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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
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.
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
) 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 (
). 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 (
)). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
), 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 (
). 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 (
). 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 (
) 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 (
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 (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
). 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 (
), 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.
Table 2Structures of HIV TAR in complex with peptides or proteins
). 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) (
). 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 (
). 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 (
), 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 (
) 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) (
). 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 (
). 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 (
). 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 (
). 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 (
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 (
). 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 (
). 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 (
). 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 (
)—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 (