulwerdi F.A.
      • Shortridge M.D.
      • Sztuba-Solinska J.
      • Wilson R.
      • Le Grice S.F.
      • Varani G.
      • Schneekloth Jr., J.S.
      Development of small molecules with a noncanonical binding mode to HIV-1 trans activation response (TAR) RNA.
      ). To gain perspective about the successes and ongoing challenges, it is instructive to examine the handful of structurally characterized TAR–small molecule complexes to assess compound localization and commonalities in their modes of molecular recognition. A survey of such complexes (Table 1) reveals common chemical features, including positively charged alkylamine or guanidinium groups and planar heteroaromatic groups, such as naphthyl, indole, phenyl, or phenothiazine moieties. Although neomycin and derivatives thereof are known to bind TAR (
      • Faber C.
      • Sticht H.
      • Schweimer K.
      • Rösch P.
      Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B.
      ), we will not consider aminoglycosides here, due to their promiscuous RNA binding resulting from many positively charged amines (Fig. 3A), their toxicity (
      • Stelzer A.C.
      • Frank A.T.
      • Kratz J.D.
      • Swanson M.D.
      • Gonzalez-Hernandez M.J.
      • Lee J.
      • Andricioaei I.
      • Markovitz D.M.
      • Al-Hashimi H.M.
      Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble.
      ,
      • Vicens Q.
      • Westhof E.
      RNA as a drug target: the case of aminoglycosides.
      ,
      • Chittapragada M.
      • Roberts S.
      • Ham Y.W.
      Aminoglycosides: molecular insights on the recognition of RNA and aminoglycoside mimics.
      • Davidson A.
      • Begley D.W.
      • Lau C.
      • Varani G.
      A small-molecule probe induces a conformation in HIV TAR RNA capable of binding drug-like fragments.
      ), and the recent focus on compounds with “drug-like” properties in terms of potency, solubility, selectivity, and distribution, as well as RNA targeting by use of specific modes of molecular recognition (
      • Connelly C.M.
      • Moon M.H.
      • Schneekloth Jr., J.S.
      The emerging role of RNA as a therapeutic target for small molecules.
      ).
      Table 1Structural complexes of TAR in complex with small molecules
      TAR typeLigandLigand scaffoldMethodPBD codesRefs.
      HIV-1ArginineAmino acidNMR1arj
      • Aboul-ela F.
      • Karn J.
      • Varani G.
      The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein.
      HIV-2ArgininamideAmino acid derivativeNMR1akx and 1aju
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      HIV-1 (Δ apical loop)Calcium ionDivalent ionX-ray397d
      • Ippolito J.A.
      • Steitz T.A.
      A 1.3-A resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation.
      HIV-1NeomycinAminoglycosideNMR1qd3
      • Faber C.
      • Sticht H.
      • Schweimer K.
      • Rösch P.
      Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B.
      HIV-1AcetylpromazinePhenothiazine drugNMR1lvj
      • Du Z.
      • Lind K.E.
      • James T.L.
      Structure of TAR RNA complexed with a Tat–TAR interaction nanomolar inhibitor that was identified by computational screening.
      HIV-1RBT-550Benzyl indoleNMR1uts
      • Murchie A.I.
      • Davis B.
      • Isel C.
      • Afshar M.
      • Drysdale M.J.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.M.
      • Mirza S.
      • Prescott C.D.
      • Vaglio P.
      • Aboul-ela F.
      • Karn J.
      Structure-based drug design targeting an inactive RNA conformation: exploiting the flexibility of HIV-1 TAR RNA.
      HIV-1RBT-203Methoxy phenoxyNMR1uud
      • Davis B.
      • Afshar M.
      • Varani G.
      • Murchie A.I.
      • Karn J.
      • Lentzen G.
      • Drysdale M.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.
      • Aboul-ela F.
      Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic “hot spots”.
      HIV-1RBT-158BenzylpiperazineNMR1uui
      • Davis B.
      • Afshar M.
      • Varani G.
      • Murchie A.I.
      • Karn J.
      • Lentzen G.
      • Drysdale M.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.
      • Aboul-ela F.
      Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic “hot spots”.
      HIV-1Chemical probe4-Methoxynaphthalene-2-amineNMR2l8h
      • Davidson A.
      • Begley D.W.
      • Lau C.
      • Varani G.
      A small-molecule probe induces a conformation in HIV TAR RNA capable of binding drug-like fragments.
      Figure thumbnail gr3
      Figure 3Chemical structures, interaction properties, and representative modes of small-molecule binding to HIV TAR. A, chemical diagrams for various small molecules that bind TAR and have been characterized structurally by experimental approaches. Positively charged groups are light blue, and aromatic rings are pale yellow. Equilibrium KD values for TAR binding to neomycin and argininamide were derived from NMR (
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ,
      • Faber C.
      • Sticht H.
      • Schweimer K.
      • Rösch P.
      Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B.
      ). Ki values for RBT-203 and RBT-550 were measured for the ability to displace a Tat-derived peptide from TAR, as monitored by FRET (
      • Murchie A.I.
      • Davis B.
      • Isel C.
      • Afshar M.
      • Drysdale M.J.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.M.
      • Mirza S.
      • Prescott C.D.
      • Vaglio P.
      • Aboul-ela F.
      • Karn J.
      Structure-based drug design targeting an inactive RNA conformation: exploiting the flexibility of HIV-1 TAR RNA.
      ,
      • Davis B.
      • Afshar M.
      • Varani G.
      • Murchie A.I.
      • Karn J.
      • Lentzen G.
      • Drysdale M.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.
      • Aboul-ela F.
      Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic “hot spots”.
      ). The EC50 value of acetylproamizine was estimated based on an EMSA analysis of concentration-dependent disruption of a TAR–Tat–CycT1 complex (
      • Lind K.E.
      • Du Z.
      • Fujinaga K.
      • Peterlin B.M.
      • James T.L.
      Structure-based computational database screening, in vitro assay, and NMR assessment of compounds that target TAR RNA.
      ). Here and elsewhere, shape correlation coefficients for RNA–ligand interfaces were calculated by the program Sc on a scale of 0 to 1.0 (
      • Lawrence M.C.
      • Colman P.M.
      Shape complementarity at protein/protein interfaces.
      ). Calculations in A were applied to the following: TAR–neomycin (PDB entry 1qd3) (
      • Faber C.
      • Sticht H.
      • Schweimer K.
      • Rösch P.
      Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B.
      ); TAR–argininamide (PDB entry 1akx) (
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ); TAR–RBT-203 (PDB entry 1uub) (
      • Davis B.
      • Afshar M.
      • Varani G.
      • Murchie A.I.
      • Karn J.
      • Lentzen G.
      • Drysdale M.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.
      • Aboul-ela F.
      Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic “hot spots”.
      ); TAR–RBT550 (PDB entry 1uts) (
      • Murchie A.I.
      • Davis B.
      • Isel C.
      • Afshar M.
      • Drysdale M.J.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.M.
      • Mirza S.
      • Prescott C.D.
      • Vaglio P.
      • Aboul-ela F.
      • Karn J.
      Structure-based drug design targeting an inactive RNA conformation: exploiting the flexibility of HIV-1 TAR RNA.
      ); and TAR–acetylpromazine (PDB entry 1lvj) (
      • Lind K.E.
      • Du Z.
      • Fujinaga K.
      • Peterlin B.M.
      • James T.L.
      Structure-based computational database screening, in vitro assay, and NMR assessment of compounds that target TAR RNA.
      ). Sc values are the average derived from the reported NMR ensembles. Solvent-accessible surface areas of RNA–ligand interfaces were calculated by PISA (
      • Krissinel E.
      • Henrick K.
      Inference of macromolecular assemblies from crystalline state.
      ). B, ribbon diagram of HIV-1 TAR (PDB entry 6cmn) (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ) depicting the locations of nucleotides (green surface) that interact with various small molecules in . Most ligands bind in the major groove at the interface between s1a and s1b; neomycin binds in the minor groove (
      • Faber C.
      • Sticht H.
      • Schweimer K.
      • Rösch P.
      Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B.
      ). C, ribbon and ball–and–stick diagram of HIV-2 TAR in complex with argininamide. D, ribbon and ball–and–stick diagram of HIV-1 TAR in complex with RBT-550.
      Historically, arginine is one of the first small molecules shown to bind TAR, leading to a conformational change (
      • Tao J.
      • Frankel A.D.
      Specific binding of arginine to TAR RNA.
      ,
      • Zacharias M.
      • Hagerman P.J.
      The bend in RNA created by the trans-activation response element bulge of human immunodeficiency virus is straightened by arginine and by Tat-derived peptide.
      ). This amino acid—and derivatives thereof—has served as a proxy for selective binding of the Tat protein to TAR. This key interaction arises from specific contacts at Uri23, Gua26, and Ade27 (
      • Tao J.
      • Frankel A.D.
      Specific binding of arginine to TAR RNA.
      ,
      • Calnan B.J.
      • Tidor B.
      • Biancalana S.
      • Hudson D.
      • Frankel A.D.
      Arginine-mediated RNA recognition: the arginine fork.
      ,
      • Tao J.
      • Frankel A.D.
      Electrostatic interactions modulate the RNA-binding and transactivation specificities of the human immunodeficiency virus and simian immunodeficiency virus Tat proteins.
      • Long K.S.
      • Crothers D.M.
      Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA.
      ). Because more structural restraints were discernible for the HIV-2 TAR–argininamide complex compared with HIV-1 TAR–argininamide, the former analysis is considered to provide a definitive basis to evaluate this RNA–ligand interaction (
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ). Indeed, HIV-2 TAR differs from HIV-1 by deletion of Cyt24 in the UCU bulge (Fig. 1C). Both HIV TAR variants have similar KD values of ∼2 mm for argininamide (Fig. 3A). The HIV-2 TAR–argininamide NMR ensemble reveals that the ligand localizes to the major groove near the central bulge, where the guanidinium moiety engages in cation–π stacking between Ade22 and Uri23 (Fig. 3, B and C). Although NMR spectra did not provide direct evidence for hydrogen bonding between argininamide and Gua26 (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ), the guanidinium position and orientation are consistent with co-planar hydrogen bonding predicted by theoretical calculations (
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ). One metric of surface complementarity at the receptor–ligand interface is the shape correlation statistic (Sc) (
      • Lawrence M.C.
      • Colman P.M.
      Shape complementarity at protein/protein interfaces.
      ). The calculated Sc of 0.67 for the HIV-2 TAR–argininamide interaction (
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ) suggests substantial surface complementarity. The complex buries 229 Å2 of the argininamide solvent-accessible surface, which is 65% of the total ligand surface area. The observation that this RNA–ligand complex shares NOEs with the HIV-1 TAR–argininamide complex suggested similar modes of effector binding (
      • Puglisi J.D.
      • Tan R.
      • Calnan B.J.
      • Frankel A.D.
      • Williamson J.R.
      Conformation of the TAR RNA-arginine complex by NMR spectroscopy.
      ,
      • Aboul-ela F.
      • Karn J.
      • Varani G.
      The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein.
      ). Although the hallmark Uri23·Ade27–Uri38 triple of the bound state was absent in NOE assignments in one study (
      • Aboul-ela F.
      • Karn J.
      • Varani G.
      The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein.
      ), another study revealed that an isomorphic C+·G-C mutant interacts with argininamide in a pH-dependent manner, suggesting base-triple formation is needed for amino acid binding (
      • Puglisi J.D.
      • Chen L.
      • Frankel A.D.
      • Williamson J.R.
      Role of RNA structure in arginine recognition of TAR RNA.
      ).
      Argininamide binding to TAR provided several insights in terms of ligand localization and the determinants of binding (Fig. 3, B and C). As we will see, this mode of binding—known as an arginine sandwich motif (ASM)— was observed next in the context of TAR–Tat interactions (described below). Of course, high-affinity Tat-mediated recognition requires multiple arginines (
      • Long K.S.
      • Crothers D.M.
      Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA.
      ,
      • Churcher M.J.
      • Lamont C.
      • Hamy F.
      • Dingwall C.
      • Green S.M.
      • Lowe A.D.
      • Butler J.G.
      • Gait M.J.
      • Karn J.
      High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region.
      ,
      • Wang Z.
      • Shah K.
      • Rana T.M.
      Probing Tat peptide–TAR RNA interactions by psoralen photo-cross-linking.
      ). This knowledge and the application of electrostatic analysis to the TAR–argininamide complex prompted high-throughput screening of bis-guanidine compounds designed to mimic argininamide binding. Based on Tat–peptide-displacement assays, a top hit, RBT-203 (Fig. 3A), showed a Ki of 1.5 μm by FRET displacement, evidence of binding by surface plasmon resonance (SPR), as well as inhibition of Tat-mediated transcription in cell-free extracts at levels of 5–15 μm (
      • Murchie A.I.
      • Davis B.
      • Isel C.
      • Afshar M.
      • Drysdale M.J.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.M.
      • Mirza S.
      • Prescott C.D.
      • Vaglio P.
      • Aboul-ela F.
      • Karn J.
      Structure-based drug design targeting an inactive RNA conformation: exploiting the flexibility of HIV-1 TAR RNA.
      ,
      • Davis B.
      • Afshar M.
      • Varani G.
      • Murchie A.I.
      • Karn J.
      • Lentzen G.
      • Drysdale M.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.
      • Aboul-ela F.
      Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic “hot spots”.
      ). NMR analysis revealed the compound induces a conformation similar to that of the TAR–argininamide complex, although neither guanidinium group of RBT-203 interacts with a guanine base (
      • Davis B.
      • Afshar M.
      • Varani G.
      • Murchie A.I.
      • Karn J.
      • Lentzen G.
      • Drysdale M.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.
      • Aboul-ela F.
      Rational design of inhibitors of HIV-1 TAR RNA through the stabilisation of electrostatic “hot spots”.
      ). Addition of an indole ring into the RBT-203 benzyl scaffold and replacement of the guanidinium groups by piperazine and a primary amine improved the Ki to 39 nm, although antiviral activity was not assessed (
      • Murchie A.I.
      • Davis B.
      • Isel C.
      • Afshar M.
      • Drysdale M.J.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.M.
      • Mirza S.
      • Prescott C.D.
      • Vaglio P.
      • Aboul-ela F.
      • Karn J.
      Structure-based drug design targeting an inactive RNA conformation: exploiting the flexibility of HIV-1 TAR RNA.
      ). This new compound, RBT-550 (Fig. 3A), was shown by NMR to bind TAR in a fundamentally different manner compared with argininamide. The indole ring appears to intercalate adjacent to the UCU bulge between the Gua26–Cyt39 and Ade22–Uri40 base pairs (Fig. 3D). Uri23 does not form the hallmark base triple, and the primary amine of RBT-550 interacts with the Gua26 backbone; the piperazine moiety protrudes into solvent but appears to restrict the propylamine conformation in some members of the structural ensemble. Intercalation produces a high degree of shape complementarity (average Sc of 0.67), and 290 Å2 of the ligand is buried in the interface, representing 44% of its solvent-accessible surface. The observation that RBT-550 promotes a TAR conformation that differs from the argininamide-bound complex lends support to the idea that some small molecules can shift the RNA conformational equilibrium to an “inactive” state, which is a generally accepted drug strategy (
      • Dethoff E.A.
      • Petzold K.
      • Chugh J.
      • Casiano-Negroni A.
      • Al-Hashimi H.M.
      Visualizing transient low-populated structures of RNA.
      ,
      • Faber C.
      • Sticht H.
      • Schweimer K.
      • Rösch P.
      Structural rearrangements of HIV-1 Tat-responsive RNA upon binding of neomycin B.
      ,
      • Murchie A.I.
      • Davis B.
      • Isel C.
      • Afshar M.
      • Drysdale M.J.
      • Bower J.
      • Potter A.J.
      • Starkey I.D.
      • Swarbrick T.M.
      • Mirza S.
      • Prescott C.D.
      • Vaglio P.
      • Aboul-ela F.
      • Karn J.
      Structure-based drug design targeting an inactive RNA conformation: exploiting the flexibility of HIV-1 TAR RNA.
      ,
      • Mei H.Y.
      • Cui M.
      • Heldsinger A.
      • Lemrow S.M.
      • Loo J.A.
      • Sannes-Lowery K.A.
      • Sharmeen L.
      • Czarnik A.W.
      Inhibitors of protein–RNA complexation that target the RNA: specific recognition of human immunodeficiency virus type 1 TAR RNA by small organic molecules.
      ).
      Computational screening of a small molecule library identified acetylpromazine (Fig. 3A) as a TAR binder, providing an early example of how this approach could be used to target RNA. Electrophoretic mobility shift assays suggested that the compound blocks formation of a TAR–Tat–CycT1 complex at ∼100 nm (
      • Lind K.E.
      • Du Z.
      • Fujinaga K.
      • Peterlin B.M.
      • James T.L.
      Structure-based computational database screening, in vitro assay, and NMR assessment of compounds that target TAR RNA.
      ). NMR analysis indicated that acetylpromazine localizes within the bulged loop of TAR in a manner analogous to RBT-550 (
      • Du Z.
      • Lind K.E.
      • James T.L.
      Structure of TAR RNA complexed with a Tat–TAR interaction nanomolar inhibitor that was identified by computational screening.
      ). Binding appears to be conferred primarily by stacking between the Gua26–Cyt39 and Ade22–Uri40 base pairs like RBT-550 and is accompanied by dissolution of the Uri23·Ade27–Uri38 base-triple. Like RBT-203 and RBT-550, there are no base-specific interactions comparable with Hoogsteen-edge readout by argininamide (Fig. 3C). The RNA–ligand interface buries 428 Å2 of solvent-accessible surface area (83% of the total), and the average Sc is 0.62, suggesting a modest degree of shape complementarity.

      Model ncRNA–inhibitor interactions: base pairing and shape complementary

      Small molecules that strongly target a specific RNA are uncommon, and these are likely to engage multiple unintended partners (
      • Abulwerdi F.A.
      • Shortridge M.D.
      • Sztuba-Solinska J.
      • Wilson R.
      • Le Grice S.F.
      • Varani G.
      • Schneekloth Jr., J.S.
      Development of small molecules with a noncanonical binding mode to HIV-1 trans activation response (TAR) RNA.
      ). At the outset of screening, effective approaches strive to limit off-target recognition by conducting binding assays in the presence of a molar excess of tRNA (
      • Crawford D.W.
      • Blakeley B.D.
      • Chen P.H.
      • Sherpa C.
      • Le Grice S.F.
      • Laird-Offringa I.A.
      • McNaughton B.R.
      An evolved RNA recognition Motif that suppresses HIV-1 Tat/TAR-dependent transcription.
      ), or by gauging nonspecific binding by use of decoy RNAs (
      • Crawford D.W.
      • Blakeley B.D.
      • Chen P.H.
      • Sherpa C.
      • Le Grice S.F.
      • Laird-Offringa I.A.
      • McNaughton B.R.
      An evolved RNA recognition Motif that suppresses HIV-1 Tat/TAR-dependent transcription.
      ,
      • Stelzer A.C.
      • Frank A.T.
      • Kratz J.D.
      • Swanson M.D.
      • Gonzalez-Hernandez M.J.
      • Lee J.
      • Andricioaei I.
      • Markovitz D.M.
      • Al-Hashimi H.M.
      Discovery of selective bioactive small molecules by targeting an RNA dynamic ensemble.
      ), RNase footprinting (
      • Parsons J.
      • Castaldi M.P.
      • Dutta S.
      • Dibrov S.M.
      • Wyles D.L.
      • Hermann T.
      Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA.
      ), or whole transcriptome analysis (
      • Hilimire T.A.
      • Chamberlain J.M.
      • Anokhina V.
      • Bennett R.P.
      • Swart O.
      • Myers J.R.
      • Ashton J.M.
      • Stewart R.A.
      • Featherston A.L.
      • Gates K.
      • Helms E.D.
      • Smith H.C.
      • Dewhurst S.
      • Miller B.L.
      HIV-1 frameshift RNA-targeted triazoles inhibit propagation of replication-competent and multi-drug-resistant HIV in human cells.
      ). Even after the identification of a tight-binding RNA inhibitor, the structure determination of such a complex is even more extraordinary. As we noted, many technical obstacles were overcome to obtain reliable experimental structures of TAR (
      • Brodsky A.S.
      • Williamson J.R.
      Solution structure of the HIV-2 TAR–argininamide complex.
      ,
      • Ippolito J.A.
      • Steitz T.A.
      A 1.3-A resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation.
      ). To improve such outcomes, the analysis of TAR binding to various small-molecule ligands would have benefitted from complementary biophysical approaches to rigorously and reproducibly assess the binding determinants of hit compounds (
      • Hellman L.M.
      • Fried M.G.
      Electrophoretic mobility shift assay (EMSA) for detecting protein–nucleic acid interactions.
      ,
      • Pollard T.D.
      A guide to simple and informative binding assays.
      • Cer R.Z.
      • Mudunuri U.
      • Stephens R.
      • Lebeda F.J.
      IC50-to-Ki: a web-based tool for converting IC50 to Ki values for inhibitors of enzyme activity and ligand binding.
      ). Methods that provide thermodynamic parameters (ΔG, ΔH, and −TΔS) and KD values have proven especially useful to relate structural observations to specific modes of binding (
      • Freire E.
      A thermodynamic approach to the affinity optimization of drug candidates.
      ). SPR is also considered a rigorous secondary screen to validate high-throughput approaches, while providing kinetic constants kon and koff for lead optimization (
      • Huber W.
      A new strategy for improved secondary screening and lead optimization using high-resolution SPR characterization of compound-target interactions.
      ). A future challenge for inhibitor studies of TAR will be to relate quantitatively vetted molecular-recognition attributes of ligand binding to the drug-discovery process. Accordingly, we now consider examples of well-defined ncRNA–effector complexes with distinct RNA recognition features, supporting equilibrium binding constants, and analyses of downstream inhibitor effects on antiviral or antibacterial function.
      Benzimidazole derivatives have been identified by MS-based screening that target the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) genomic RNA (
      • Seth P.P.
      • Miyaji A.
      • Jefferson E.A.
      • Sannes-Lowery K.A.
      • Osgood S.A.
      • Propp S.S.
      • Ranken R.
      • Massire C.
      • Sampath R.
      • Ecker D.J.
      • Swayze E.E.
      • Griffey R.H.
      SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.
      ). The IRES features a series of folded domains, including conserved domain II. This region comprises a bent, bulged loop that is key for positioning the viral mRNA initiation codon and activation of the host ribosome (
      • Davis D.R.
      • Seth P.P.
      Therapeutic targeting of HCV internal ribosomal entry site RNA.
      ). Biophysical analysis demonstrated benzimidazole compounds straighten domain IIa and reorder the bulge (
      • Parsons J.
      • Castaldi M.P.
      • Dutta S.
      • Dibrov S.M.
      • Wyles D.L.
      • Hermann T.
      Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA.
      ,
      • Paulsen R.B.
      • Seth P.P.
      • Swayze E.E.
      • Griffey R.H.
      • Skalicky J.J.
      • Cheatham 3rd., T.E.
      • Davis D.R.
      Inhibitor-induced structural change in the HCV IRES domain IIa RNA.
      ), which has parallels to the ligand-bound and apo-states of TAR (Fig. 2, A and D). Significantly, the restructured S-shape of domain IIa produces a cavity suited to small-molecule binding.
      Lead optimization led to compound 12 that binds HCV IRES domain IIa with a KD of 860 nm (Fig. 4A) (
      • Seth P.P.
      • Miyaji A.
      • Jefferson E.A.
      • Sannes-Lowery K.A.
      • Osgood S.A.
      • Propp S.S.
      • Ranken R.
      • Massire C.
      • Sampath R.
      • Ecker D.J.
      • Swayze E.E.
      • Griffey R.H.
      SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.
      ). A 2.2 Å resolution co-crystal structure reveals the mode of RNA recognition by 12 (Fig. 4B). Specifically, the 2-aminoimidazole moiety donates hydrogen bonds to the Hoogsteen edge of Gua110, like argininamide (Fig. 3C). The dimethylamino-propyl group makes an electrostatic interaction with a nonbridging oxygen of Ade109, whereas the dimethylamino-methyl group forms a water-mediated contact to a nonbridging oxygen of Ade53. The benzimidazole moiety engages in π-stacking between purines Gua52 and Ade53. These features are corroborated strongly by structure–activity relationships (
      • Seth P.P.
      • Miyaji A.
      • Jefferson E.A.
      • Sannes-Lowery K.A.
      • Osgood S.A.
      • Propp S.S.
      • Ranken R.
      • Massire C.
      • Sampath R.
      • Ecker D.J.
      • Swayze E.E.
      • Griffey R.H.
      SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.
      ). Compound 12 sequesters 384 Å2 of its surface in the RNA pocket or 71% of the ligand’s solvent-accessible area. As expected from the structure, the shape correlation between the RNA and ligand surfaces is high with an Sc value of 0.82. Importantly, compound 12 was also active in HCV-replicon assays. The inhibitor reduced HCV RNA levels in cells with an EC50 of 3.9 μm (
      • Seth P.P.
      • Miyaji A.
      • Jefferson E.A.
      • Sannes-Lowery K.A.
      • Osgood S.A.
      • Propp S.S.
      • Ranken R.
      • Massire C.
      • Sampath R.
      • Ecker D.J.
      • Swayze E.E.
      • Griffey R.H.
      SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.
      ), similar to levels needed to inhibit translation from subgenomic replicons (2.8 ± 0.4 μm) and full-length virus (3.4 ± 0.5 μm) (
      • Dibrov S.M.
      • Ding K.
      • Brunn N.D.
      • Parker M.A.
      • Bergdahl B.M.
      • Wyles D.L.
      • Hermann T.
      Structure of a hepatitis C virus RNA domain in complex with a translation inhibitor reveals a binding mode reminiscent of riboswitches.
      ). Although a related compound 11 showed slightly poorer binding affinity (KD 1.7 μm), it performed better in the replicon assay (EC50 of 1.5 μm). Compound 11 replaces the tetrahydropyran ring with a smaller tetrahydrofuran. This subtle difference has been attributed to differences in cellular penetration (
      • Seth P.P.
      • Miyaji A.
      • Jefferson E.A.
      • Sannes-Lowery K.A.
      • Osgood S.A.
      • Propp S.S.
      • Ranken R.
      • Massire C.
      • Sampath R.
      • Ecker D.J.
      • Swayze E.E.
      • Griffey R.H.
      SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.
      ), which is a major consideration beyond a tight-binding KD.
      Figure thumbnail gr4
      Figure 4Chemical properties and modes of drug binding to representative ncRNAs. A, chemical structure of benzimidazole variant “compound 12”. The KD of binding to HCV domain IIa and EC50 from replicon assays are shown (
      • Seth P.P.
      • Miyaji A.
      • Jefferson E.A.
      • Sannes-Lowery K.A.
      • Osgood S.A.
      • Propp S.S.
      • Ranken R.
      • Massire C.
      • Sampath R.
      • Ecker D.J.
      • Swayze E.E.
      • Griffey R.H.
      SAR by MS: discovery of a new class of RNA-binding small molecules for the hepatitis C virus: internal ribosome entry site IIA subdomain.
      ). The chiral center is labeled with an asterisk. B, ball–and–stick diagram of HCV domain IIa (purple) bound to compound 12 (yellow) (PDB entry 3tzr) (
      • Dibrov S.M.
      • Ding K.
      • Brunn N.D.
      • Parker M.A.
      • Bergdahl B.M.
      • Wyles D.L.
      • Hermann T.
      Structure of a hepatitis C virus RNA domain in complex with a translation inhibitor reveals a binding mode reminiscent of riboswitches.
      ). The stereochemistry was not resolved in electron density maps. C, chemical structure of the FMN analogue BRX1555. The KD value of drug binding, the EC50 value from single-round transcription assays, and the IC50 value for bacterial growth inhibition are provided (
      • Vicens Q.
      • Mondragón E.
      • Reyes F.E.
      • Coish P.
      • Aristoff P.
      • Berman J.
      • Kaur H.
      • Kells K.W.
      • Wickens P.
      • Wilson J.
      • Gadwood R.C.
      • Schostarez H.J.
      • Suto R.K.
      • Blount K.F.
      • Batey R.T.
      Structure–activity relationship of flavin analogues that target the flavin mononucleotide riboswitch.
      ). D, ball-and-stick diagram of the Fusobacterium nucleatum FMN riboswitch in complex with BRX1555 (PDB entry 6dn3) (
      • Vicens Q.
      • Mondragón E.
      • Reyes F.E.
      • Coish P.
      • Aristoff P.
      • Berman J.
      • Kaur H.
      • Kells K.W.
      • Wickens P.
      • Wilson J.
      • Gadwood R.C.
      • Schostarez H.J.
      • Suto R.K.
      • Blount K.F.
      • Batey R.T.
      Structure–activity relationship of flavin analogues that target the flavin mononucleotide riboswitch.
      ). The respective ncRNA–inhibitor structures were chosen based on visual inspection of ligand fit to electron-density maps and associated quality-control indicators.
      Riboswitches represent another class of structured ncRNAs that change their conformations in response to the binding of cognate ions or small molecules (
      • Wedekind J.E.
      • Dutta D.
      • Belashov I.A.
      • Jenkins J.L.
      Metalloriboswitches: RNA-based inorganic ion sensors that regulate genes.
      ,
      • McCown P.J.
      • Corbino K.A.
      • Stav S.
      • Sherlock M.E.
      • Breaker R.R.
      Riboswitch diversity and distribution.
      • Hiller D.A.
      • Strobel S.A.
      The chemical versatility of RNA.
      ). Such sensing results in mRNA regulatory feedback that controls downstream genes (
      • Breaker R.R.
      Riboswitches and translation control.
      ,
      • Sherwood A.V.
      • Henkin T.M.
      Riboswitch-mediated gene regulation: novel RNA architectures dictate gene expression responses.
      • Serganov A.
      • Nudler E.
      A decade of riboswitches.
      ). The flavin mononucleotide (FMN) riboswitch is notable because it senses the cofactor FMN as well as the natural product roseoflavin, which acts as an antibacterial (
      • Lee E.R.
      • Blount K.F.
      • Breaker R.R.
      Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression.
      ,
      • Otani S.
      • Takatsu M.
      • Nakano M.
      • Kasai S.
      • Miura R.
      Letter: Roseoflavin, a new antimicrobial pigment from Streptomyces.
      ). This vulnerability has fostered efforts to target the FMN riboswitch with novel antibiotics (
      • Blount K.F.
      • Megyola C.
      • Plummer M.
      • Osterman D.
      • O'Connell T.
      • Aristoff P.
      • Quinn C.
      • Chrusciel R.A.
      • Poel T.J.
      • Schostarez H.J.
      • Stewart C.A.
      • Walker D.P.
      • Wuts P.G.
      • Breaker R.R.
      Novel riboswitch-binding flavin analog that protects mice against Clostridium difficile infection without inhibiting cecal flora.
      ,
      • Howe J.A.
      • Wang H.
      • Fischmann T.O.
      • Balibar C.J.
      • Xiao L.
      • Galgoci A.M.
      • Malinverni J.C.
      • Mayhood T.
      • Villafania A.
      • Nahvi A.
      • Murgolo N.
      • Barbieri C.M.
      • Mann P.A.
      • Carr D.
      • Xia E.
      • et al.
      Selective small-molecule inhibition of an RNA structural element.
      ,
      • Howe J.A.
      • Xiao L.
      • Fischmann T.O.
      • Wang H.
      • Tang H.
      • Villafania A.
      • Zhang R.
      • Barbieri C.M.
      • Roemer T.
      Atomic resolution mechanistic studies of ribocil: a highly selective unnatural ligand mimic of the E. coli FMN riboswitch.
      ,
      • Vicens Q.
      • Mondragón E.
      • Reyes F.E.
      • Coish P.
      • Aristoff P.
      • Berman J.
      • Kaur H.
      • Kells K.W.
      • Wickens P.
      • Wilson J.
      • Gadwood R.C.
      • Schostarez H.J.
      • Suto R.K.
      • Blount K.F.
      • Batey R.T.
      Structure–activity relationship of flavin analogues that target the flavin mononucleotide riboswitch.
      ,
      • Wang H.
      • Mann P.A.
      • Xiao L.
      • Gill C.
      • Galgoci A.M.
      • Howe J.A.
      • Villafania A.
      • Barbieri C.M.
      • Malinverni J.C.
      • Sher X.
      • Mayhood T.
      • McCurry M.D.
      • Murgolo N.
      • Flattery A.
      • Mack M.
      • Roemer T.
      Dual-targeting small-molecule inhibitors of the Staphylococcus aureus FMN riboswitch disrupt riboflavin homeostasis in an infectious setting.
      ). Recent structure-guided design led to the discovery of compound BRX1555—an FMN analogue that binds with a KD of 39.0 ± 0.7 nm based on in-line probing (Fig. 4C) (
      • Vicens Q.
      • Mondragón E.
      • Reyes F.E.
      • Coish P.
      • Aristoff P.
      • Berman J.
      • Kaur H.
      • Kells K.W.
      • Wickens P.
      • Wilson J.
      • Gadwood R.C.
      • Schostarez H.J.
      • Suto R.K.
      • Blount K.F.
      • Batey R.T.
      Structure–activity relationship of flavin analogues that target the flavin mononucleotide riboswitch.
      ). The ligand has an in vitro EC50 of 1.70 ± 0.18 μm in single-turnover transcription termination assays and an IC50 of 0.49 ± 0.09 μm in bacterial growth inhibition assays (
      • Vicens Q.
      • Mondragón E.
      • Reyes F.E.
      • Coish P.
      • Aristoff P.
      • Berman J.
      • Kaur H.
      • Kells K.W.
      • Wickens P.
      • Wilson J.
      • Gadwood R.C.
      • Schostarez H.J.
      • Suto R.K.
      • Blount K.F.
      • Batey R.T.
      Structure–activity relationship of flavin analogues that target the flavin mononucleotide riboswitch.
      ).
      The 2.80 Å resolution co-crystal structure of the FMN riboswitch in complex with BRX1555 reveals key details about its mode of molecular recognition. As expected, the inhibitor overlaps with the binding site of the natural ligand, which resides at the center of a six-way helical junction comprising two pairs of stabilizing loops (
      • Serganov A.
      • Huang L.
      • Patel D.J.
      Coenzyme recognition and gene regulation by a flavin mononucleotide riboswitch.
      ). The isoalloxazine ring of the inhibitor stacks centrally between Ade48 (junction J3-4) and Ade85 (pairing region P6) (Fig. 4D). The face of Ade99 (J6-1) hydrogen bonds to the uracil-like edge of BRX1555 in a manner similar to FMN. Gua62 (J4-5) stacks against the phenyl group of the inhibitor, reminiscent of the 2-methylaminopyrimidine moiety of ribocil—the synthetic FMN analogue discovered by Merck (
      • Howe J.A.
      • Wang H.
      • Fischmann T.O.
      • Balibar C.J.
      • Xiao L.
      • Galgoci A.M.
      • Malinverni J.C.
      • Mayhood T.
      • Villafania A.
      • Nahvi A.
      • Murgolo N.
      • Barbieri C.M.
      • Mann P.A.
      • Carr D.
      • Xia E.
      • et al.
      Selective small-molecule inhibition of an RNA structural element.
      ). In terms of binding and localization, the similarities of BRX1555 and ribocil are remarkable, especially because the former molecule was developed by structure-based design and the latter was identified by phenotypic screens that yielded a novel chemical scaffold distinct from FMN (
      • Howe J.A.
      • Wang H.
      • Fischmann T.O.
      • Balibar C.J.
      • Xiao L.
      • Galgoci A.M.
      • Malinverni J.C.
      • Mayhood T.
      • Villafania A.
      • Nahvi A.
      • Murgolo N.
      • Barbieri C.M.
      • Mann P.A.
      • Carr D.
      • Xia E.
      • et al.
      Selective small-molecule inhibition of an RNA structural element.
      ,
      • Vicens Q.
      • Mondragón E.
      • Reyes F.E.
      • Coish P.
      • Aristoff P.
      • Berman J.
      • Kaur H.
      • Kells K.W.
      • Wickens P.
      • Wilson J.
      • Gadwood R.C.
      • Schostarez H.J.
      • Suto R.K.
      • Blount K.F.
      • Batey R.T.
      Structure–activity relationship of flavin analogues that target the flavin mononucleotide riboswitch.
      ). Like FMN and ribocil, the riboswitch–BRX1555 complex buries a large amount of the inhibitor’s solvent-accessible surface in the interface (468 Å2 or 88%). The riboswitch–BRX1555 complex also shows significant shape complementarity, as indicated by an Sc value of 0.72. Interestingly, significant commonalities exist in the interactions used by HCV IRES domain IIa and the FMN riboswitch in terms of ligand recognition; these likenesses include hydrogen bonding that imparts base-specific readout, co-axial base stacking, solvent exclusion, and high shape complementarity (Fig. 4, B and D). These features also represent key molecular recognition determinants in peptide binding to TAR, which we will now explore.

      Molecular recognition of TAR by Tat peptides

      The HIV-1 Tat protein comprises multiple functional domains that are needed to complete the viral life cycle (Fig. 5A). TAR binding requires a basic ARM (
      • Weeks K.M.
      • Ampe C.
      • Schultz S.C.
      • Steitz T.A.
      • Crothers D.M.
      Fragments of the HIV-1 Tat protein specifically bind TAR RNA.
      ,
      • Calnan B.J.
      • Tidor B.
      • Biancalana S.
      • Hudson D.
      • Frankel A.D.
      Arginine-mediated RNA recognition: the arginine fork.
      ) harboring nine arginines. Of these, Arg-52 is most essential because its mutation to lysine yields a drastic loss of transactivation (
      • Calnan B.J.
      • Tidor B.
      • Biancalana S.
      • Hudson D.
      • Frankel A.D.
      Arginine-mediated RNA recognition: the arginine fork.
      ). Mutagenesis of flanking residues supports the presence of a supplemental electrostatic interaction network that modulates RNA binding as well as transactivation (
      • Tao J.
      • Frankel A.D.
      Electrostatic interactions modulate the RNA-binding and transactivation specificities of the human immunodeficiency virus and simian immunodeficiency virus Tat proteins.
      ,
      • Calnan B.J.
      • Biancalana S.
      • Hudson D.
      • Frankel A.D.
      Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA–protein recognition.
      ). Thus far, elucidation of the intact TAR–Tat–SEC complex (Fig. 1A) has remained elusive, although divide–and–conquer efforts have led to core SEC complexes in the presence of Tat’s transactivation domain. Nevertheless, these co-crystal structures currently lack the Tat ARM domain (
      • Tahirov T.H.
      • Babayeva N.D.
      • Varzavand K.
      • Cooper J.J.
      • Sedore S.C.
      • Price D.H.
      Crystal structure of HIV-1 Tat complexed with human P-TEFb.
      ,
      • Schulze-Gahmen U.
      • Hurley J.H.
      Structural mechanism for HIV-1 TAR loop recognition by Tat and the super-elongation complex.
      ), providing an incomplete picture of RNA recognition. Accordingly, we will now focus on recent structures of Tat-derived ARM peptides in complex with TAR that have led to a new understanding of this key RNA–protein interaction and how it provides a foundation for HIV inhibitor design. A structural survey of known peptides and proteins bound to TAR is presented in Table 2.
      Figure thumbnail gr5
      Figure 5Tat organization and molecular recognition of TAR by Tat ARM domains, lab-evolved proteins, and cyclic peptides. A, functional domain organization of the HIV-1 Tat protein (
      • Mousseau G.
      • Valente S.T.
      Role of host factors on the regulation of Tat-mediated HIV-1 transcription.
      ). The transactivation domain comprises an N-terminal acidic and proline-rich domain, a cysteine-rich domain that binds Zn(II) (i.e. zinc finger or ZnF), a core region, and a basic ARM. Additional domains include a glutamine-rich region and the E2 CTD. B, summary of peptide sequence interactions with TAR RNAs from C–J (blue box). Amino acids of naturally occurring BIV and HIV-1 Tat ARMs are shown; the sequence alignment is based on common recognition modes of RNA targets as follows: Gua11(26) and Gua14(28) of BIV(HIV) TAR are recognized by Arg-73(R52) and Arg-70(R49) of BIV(HIV) Tat (yellow box). Amino acids of lab-evolved proteins and cyclic peptides from structure-based design. The sequence alignment is based on common spatial recognition at Gua26 and Gua28 of HIV-1 TAR by Arg-47 and Arg-49 of TBP6.7 and Arg-3 and Arg-5 of JB181. Specific RNA–peptide interaction types are listed above each amino acid; the symbols are as follows: π indicates cation–π or aromatic stacking; H equals hydrogen-bond recognition of a guanine Hoogsteen edge; Pi indicates salt-bridge formation to the phosphate backbone; b indicates hydrogen-bond recognition to a nucleobase; a pentagon indicates a hydrogen-bond contact to ribose. Symbols of nonstandard amino acids are as follows: B equals l-2,4-diaminobutyric acid; O equals l-ornithine; backward P is d-proline. C, global view depicting BIV TAR (purple ribbon) recognition by the BIV-Tat ARM (yellow worm) (PDB entry 1biv) (
      • Ye X.
      • Kumar R.A.
      • Patel D.J.
      Molecular recognition in the bovine immunodeficiency virus Tat peptide–TAR RNA complex.
      ). The Sc value was calculated from the lowest energy NMR core structure (amino acids 67–79). D, close-up view of BIV TAR recognition by BIV Tat at the UU bulge. Despite differences in the central bulge compared with HIV TAR (C), BIV TAR exhibits a major-groove base triple at Uri10·Ade13–Uri24. The Tat peptide undergoes a sharp bend with dihedral angles of φi + 1 75°, ψi + 1 −10°, and φi + 2 −133°, ψi + 2 63° characteristic of a type V′ turn (

      Creighton, T. E., (1993) Proteins: Structures and Molecular Properties, 2nd Ed., pp. 1–507, W.H. Freeman and Co., New York

      ); the peptide has no other β-hairpin characteristics. For clarity, only amino acids engaged in peptide–RNA were included in diagrams. E, global view depicting HIV-2 TAR recognition by the HIV-1 Tat peptide (PDB entry 6mce) (
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      ). The Sc value was calculated from the lowest energy NMR core structure (amino acids 48–54). F, close-up view of HIV-2 TAR recognition at its UU bulge by HIV-1 Tat. Three nucleobases compose an ASM (cyan highlight) that engages Arg-52 of Tat. G, global view depicting HIV-1 TAR recognition by TBP6.7 (PDB entry 6cmn) (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ). The lab-evolved β2–β3 loop (yellow) recognizes the TAR major groove. The Sc value was calculated from the co-crystal structure. H, close-up view of HIV-1 TAR recognition in the central UCU bulge by TBP6.7. Arg-47 engages TAR at the ASM, similar to F. I, global view of HIV-1 TAR recognition by the cyclic peptide JB181 (PDB entry 6d2u) (
      • Shortridge M.D.
      • Wille P.T.
      • Jones A.N.
      • Davidson A.
      • Bogdanovic J.
      • Arts E.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.
      ). The Sc value was calculated from the lowest energy NMR structure. J, close-up view of HIV-1 TAR recognition at the UCU bulge by JB181. The DP13–LP14 turn is shown to emphasize the restrained peptide conformation. CTD, C-terminal domain.
      Table 2Structures of HIV TAR in complex with peptides or proteins
      TAR RNALigand sourceLigand scaffoldMethodPDB codesRefs.
      BIVBIV Tat (68–81)PeptideNMR1mnb
      • Puglisi J.D.
      • Chen L.
      • Blanchard S.
      • Frankel A.D.
      Solution structure of a bovine immunodeficiency virus Tat–TAR peptide–RNA complex.
      BIVBIV Tat (65–81)PeptideNMR1biv
      • Ye X.
      • Kumar R.A.
      • Patel D.J.
      Molecular recognition in the bovine immunodeficiency virus Tat peptide–TAR RNA complex.
      BIVBIV Tat-derived peptideCyclic peptideNMR2a9x
      • Leeper T.C.
      • Athanassiou Z.
      • Dias R.L.
      • Robinson J.A.
      • Varani G.
      TAR RNA recognition by a cyclic peptidomimetic of Tat protein.
      BIVJDV Tat ARMPeptideNMR1zbn
      • Calabro V.
      • Daugherty M.D.
      • Frankel A.D.
      A single intermolecular contact mediates intramolecular stabilization of both RNA and protein.
      EIAVEIAV CycT1-TatProtein complexX-ray2w2h
      • Anand K.
      • Schulte A.
      • Vogel-Bachmayr K.
      • Scheffzek K.
      • Geyer M.
      Structural insights into the cyclin T1-Tat–TAR RNA transcription activation complex from EIAV.
      HIV-1Designed Tat mimic L-22Cyclic peptideNMR2kdq
      • Davidson A.
      • Leeper T.C.
      • Athanassiou Z.
      • Patora-Komisarska K.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein.
      HIV-2Lead compoundArginine-pyrimidine tripeptideNMR2kmj
      • Ferner J.
      • Suhartono M.
      • Breitung S.
      • Jonker H.R.
      • Hennig M.
      • Wöhnert J.
      • Göbel M.
      • Schwalbe H.
      Structures of HIV TAR RNA–ligand complexes reveal higher binding stoichiometries.
      HIV-1Designed Tat mimic KPZ-41Cyclic peptideNMR2kx5
      • Davidson A.
      • Patora-Komisarska K.
      • Robinson J.A.
      • Varani G.
      Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein.
      HIV-1L-22 (low population)Cyclic peptideNMR5j2w
      • Borkar A.N.
      • Bardaro Jr., M.F.
      • Camilloni C.
      • Aprile F.A.
      • Varani G.
      • Vendruscolo M.
      Structure of a low-population binding intermediate in protein–RNA recognition.
      HIV-1HIV-1 Tat (1–49)-SECProtein complexX-ray5l1z
      • Schulze-Gahmen U.
      • Echeverria I.
      • Stjepanovic G.
      • Bai Y.
      • Lu H.
      • Schneidman-Duhovny D.
      • Doudna J.A.
      • Zhou Q.
      • Sali A.
      • Hurley J.H.
      Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex.
      HIV-1HIV-1 Tat (1–49)-SECProtein complexX-ray6cyt
      • Schulze-Gahmen U.
      • Hurley J.H.
      Structural mechanism for HIV-1 TAR loop recognition by Tat and the super-elongation complex.
      HIV-1TBP6.7Lab-evolved proteinX-ray6cmn
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      HIV-2HIV-1 Tat (44–60)PeptideNMR6mce
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      HIV-1Designed Tat mimic JB181Cyclic peptideNMR6d2u
      • Shortridge M.D.
      • Wille P.T.
      • Jones A.N.
      • Davidson A.
      • Bogdanovic J.
      • Arts E.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.
      To provide perspective on the recent structure of the HIV-2 TAR–Tat complex, it is important to recognize that initial high-resolution insights came from NMR studies of the BIV TAR–Tat complex (
      • Puglisi J.D.
      • Chen L.
      • Blanchard S.
      • Frankel A.D.
      Solution structure of a bovine immunodeficiency virus Tat–TAR peptide–RNA complex.
      ,
      • Ye X.
      • Kumar R.A.
      • Patel D.J.
      Molecular recognition in the bovine immunodeficiency virus Tat peptide–TAR RNA complex.
      ). Like HIV-1 Tat, the ARM domain of BIV Tat is also arginine-rich (Fig. 5B). The peptide binds BIV TAR in the major groove near the central UU bulge, where it forms a short antiparallel strand capped by a distorted type V′ β-turn (Fig. 5C) (

      Creighton, T. E., (1993) Proteins: Structures and Molecular Properties, 2nd Ed., pp. 1–507, W.H. Freeman and Co., New York

      ). Like many β-turns, the ith to i + 3rd hydrogen bond is absent, but the carbonyl oxygen of the ith residue (Arg-73) receives a hydrogen bond from the i +4th side chain (Arg-77) (Fig. 5D). The net result is a β-hairpin spanning the width of the major groove. Base-specific readout is mediated by guanidinium groups from Arg-70, Arg-73, and Arg-77, which hydrogen bonds to the Hoogsteen edges of Gua14, Gua11, and Gua9. Cation–π stacking is observed between Arg-70 and Ade13 of the central base triple and between Arg-73 and Gua9. A handful of salt-bridge and hydrogen-bond interactions occur, including Lys-75 N∈ to the pro-(Rp)-oxygen of Uri24 and the backbone amide of Gly-71 to N7 of Gua22. The complex buries 62% of the total Tat peptide (Ser-65 to Arg-81) solvent-accessible surface or 1187 Å2. The interface exhibits a substantial amount of shape complementarity, as indicated by an Sc value of 0.70. These molecular recognition properties are consistent with the KD of 1.3 ± 0.1 nm measured for this strong peptide–RNA binding interaction (
      • Tok J.B.
      • Des Jean R.C.
      • Fenker J.
      Binding of a cyclic BIV βa-Tat peptide with its TAR RNA construct.
      ).
      More recently, the solution–NMR structure of the HIV-1 Tat peptide (amino acids 44–60) was determined in complex with HIV-2 TAR (
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      ). This exciting new complex reveals unprecedented chemical details about the mode of TAR–Tat molecular recognition (Fig. 5B). Remarkably, the Tat ARM spans the length of the TAR major groove, starting with the N terminus abutting the well-ordered apical loop (Fig. 5E). This tight RNA turn is fortified by a canonical Cyt30–Gua34 base pair first observed in the HIV-1 TAR complex with the lab-evolved protein TBP6.7 (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ) (discussed below). The C terminus of the peptide extends through stem s1a (Fig. 5E) and protrudes into solvent past Arg-57. Consistent with CD spectra of the isolated peptide in solution (
      • Calnan B.J.
      • Biancalana S.
      • Hudson D.
      • Frankel A.D.
      Analysis of arginine-rich peptides from the HIV Tat protein reveals unusual features of RNA–protein recognition.
      ), the ensemble of Tat conformers in the bound state lacks regular secondary-structure features in contrast to the β-turn in BIV Tat (Fig. 5, E versus C).
      As anticipated, the determinants of TAR–Tat binding specificity include key arginines that read the Hoogsteen edges of conserved guanine bases in the TAR sequence (Fig. 5, B and F). The indispensable nature of Arg-52 (
      • Tao J.
      • Frankel A.D.
      Specific binding of arginine to TAR RNA.
      ,
      • Tao J.
      • Frankel A.D.
      Electrostatic interactions modulate the RNA-binding and transactivation specificities of the human immunodeficiency virus and simian immunodeficiency virus Tat proteins.
      ) is consistent with its recognition of Gua26 (Fig. 5F)— the site of argininamide binding (Fig. 3C). Arg-52 is sequestered by cation–π stacking of its guanidinium group between bases from Ade22 and Uri23. The latter base engages in the hallmark bound-state base triple. The constellation of bases and mode of amino acid recognition compose the specialized ASM protein–RNA interaction module (Fig. 5F) (
      • Durney M.A.
      • D'Souza V.M.
      Preformed protein-binding motifs in 7SK snRNA: structural and thermodynamic comparisons with retroviral TAR.
      )— first observed for argininamide (above). Although the ASM appears only once in the TAR–Tat complex, it is utilized four times in the 7SK–Tat complex (data not shown) (
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      ). Arg-73 of BIV Tat uses comparable ASM-like readout, although the Arg-73 guanidinium group does not stack beneath the Uri10 base (Fig. 5D).
      A different mode of TAR recognition is used by the Arg-49 group of Tat, which also hydrogen bonds to the Hoogsteen edge of a conserved guanine (i.e. Gua28), while making contacts to the 2′-OH of Uri23 (Fig. 5, B and F). Although the latter nucleobase stacks upon the Arg-49 side chain, this binding mode does not constitute an ASM because the guanidinium is not flanked by bases on both sides (i.e. it is an “open-faced” arginine sandwich). Beyond arginine, Tat uses additional stabilizing hydrogen bonds to recognize TAR in the upper and lower stems. These include the following: the ∈-amino groups of Lys-50 and Lys-51, which interact with backbone oxygens from Gua36 and Cyt37; the Gly-48 carbonyl oxygen, which interacts with the exocyclic amine of Cyt29 (Fig. 5, B and F); and Arg-53 and Arg-55 from the flexible C-terminal tail of Tat, which interact with the backbone at Cyt39 and Uri40, whereas Gln-54 recognizes atom N7 of Gua43 (data not shown). The cumulative interactions are summarized in Fig. 5B.
      The HIV-1 Tat peptide recognizes TAR with a KD of 22.5 ± 15.2 nm based on ITC (
      • Pham V.V.
      • Salguero C.
      • Khan S.N.
      • Meagher J.L.
      • Brown W.C.
      • Humbert N.
      • de Rocquigny H.
      • Smith J.L.
      • D'Souza V.M.
      HIV-1 Tat interactions with cellular 7SK and viral TAR RNAs identifies dual structural mimicry.
      ). This slightly reduced affinity compared to BIV TAR–Tat represents a change in free energy (ΔΔG) of only +1.7 kcal mol−1, e.g. the difference of 2–3 hydrogen bonds. Like the BIV TAR–Tat complex, the lowest energy peptide of the HIV TAR–Tat ensemble is significantly sequestered in the major groove with 41% of the peptide (1185 Å2) buried from solvent. This degree of similarity is striking, considering that the HIV-1 Tat peptide adopts an extended conformation compared with the BIV U-shaped polypeptide path (Fig. 5, E versus C). As expected, the HIV TAR–Tat interface exhibits substantial shape complementarity in its core, as indicated by an Sc value of 0.66—comparable with antibody–antigen interfaces and peptides designed to inhibit β-amyloid aggregation (
      • Lawrence M.C.
      • Colman P.M.
      Shape complementarity at protein/protein interfaces.
      ,
      • Lu J.
      • Cao Q.
      • Wang C.
      • Zheng J.
      • Luo F.
      • Xie J.
      • Li Y.
      • Ma X.
      • He L.
      • Eisenberg D.
      • Nowick J.
      • Jiang L.
      • Li D.
      Structure-based peptide inhibitor design of amyloid-β aggregation.
      ).

      Lab-evolved proteins for HIV-1 TAR recognition

      Advances in protein engineering have facilitated the design of novel RNA-binding proteins with distinct functions (
      • Chen Y.
      • Varani G.
      Engineering RNA-binding proteins for biology.
      ). In this regard, the TAR-binding protein (TBP) is a model system that was “evolved” from RRM1 of the U1A spliceosomal protein by combining saturation mutagenesis, yeast display, and cell sorting (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ,
      • Crawford D.W.
      • Blakeley B.D.
      • Chen P.H.
      • Sherpa C.
      • Le Grice S.F.
      • Laird-Offringa I.A.
      • McNaughton B.R.
      An evolved RNA recognition Motif that suppresses HIV-1 Tat/TAR-dependent transcription.
      ). The unique mode of HIV-1 TAR recognition by variant TBP6.7 was visualized recently by a co-crystal structure determined to 1.80 Å resolution (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ). Unexpectedly, TAR recognition by TBP6.7 entails doubled-stranded RNA recognition of s1b and the UCU bulge (Fig. 5G). This mode of binding differs entirely from the parental U1A protein, which binds to a single-stranded loop within the U1 small nuclear RNA (
      • Oubridge C.
      • Ito N.
      • Evans P.R.
      • Teo C.H.
      • Nagai K.
      Crystal structure at 1.92 A resolution of the RNA-binding domain of the U1A spliceosomal protein complexed with an RNA hairpin.
      ). The major determinants of TAR RNA recognition by TBP6.7 are attributable to residues in the evolved β2–β3 loop. For rigor, every amino acid in the loop was mutated and analyzed for TAR binding by ITC, thereby relating structure and recognition in terms of free-energy changes. Arg-47, Arg-49, and Arg-52 are the most energetically significant residues as reflected by their ΔΔG values of +3.8, +3.2, and +2.8 kcal mol−1 for Arg–to–Ala mutations. These observations agree well with the structure wherein each residue penetrates deeply into the major groove to recognize a conserved guanine. Like the HIV-1 TAR–Tat interaction, Arg-47 utilizes the ASM in which its guanidinium group stacks between Ade22 and Uri23, while forming hydrogen bonds to the Hoogsteen edge of Gua26 (Fig. 5, B and H). Unlike the modes of TAR RNA recognition by BIV and HIV Tat peptides (Fig. 5, D and F), Arg-47 simultaneously makes two electrostatic contacts to Uri23 phosphate. The collective interactions appear to be a variation of a hypothetical “arginine fork” interaction, wherein both edges of the Tat-derived guanidinium group were hypothesized to bind TAR’s phosphate backbone (
      • Calnan B.J.
      • Tidor B.
      • Biancalana S.
      • Hudson D.
      • Frankel A.D.
      Arginine-mediated RNA recognition: the arginine fork.
      ). Otherwise, the TAR–TBP6.7 complex typifies the TAR-bound conformation featuring the hallmark Uri23·Ade27–Uri38 base triple and canonical Cyt30–Gua34 pair in the apical loop (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ).
      Other similarities exist between the modes of HIV-1 Tat and TBP6.7 recognition of TAR. Specifically, Arg-49 of TBP6.7 stacks upon Ade27 while hydrogen bonding and engaging in electrostatic interactions with the Gua28 Hoogsteen edge and phosphate group (Fig. 5H). Arg-49 of HIV-1 Tat forms similar stacking and base-pairing interactions but hydrogen bonds to the 2′-OH of Uri23 (Fig. 5F). Unlike the HIV-1 TAR–Tat complex, TBP6.7 uses a third arginine for guanine recognition. Arg-52 of TBP6.7 reads the Hoogsteen edge of Gua36 while stacking beneath Gua34. Beyond TBP6.7, BIV Tat is the only other example of major-groove guanine recognition by three peptide arginines (Fig. 5, B and D). Despite similarities in TAR recognition among TBP6.7, HIV-1 Tat, and BIV Tat, the commonalities are entirely local and do not reflect common polypeptide folds (Fig. 5, C, E, and G). In terms of the buried surface area and shape complementarity of the TAR–TBP6.7 interface, a total of 718 Å2 of TAR is sequestered, wherein 384 Å2 is attributable to the β2–β3 loop. Recognition of TAR by TBP6.7 gives an Sc value of 0.79 (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ), which is comparable with Sc values of peptides selected by phage display to bind the insulin receptor ectodomain (
      • Lawrence C.F.
      • Margetts M.B.
      • Menting J.G.
      • Smith N.A.
      • Smith B.J.
      • Ward C.W.
      • Lawrence M.C.
      Insulin mimetic peptide disrupts the primary binding site of the insulin receptor.
      ). Overall, these properties closely resemble comparable metrics for the BIV and HIV TAR–Tat complexes (Fig. 5, C, E, and G).
      The observation that the major determinants of TAR recognition by TBP6.7 are localized mostly to the lab-evolved β2–β3 loop has ramifications for inhibitor design using a short peptide that comprises the isolated β2–β3 loop. Indeed, a series of complementary experiments demonstrated that the β2–β3–loop sequence could be removed from the context of TBP6.7 and was still capable of TAR binding. When synthesized as a stapled peptide, the restrained β2–β3 loop still exhibited affinity for TAR (KD of 1.8 ± 0.5 μm) and was capable of inhibiting TAR–Tat-dependent transcription in HeLa nuclear lysate (
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.
      • Beardslee P.C.
      • Mathews D.H.
      • Pentelute B.L.
      • McNaughton B.R.
      • Wedekind J.E.
      Structure of HIV TAR in complex with a Lab-evolved RRM provides insight into duplex RNA recognition and synthesis of a constrained peptide that impairs transcription.
      ). At present, it is unknown whether stapled β2–β3-loop peptides enter cells or whether they possess antiviral activity. Nevertheless, this work provides proof–of–principle that small peptides can be derived from proteins evolved in the lab to recognize TAR.

      TAR recognition by structure-based design of cyclic peptides

      A more traditional approach to disrupt the SEC–TAR interaction (Fig. 1A) is to exploit existing knowledge of TAR–Tat molecular recognition to guide design of restrained, inhibitory peptides (
      • Davidson A.
      • Patora-Komisarska K.
      • Robinson J.A.
      • Varani G.
      Essential structural requirements for specific recognition of HIV TAR RNA by peptide mimetics of Tat protein.
      ,
      • Davidson A.
      • Leeper T.C.
      • Athanassiou Z.
      • Patora-Komisarska K.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein.
      ). Past studies leveraged structural information from the BIV TAR–Tat interaction (Fig. 5, C and D) to produce a number of cyclic peptides (
      • Leeper T.C.
      • Athanassiou Z.
      • Dias R.L.
      • Robinson J.A.
      • Varani G.
      TAR RNA recognition by a cyclic peptidomimetic of Tat protein.
      ,
      • Athanassiou Z.
      • Dias R.L.
      • Moehle K.
      • Dobson N.
      • Varani G.
      • Robinson J.A.
      Structural mimicry of retroviral Tat proteins by constrained, β-hairpin peptidomimetics: ligands with high affinity and selectivity for viral TAR RNA regulatory elements.
      ,
      • Athanassiou Z.
      • Patora K.
      • Dias R.L.
      • Moehle K.
      • Robinson J.A.
      • Varani G.
      Structure-guided peptidomimetic design leads to nanomolar β-hairpin inhibitors of the Tat–TAR interaction of bovine immunodeficiency virus.
      ) that culminated recently in an “ultrahigh affinity” cyclic peptidomimetic. This inhibitor, JB181, binds HIV-1 TAR with an unprecedented KD of 28 ± 4 pm (
      • Shortridge M.D.
      • Wille P.T.
      • Jones A.N.
      • Davidson A.
      • Bogdanovic J.
      • Arts E.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      An ultra-high affinity ligand of HIV-1 TAR reveals the RNA structure recognized by P-TEFb.
      ). NMR solution analysis revealed that JB181 recognizes TAR in the s1b major groove and bulge (Fig. 5I). However, rather than adopting an elongated peptide as observed for the HIV-2 TAR–Tat complex, the designed peptide forms a β-hairpin comprising 14 residues (Fig. 5B). To reduce conformational flexibility, the peptide termini are linked by an innovative l- and d-proline turn that covalently cyclizes the inhibitor (Fig. 5J). The RNA recognition-end of the peptide adopts a distorted type II β-turn wherein the carbonyl oxygen of Arg-5 (ith amino acid) accepts a hydrogen bond from the backbone amide of Arg-8 (i + 3rd) (data not shown). Overall, cyclization stabilizes the antiparallel β-strand structure and positions the ith and i +1st amino acids to interact with the major groove and UCU bulge.
      Combining natural and unnatural amino acids in the cyclic peptide offers advantages to elicit desired RNA–peptide interactions. Placement of l-2,4-diaminobutyric acid (B) at position 1—as opposed to Arg-1 used in precursor peptide L-22 (
      • Davidson A.
      • Leeper T.C.
      • Athanassiou Z.
      • Patora-Komisarska K.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein.
      )—induces favorable salt bridges between the B1 amino group and phosphates at Gua21 and Ade22 (Fig. 5J). This pairing serves to anchor the peptide in the major groove and promotes electrostatic binding by other basic groups introduced to recognize both bulge and major-groove features. For example, the guanidinium groups of Arg-3 and Arg-5 interact with Gua26 and Gua28, and the Lys-6 Nζ group hydrogen bonds to the carbonyl oxygen of Uri25.
      In some respects, the determinants of TAR molecular recognition by JB181 are comparable with naturally occurring modes of TAR recognition by the Tat ARM domains from BIV and HIV. JB181 buries 920 Å2 or 55% of its solvent-accessible surface area in the RNA–inhibitor interface. This level of sequestration is comparable with Tat binding to BIV or HIV-2 TAR (∼1200 Å2). Recognition of BIV TAR Gua9 and Gua11 by Arg-77 and Arg-73 of BIV Tat are analogous to JB181’s use of Arg-5 to recognize Gua28 because both sets of interactions involve favorable co-planar positioning of a guanidinium group to donate two hydrogen bonds to O6 and N7 of the base Hoogsteen edge (Fig. 5, D and J). HIV-1 Tat similarly employs a single imino group of Arg-52 and Arg-49 to recognize the Hoogsteen edges of Gua26 and Gua28 within HIV-2 TAR, akin to JB181’s use of Arg-3 and Lys-6 to recognize O6 and O4 of Gua26 and Uri25—albeit JB181 does not utilize the ASM. Although JB181 binds TAR with 100–1000-fold greater affinity than HIV and BIV Tat, it uses fewer specific interactions to recognize TAR (Fig. 5B). Whereas BIV and HIV-1 Tat peptides use every arginine of the ARM sequence for RNA binding, JB181 utilizes half of its complement. This attribute may be indicative of a greater role for JB181’s charged residues in general electrostatic recognition of the RNA. Notably, the Sc value of 0.59 for the TAR–JB181 complex agrees well with that of a similar antiviral cyclic peptide, L-22, whose shape complementarity score is 0.60 in the context of the TAR complex (
      • Davidson A.
      • Leeper T.C.
      • Athanassiou Z.
      • Patora-Komisarska K.
      • Karn J.
      • Robinson J.A.
      • Varani G.
      Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein.
      ,
      • Belashov I.A.
      • Crawford D.W.
      • Cavender C.E.
      • Dai P.