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NECA derivatives exploit the paralog-specific properties of the site 3 side pocket of Grp94, the endoplasmic reticulum Hsp90

Open AccessPublished:September 09, 2019DOI:https://doi.org/10.1074/jbc.RA119.009960
      The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90β, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5′N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5′ moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.

      Introduction

      Hsp90 chaperones are required for the conformational maturation and late-stage activation of hundreds of client proteins, many of which are essential for cell viability (
      • Marzec M.
      • Eletto D.
      • Argon Y.
      GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum.
      ,
      • Johnson J.L.
      Evolution and function of diverse Hsp90 homologs and cochaperone proteins.
      • Taipale M.
      • Jarosz D.F.
      • Lindquist S.
      HSP90 at the hub of protein homeostasis: emerging mechanistic insights.
      ). Grp94, the endoplasmic reticulum Hsp90 paralog, is required for localization of cell surface receptors, including Toll-like receptors, Lrp6, Her2, and integrins (
      • Patel P.D.
      • Yan P.
      • Seidler P.M.
      • Patel H.J.
      • Sun W.
      • Yang C.
      • Que N.S.
      • Taldone T.
      • Finotti P.
      • Stephani R.A.
      • Gewirth D.T.
      • Chiosis G.
      Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2.
      • Randow F.
      • Seed B.
      Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability.
      ,
      • Liu B.
      • Staron M.
      • Hong F.
      • Wu B.X.
      • Sun S.
      • Morales C.
      • Crosson C.E.
      • Tomlinson S.
      • Kim I.
      • Wu D.
      • Li Z.
      Essential roles of grp94 in gut homeostasis via chaperoning canonical Wnt pathway.
      • Yang Y.
      • Liu B.
      • Dai J.
      • Srivastava P.K.
      • Zammit D.J.
      • Lefrançois L.
      • Li Z.
      Heat shock protein gp96 is a master chaperone for Toll-like receptors and is important in the innate function of macrophages.
      ), as well as secreted proteins, such as insulin-like growth factors (
      • Wanderling S.
      • Simen B.B.
      • Ostrovsky O.
      • Ahmed N.T.
      • Vogen S.M.
      • Gidalevitz T.
      • Argon Y.
      GRP94 is essential for mesoderm induction and muscle development because it regulates insulin-like growth factor secretion.
      ). Overexpression of Grp94 occurs in various cancers, including multiple myeloma and Her2+ breast cancer, and correlates with a poor prognosis (
      • Wu B.X.
      • Hong F.
      • Zhang Y.
      • Ansa-Addo E.
      • Li Z.
      GRP94/gp96 in Cancer: Biology, Structure, Immunology, and Drug Development.
      ,
      • Ansa-Addo E.A.
      • Thaxton J.
      • Hong F.
      • Wu B.X.
      • Zhang Y.
      • Fugle C.W.
      • Metelli A.
      • Riesenberg B.
      • Williams K.
      • Gewirth D.T.
      • Chiosis G.
      • Liu B.
      • Li Z.
      Clients and oncogenic roles of molecular chaperone gp96/grp94.
      ). Recent studies have shown that blocking Grp94 activity in these cancer cells leads to client degradation and reduces cancer cell viability (
      • Patel P.D.
      • Yan P.
      • Seidler P.M.
      • Patel H.J.
      • Sun W.
      • Yang C.
      • Que N.S.
      • Taldone T.
      • Finotti P.
      • Stephani R.A.
      • Gewirth D.T.
      • Chiosis G.
      Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2.
      ,
      • Hua Y.
      • White-Gilbertson S.
      • Kellner J.
      • Rachidi S.
      • Usmani S.Z.
      • Chiosis G.
      • Depinho R.
      • Li Z.
      • Liu B.
      Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma.
      ).
      Over a dozen Hsp90 inhibitors that target the ATP-binding pocket in the N-terminal domain (NTD)
      The abbreviations used are: NTD
      N-terminal domain
      PU
      purine
      NECA
      5′N-ethylcarboxamido adenosine
      NPCA
      N-propylcarboxamido adenosine
      NEoCA
      N-hydroxyethylcarboxamido adenosine
      NEaCA
      N-aminoethylcarboxamido adenosine
      ITC
      isothermal titration calorimetry
      FP
      fluorescence polarization.
      have been tested in clinical trials, but a lack of biomarkers for patient selection, compound- or target-specific toxicities, and, under some conditions, up-regulation of compensatory chaperone systems have hindered their progress to approval (
      • Jhaveri K.
      • Taldone T.
      • Modi S.
      • Chiosis G.
      Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers.
      • Neckers L.
      • Workman P.
      Hsp90 molecular chaperone inhibitors: are we there yet?.
      ,
      • Joshi S.
      • Wang T.
      • Araujo T.L.S.
      • Sharma S.
      • Brodsky J.L.
      • Chiosis G.
      Adapting to stress: chaperone networks in cancer.
      ,
      • Rodina A.
      • Wang T.
      • Yan P.
      • Gomes E.D.
      • Dunphy M.P.
      • Pillarsetty N.
      • Koren J.
      • Gerecitano J.F.
      • Taldone T.
      • Zong H.
      • Caldas-Lopes E.
      • Alpaugh M.
      • Corben A.
      • Riolo M.
      • Beattie B.
      • et al.
      The epichaperome is an integrated chaperome network that facilitates tumour survival.
      ,
      • Taldone T.
      • Wang T.
      • Rodina A.
      • Pillarsetty N.V.K.
      • Digwal C.S.
      • Sharma S.
      • Yan P.
      • Joshi S.
      • Pagare P.P.
      • Bolaender A.
      • Roboz G.J.
      • Guzman M.L.
      • Chiosis G.
      A chemical biology approach to the chaperome in cancer-hsp90 and beyond.
      • Wang T.
      • Rodina A.
      • Dunphy M.P.
      • Corben A.
      • Modi S.
      • Guzman M.L.
      • Gewirth D.T.
      • Chiosis G.
      Chaperome heterogeneity and its implications for cancer study and treatment.
      ). In addition to Grp94, humans possess two Hsp90 paralogs in the cytosol (Hsp90α and Hsp90β) and one in the mitochondria (Trap1). Because Hsp90 paralogs have distinct sets of client proteins with widely diverse cellular functions, selective inhibition (targeting just one of the four paralogs) may mitigate the limitations of some of the pan-hsp90 inhibitors and improve drug efficacy in cancer treatment (
      • Gewirth D.T.
      paralog-specific Hsp90 inhibitors: a brief history and a bright future.
      ).
      The residues that comprise the ATP-binding pocket of the NTDs are almost universally conserved, making hsp90 paralog selective drug discovery a challenge. Three distinct side pockets, however, adjoin the central ATP-binding cavity of hsp90s (Fig. 1), and paralog selectivity can be achieved by accessing these side pockets in one paralog but not another. Although the residues that line the side pockets are also conserved between paralogs, selective access is governed by intrinsic conformational differences between the NTDs of each paralog as well as by ligand-driven rearrangements (
      • Patel P.D.
      • Yan P.
      • Seidler P.M.
      • Patel H.J.
      • Sun W.
      • Yang C.
      • Que N.S.
      • Taldone T.
      • Finotti P.
      • Stephani R.A.
      • Gewirth D.T.
      • Chiosis G.
      Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2.
      ,
      • Ernst J.T.
      • Liu M.
      • Zuccola H.
      • Neubert T.
      • Beaumont K.
      • Turnbull A.
      • Kallel A.
      • Vought B.
      • Stamos D.
      Correlation between chemotype-dependent binding conformations of HSP90α/β and isoform selectivity: implications for the structure-based design of HSP90α/β-selective inhibitors for treating neurodegenerative diseases.
      • Ernst J.T.
      • Neubert T.
      • Liu M.
      • Sperry S.
      • Zuccola H.
      • Turnbull A.
      • Fleck B.
      • Kargo W.
      • Woody L.
      • Chiang P.
      • Tran D.
      • Chen W.
      • Snyder P.
      • Alcacio T.
      • Nezami A.
      • et al.
      Identification of novel HSP90α/β isoform selective inhibitors using structure-based drug design. demonstration of potential utility in treating CNS disorders such as Huntington’s disease.
      ,
      • Patel H.J.
      • Patel P.D.
      • Ochiana S.O.
      • Yan P.
      • Sun W.
      • Patel M.R.
      • Shah S.K.
      • Tramentozzi E.
      • Brooks J.
      • Bolaender A.
      • Shrestha L.
      • Stephani R.
      • Finotti P.
      • Leifer C.
      • Li Z.
      • Gewirth D.T.
      • et al.
      Structure-activity relationship in a purine-scaffold compound series with selectivity for the endoplasmic reticulum Hsp90 paralog Grp94.
      ,
      • Que N.L.S.
      • Crowley V.M.
      • Duerfeldt A.S.
      • Zhao J.
      • Kent C.N.
      • Blagg B.S.J.
      • Gewirth D.T.
      Structure-based design of a Grp94-selective inhibitor: exploiting a key residue in Grp94 to optimize paralog-selective binding.
      • Soldano K.L.
      • Jivan A.
      • Nicchitta C.V.
      • Gewirth D.T.
      Structure of the N-terminal domain of GRP94: basis for ligand specificity and regulation.
      ). In one of the best-studied examples, two of these side pockets, site 1 and site 2, have been shown to distinguish between cytosolic Hsp90 and Grp94. Crystal structures of each paralog bound to PU-H54, a purine-based (PU) Grp94-selective inhibitor, revealed that the flexibility of the Grp94 lid (helices 4 and 5), compared with its Hsp90 counterpart, permits conditional access to site 2 upon inhibitor binding. In Grp94:PU-H54, rearrangement of the lid causes Phe-199 to swing toward site 1, exposing the hydrophobic cleft of site 2. This movement allows the nonpolar 8-aryl moiety of PU-H54 to enter site 2 in an energetically favorable conformation. On the other hand, the structure of Hsp90:PU-H54 showed that the lid is unaffected by inhibitor binding and that Hsp90 Phe-138, the equivalent of Grp94 Phe-199, blocks site 2, forcing the 8-aryl moiety of PU-H54 to remain in the energetically less favorable site 1.
      Figure thumbnail gr1
      Figure 1Side pockets of the ATP-binding cavity in the Grp94 NTD. Residue numbers of side pocket side chains are indicated.
      While differential access to site 1 and site 2 offers selectivity within the PU inhibitor scaffold, less is known about how other regions of the ATP-binding pocket could be exploited for paralog selectivity. 5′N-ethylcarboxamido adenosine (NECA) is an ATP mimetic that selectively binds Grp94 in pulldown experiments (
      • Hutchison K.A.
      • Fox I.H.
      Purification and characterization of the adenosine A2-like binding site from human placental membrane.
      ,
      • Hutchison K.A.
      • Nevins B.
      • Perini F.
      • Fox I.H.
      Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins.
      ) and shows higher affinity for Grp94 than for Hsp90 (
      • Rosser M.F.
      • Nicchitta C.V.
      Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94: I: evidence for allosteric regulation of ligand binding.
      ). The structure of Grp94:NECA revealed that the 5′N-ethylcarboxamido moiety occupies the side pocket called site 3, which sits adjacent to the termini of the helix 4/5 lid (
      • Soldano K.L.
      • Jivan A.
      • Nicchitta C.V.
      • Gewirth D.T.
      Structure of the N-terminal domain of GRP94: basis for ligand specificity and regulation.
      ). Modeling the NECA binding pose into the structure of unliganded yeast Hsp90 showed that access to site 3 is disfavored by a predicted clash between the terminal methyl group of the 5′ moiety and the backbone carbonyl oxygen of Hsp90 Gly-121. In Grp94:NECA, the equivalent Gly-196 is displaced by 3.6 Å compared with Hsp90, allowing the 5′ moiety to enter site 3. Conformational remodeling of the Hsp90 lid or the entering ligand would therefore be required to accommodate NECA in Hsp90. Furthermore, the structure of Grp94:NECA revealed that the volume of site 3 is not fully occupied by the 5′ moiety of NECA, suggesting that inhibitors could be designed to fill site 3 and enhance Grp94 selectivity and affinity.
      To explore the ability of ligands to more completely occupy site 3 in Grp94 and to characterize the selectivity of NECA-based ligands, we designed 5′-modified derivatives of NECA that are predicted to extend further into site 3. We found that one of these derivatives exhibits improved selectivity for Grp94 over Hsp90. In addition, crystal structures of Grp94 and Hsp90 bound to NECA and its derivatives show that subtle energetic differences between the 5′ moieties of these ligands, leading to partial remodeling of site 3, dictate the selective preference for Grp94 over Hsp90. Taken together, our data demonstrate that site 3 is a promising target for the design of Grp94 selective inhibitors.

      Results

      NECA and its 5′-modified derivatives bind selectively to Grp94 over Hsp90

      Analysis of the structure of Grp94 in complex with NECA (
      • Soldano K.L.
      • Jivan A.
      • Nicchitta C.V.
      • Gewirth D.T.
      Structure of the N-terminal domain of GRP94: basis for ligand specificity and regulation.
      ) showed that the 5′N-ethylcarboxamido moiety does not completely fill site 3 of the ATP-binding pocket. Site 3, which is formed by Met-85, Asn-162, Leu-163, Thr-165, Ala-167, Thr-171, Gly-196, Val-197, Phe-199, and Tyr-200, sits adjacent to the central adenine-binding cavity of the N-terminal domain. We designed 3 ligands, N-propylcarboxamido adenosine (NPCA), N-hydroxyethylcarboxamido adenosine (NEoCA), and N-aminoethycarboxamido adenosine (NEaCA), which lengthen the 5′ moiety of NECA by an additional methyl, hydroxyl, or amino group, respectively (Fig. 2). We assessed the binding properties of these ligands to Grp94 and Hsp90 using isothermal titration calorimetry (ITC). As seen in Fig. 3 and Table 1, NECA, NPCA, and NEoCA bind to Grp94 with Kd values of 2.8 μm, 6.3 μm, and 9.1 μm, respectively. The measured affinity of NEaCA was considerably worse, with a Kd value of 130 μm.
      Figure thumbnail gr2
      Figure 2NECA and derivatives used in this study. Binding pockets of scaffold and substituent moieties in Hsp90 and Grp94 are indicated schematically.
      Figure thumbnail gr3
      Figure 3ITC analysis of NECA and derivatives binding to Grp94 and Hsp90. Titrations were carried out at 25 °C. Calculated dissociation constants are given on each thermogram. Errors in Kds are standard error of the mean of two replicate measurements.
      Table 1Thermodynamic parameters of ligand binding
      LigandProteinKdΔGΔHTΔS
      μmkcal/molkcal/molkcal/mol
      NECAGrp94N2.8 ± 0.1−7.545 ± 0.030−18.8 ± 0.6−11.26 ± 0.54
      Hsp90N14.0 ± 2.8−6.601 ± 0.120−11.4 ± 0.0−4.78 ± 0.15
      NPCAGrp94N6.3 ± 0.1−7.071 ± 0.006−16.3 ± 0.6−9.23 ± 0.57
      Hsp90N57.5 ± 2.1−5.762 ± 0.022−8.3 ± 0.0−2.54 ± 0.02
      NEoCAGrp94N9.1 ± 0.3−6.849 ± 0.018−20.1 ± 0.3−13.25 ± 0.30
      Hsp90N67.5 ± 3.5−5.667 ± 0.031−13.1 ± 2.0−7.43 ± 1.95
      NEaCAGrp94N130.0 ± 11.0−5.281 ± 0.052−12.0 ± 1.0−6.72 ± 1.04
      Hsp90N708.0 ± 88−4.283 ± 0.074−7.3 ± 0.6−3.02 ± 0.64
      PU-H71Grp94N0.063 ± 0.033−9.867 ± 0.330−15.4 ± 1.1−5.48 ± 1.39
      Hsp90N0.0049 ± 0.0020−11.440 ± 0.339−19.9 ± 2.8−8.43 ± 3.08
      NECA and its derivatives exhibit preferential binding to Grp94 over Hsp90. As seen in Fig. 3 and Table 1, NECA, NPCA, NEoCA, and NEaCA bind to human Hsp90α with Kd values of 14, 58, 68, and 708 μm, respectively. When comparing the ratio of Kd values for Grp94 and Hsp90, it is apparent that NECA binds with 5-fold greater affinity to Grp94 than to Hsp90, whereas NPCA, NEoCA, and NEaCA exhibit 9.1-, 7.4-, and 5.4-fold higher affinity for Grp94 than for Hsp90. In this series, the order of -fold selectivity of compounds for Grp94 over Hsp90 is NPCA > NEoCA > NEaCA ∼ NECA.
      We compared the Kd values determined by ITC with Ki values calculated from a fluorescence polarization (FP) competition displacement assay using geldanamycin-Cy3b as the tracer (
      • Kim J.
      • Felts S.
      • Llauger L.
      • He H.
      • Huezo H.
      • Rosen N.
      • Chiosis G.
      Development of a fluorescence polarization assay for the molecular chaperone Hsp90.
      ,
      • Taldone T.
      • Patel P.D.
      • Patel M.
      • Patel H.J.
      • Evans C.E.
      • Rodina A.
      • Ochiana S.
      • Shah S.K.
      • Uddin M.
      • Gewirth D.
      • Chiosis G.
      Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series.
      ). As seen in Fig. 4 and Table 2, the Ki values for NECA, NPCA, NEoCA, and NEaCA, calculated from the IC50 measurements according to the method of Nikolovska-Coleska et al. (
      • Nikolovska-Coleska Z.
      • Wang R.
      • Fang X.
      • Pan H.
      • Tomita Y.
      • Li P.
      • Roller P.P.
      • Krajewski K.
      • Saito N.G.
      • Stuckey J.A.
      • Wang S.
      Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization.
      ) yielded values of 1.0, 2.0, 3.9, and >20 μm for Grp94 and 7.9, 32.8, >40, and >40 μm for Hsp90. The Ki data agree with the measured Kd values and yield Grp94 selectivity factors of 16.6 for NPCA, >10 for NEoCA, and 8.0 for NECA, in good agreement with the Kd data calculated from the ITC measurements.
      Figure thumbnail gr4
      Figure 4FP binding assay of NECA and derivatives. A and B, the assay contained 10 nm full-length hHsp90α (A) or Grp94 (B) and 6 nm geldanamycin-Cy3b tracer. Graphs represent the average of two independent measurements.
      Table 2Comparison of FP IC50, FP Ki, ITC Kd, and ATPase IC50 values
      LigandProteinFP IC50FP KiITC KdATPase IC50Fold Grp94 Selectivity
      IC50KiKd
      μmμmμmμm
      NECAGrp944.8 ± 0.71.0 ± 0.12.8 ± 0.1410 ± 4.520.7 ± 4.57.9 ± 1.55.0 ± 1.0
      Hsp9099.6 ± 16.27.9 ± 1.314.0 ± 2.8ND
      NPCAGrp949.7 ± 1.62.0 ± 0.36.3 ± 0.117 ± 3.542.8 ± 7.916.4 ± 2.89.1 ± 0.4
      Hsp90414.9 ± 33.632.8 ± 2.757.5 ± 2.1ND
      NEoCAGrp9418.9 ± 2.63.9 ± 0.59.1 ± 0.310 ± 2.4>26 ± 4>10 ± 1.37.4 ± 0.5
      Hsp90>500>4067.5 ± 3.5ND
      NEaCAGrp94>100>20130 ± 11>100>5>25.4 ± 0.8
      Hsp90>500>40708 ± 88ND
      Compounds that target the ATP-binding pocket are predicted to competitively displace ATP, leading to loss of ATP hydrolysis activity. To verify that the NECA inhibitors displace ATP in Grp94, we monitored the ATP hydrolysis activity of Grp94 in response to increasing amounts of the NECA derivative. As seen in Table 2 and Fig. S3, all of the compounds suppressed the ATP hydrolysis activity of Grp94. IC50 values calculated from the inhibition titration yielded values of 10 μm for NECA and NEoCA, 17 μm for NPCA, and >100 μm for NEaCA. These values agree approximately with the Kd values for these compounds measured by ITC.
      From the binding data, it appears that increasing the length of the 5′ substituent improves the relative binding of NECA derivatives to Grp94 compared with Hsp90. Thus, targeting site 3 is a potential mechanism of paralog selectivity.

      Structures of Grp94 and Hsp90 bound to NECA derivatives

      To understand the structural basis of the observed differences in affinity between NECA and its derivatives for Grp94 and Hsp90, we determined the crystal structures of the N-terminal domains of Grp94 and Hsp90 in complex with these compounds. Structures of the Grp94 N-terminal domain with (Grp94N) and without (Grp94NΔ41) the charged linker in complex with NECA have been reported previously (PDB codes 1U2O, 1YSZ, and 6D28) (
      • Soldano K.L.
      • Jivan A.
      • Nicchitta C.V.
      • Gewirth D.T.
      Structure of the N-terminal domain of GRP94: basis for ligand specificity and regulation.
      ,
      • Dollins D.E.
      • Immormino R.M.
      • Gewirth D.T.
      Structure of unliganded GRP94, the endoplasmic reticulum Hsp90: basis for nucleotide-induced conformational change.
      ). Crystals of ligand complexes containing Grp94N were determined here for complexes containing NPCA and NEoCA (PDB codes 6D1X and 6CYI). Crystals of ligand complexes containing Grp94NΔ41 were determined for NPCA, NEoCA, and NEaCA (PDB codes 2GQP, 2HG1, and 2HCH). Data collection and refinement statistics are presented in Table 3.
      Table 3Data collection and refinement statistics
      Grp94NΔ41:NPCAGrp94N:NPCAGrp94 NΔ41:NEoCAGrp94 N:NEoCAGrp94 NΔ41:NEaCA
      PDB code2GQP6D1X2HG16CYI2HCH
      Data collection
      X-ray source/detectorAPS 22-BM/MarCCDRU200/R-AxisRU200/R-AxisAPS 23-IDD/PilatusRU200/R-Axis
      Space groupP212121C2221P212121C2221P212121
      Cell dimensions
      a, b, c (Å)65.69, 84.84, 95.7489.47, 99.65, 63.1965.19, 84.43, 94.8289.06, 100.21, 63.4365.66, 84.77, 94.92
      Resolution (Å) (last shell)1.50 (1.56–1.50)2.30 (2.38–2.30)2.30 (2.38–2.30)1.75 (1.78–1.75)2.30 (2.38–2.30)
      Rmerge (%)0.076 (0.588)0.07 (0.227)0.111 (0.344)0.087 (0.585)0.099 (0.348)
      Average II14.3 (3.64)n/a (2.98)10.1 (4.05)28.2 (1.2)16.6 (5.47)
      Completeness (%)99.9 (100.0)91.8 (93.6)99.9 (100.0)86.1 (42.4)99.9 (99.2)
      Redundancy7.32.63.63.75.2
      CC(1/2)NANANA0.779NA
      Refinement
      Resolution (Å)38.8–1.531.1–2.3047.41–2.3045.92–1.7647.46–2.30
      Unique reflections86,23011,51323,79324,39824,177
      Rwork/Rfree0.215/0.2350.174/0.2380.214/0.2430.204/0.2430.220/0.254
      Nonhydrogen atoms36141906374119103632
      Water molecules56311822599193
      RMSDs
      Bond lengths (Å)0.0060.0070.0060.0060.006
      Bond angles (°)1.40.891.21.011.5
      Ramachandran outliers (%)0.00.00.20.00.0
      Clash score413846
      Hsp90αN:NECAHsp90αN:NPCAHsp90αN:NEaCAHsp90αN:NEoCA
      PDB code6B996B9A6CYG6CYH
      Data collection
      X-ray source/detectorSSRL 12–2/PilatusSSRL 12–2/PilatusAPS 17ID/PilatusAPS 17ID/Pilatus
      Space groupI222P212121P21212P21212
      Cell dimensions
      a, b, c (Å)64.55, 90.08, 98.4164.45, 89.39, 99.2964.40, 89.27, 99.0764.43, 88.87, 99.00
      Resolution (Å) (last shell)1.60 (1.63–1.60)1.65 (1.68–1.65)1.50 (1.53–1.50)1.50 (1.53–1.50)
      Rmerge (%)0.105 (0.561)0.081 (0.549)0.048 (0.633)0.050 (0.413)
      Average II26.6 (2.8)29.8 (1.9)31.5 (2.5)32.6 (2.1)
      Completeness (%)99.2 (99.2)99.2 (99.5)99.9 (99.7)99.8 (97.4)
      Redundancy6.06.26.46.1
      CC(1/2)0.7910.8400.7560.818
      Refinement
      Resolution (Å)35.89–1.6043.4–1.6535.94–1.5035.94–1.50
      Unique reflections37,66068,39890,96190,962
      Rwork/Rfree0.164/0.1830.164/0.1910.179/0.2060.181/0.208
      Nonhydrogen atoms1695326132903280
      Water molecules280478636617
      RMSDs
      Bond lengths (Å)0.0060.0130.0060.007
      Bond angles (°)1.031.681.0531.107
      Ramachandran outliers (%)0.00.00.00.0
      Clash score0.6234
      The Grp94NΔ41:ligand complexes crystallize in a different space group than the Grp94N:ligand complexes, and in the Grp94NΔ41:ligand complexes, the residues corresponding to the junction between helices 3 and 4 (167–169) and some portions of helix 4 (residues 170–186) are disordered. Because these disordered regions include two residues that form part of site 3 (Ala-167 and Thr-171), we compared corresponding pairs of Grp94N and Grp94NΔ41:ligand complexes to see whether the disordered residues affected the pose or placement of the bound ligand. In all pairs, the electron density for the bound ligand is strong (Fig. S1A) and shows that the pose of the ligand in the paired Grp94N and Grp94NΔ41 complexes is nearly identical (Fig. S1B). In the results that follow, we refer to the higher-resolution structure of the N/NΔ41 pair when describing the ligand pose and reference the Grp94N structure when describing the site 3 interactions.
      The protein components of the Grp94:NECA, Grp94:NPCA, Grp94:NEoCA, and Grp94:NEaCA structures are nearly identical, with average pairwise root mean square deviations between the Cα atoms of each protein of 0.205 ± 0.038 Å. In each structure, the adenine and ribose moieties of the adenosine occupy identical positions within the ATP-binding cavity of Grp94, with the adenine resting in the central binding cavity and the hydroxyls of the pendant ribose pointing out of the mouth of the cavity (Fig. 5A). Stabilization of the bound ligand is achieved in part by 10 direct or water-mediated hydrogen bonds between the adenosine moiety and the protein, which is in agreement with the enthalpy-driven binding observed by ITC. Within the 5′ carboxamido moiety, the additional methyl, hydroxyl, or amino groups of NPCA, NEoCA, and NEaCA, respectively, are essentially superimposable and sit across the width of the site 3 pocket. Although they would be free to rotate in solution, the longer 5′ moieties of NPCA, NEoCA, and NEaCA (Fig. 5B) fit tightly into site 3 and effectively restrict the dihedral torsion angle of the amido-carbon bond to a gauche conformation (Fig. 5C). The gauche conformation is energetically less favored compared with the anti configuration seen in Grp94:NECA (Fig. 5D). This steric constraint may account for the slightly higher KD of NPCA and NEoCA to Grp94 compared with NECA.
      Figure thumbnail gr5
      Figure 5NECA and derivatives occupy site 3 in Grp94. A, overlay of Grp94:NECA (PDB code 1QY5), Grp94:NPCA, Grp94:NEoCA, and Grp94:NeaCA, showing close overlap of binding pocket residues and ligands. Carbon atoms: yellow, NECA; blue, NPCA; green, NeoCA; magenta, NEaCA. B, surface representation of the site 3 binding pocket. C, the 5′ moieties of NPCA, NEoCA, and NEaCA adopt a gauche conformation at their ends. D, shown in the same orientation as C; the 5′ moiety of NECA adopts an anti conformation.
      We also determined the structures of human Hsp90α in complex with NECA, NPCA, NEoCA, and NEaCA. Crystals of apoHsp90N were soaked with the ligand prior to freezing and data collection. Crystals of Hsp90:NECA and Hsp90:NPCA diffracted to a resolution of 1.6 Å and 1.65 Å, respectively, whereas crystals of Hsp90:NEoCA and Hsp90:NEaCA each diffracted to a resolution of 1.5 Å. All structures were solved by molecular replacement. Data collection and refinement statistics are presented in Table 3. The position of the bound ligands is supported by strong electron density (Fig. S1A).
      In the Hsp90:NECA complex, the NECA ligand occupies the ATP-binding pocket. Comparison of the structures of Hsp90:NECA and Grp94:NECA shows that the adenine and ribose moieties of NECA make similar interactions within the ATP-binding pocket but differ significantly in the poses of the 5′ moieties of the bound NECA. As seen in Fig. 6A, compared with Grp94-bound NECA (NECAGrp), the dihedral angle of the amido-carbon bond of NECAHsp undergoes a 190° rotation, resulting in the ∈ methyl group pointing toward the back face of Hsp90 site 3. In contrast, the pose of NECAGrp places the ∈ methyl closer to the front face of site 3. The entrance to site 3 in Hsp90 is constricted compared with the entrance to site 3 in Grp94. This constriction is due to differences in the position of the carbonyl oxygen of Gly-135 compared with the equivalent Gly-196 in Grp94. As described previously for yeast Hsp90 (
      • Soldano K.L.
      • Jivan A.
      • Nicchitta C.V.
      • Gewirth D.T.
      Structure of the N-terminal domain of GRP94: basis for ligand specificity and regulation.
      ), modeling the NECAGrp ligand into the structure of human Hsp90:NECA shows that the NECAGrp would clash with the carbonyl oxygen of Gly-135 (Fig. 6B). The rearranged pose of NECAHsp thus reflects the need to avoid steric clashes with Gly-135 in the Hsp90 pocket.
      Figure thumbnail gr6
      Figure 6NECA and derivatives bound to Hsp90. A, comparison of NECA poses when bound to Hsp90 (NECAHsp) and Grp94 (NECAGrp). The Hsp90-binding pocket is shown. B, modeling NECAGrp into Hsp90, showing a potential clash with the δ carbon and the carbonyl oxygen of Gly-135. C, structure of Hsp90:NPCA. Two rotamers for the side chain of Tyr-139 were observed in the crystal structure. D, structure of Hsp90:NEoCA. The ∈ oxygen of NEoCA rotates to avoid a clash with Tyr-139, obviating the need to adopt the distal rotamer. E, structure of Hsp90:NEaCA. The ∈ nitrogen adopts two poses and forms an extensive hydrogen bond network with the site 3 binding pocket.
      NPCA binds to Hsp90 with a 3.5-fold lower affinity than NECA. Examination of the structure of the Hsp90:NPCA complex shows that, although the adenine and ribose again occupy their expected positions, the 5′ moiety fits deeper into site 3 of Hsp90 compared with NECA and packs closely against the side chain of Tyr-139 (Fig. 6C). In the high-resolution structure of Hsp90:NPCA, the electron density for Tyr-139 is consistent with two side-chain rotamers. In the more favored “distal” rotamer, the side chain of Tyr-139 is 3.5 Å away from the epsilon methyl of NPCA. In the “proximal” rotamer, on the other hand, the side chain of Tyr-139 is rotated 19.6° away from the distal position. This decreases the distance from the ∈ methyl of NPCA to Tyr-139 to 2.4 Å, a distance that is too close to be energetically favorable (Fig. 6C). The distal rotamer conformation appears to be a consequence of the longer 5′ moiety of NPCA because comparison of the high-resolution Hsp90:NECA and Hsp90:NPCA structures shows that, in Hsp90:NECA, Tyr-139 occupies only the “normal” proximal rotamer conformation.
      The distal rotamer conformation of Tyr-139 enlarges site 3 in Hsp90:NPCA and prevents a van der Waals clash with the longer 5′ moiety of NPCA. The distal rotamer of Tyr-139 seen in Hsp90:NPCA, however, pushes the side chain of the residue deeper into the hydrophobic core of Hsp90. Although the energetic consequences of this movement are unknown, an analysis of the 194 human Hsp90α N-terminal domain structures in the PDB shows that only 10 other structures exhibit this Tyr-139 conformation (Fig. S2). This set includes eight related complexes of Hsp90 bound to tricyclic imidazo pyridines (
      • Vallée F.
      • Carrez C.
      • Pilorge F.
      • Dupuy A.
      • Parent A.
      • Bertin L.
      • Thompson F.
      • Ferrari P.
      • Fassy F.
      • Lamberton A.
      • Thomas A.
      • Arrebola R.
      • Guerif S.
      • Rohaut A.
      • Certal V.
      • et al.
      Tricyclic series of heat shock protein 90 (Hsp90) inhibitors part I: discovery of tricyclic imidazo[4,5-c]pyridines as potent inhibitors of the Hsp90 molecular chaperone.
      ) and two structures of complexes with benzimidazol-substituted pyrrolo pyridine carboxamides. These ligands insert into site 1 of the Hsp90-binding pocket and make direct hydrogen bond interactions between the Tyr-139 OH and nitrogen substituents of the ligand. In contrast, in Hsp90:NPCA, Tyr-139 makes no stabilizing ligand interactions and instead moves only to accommodate the impinging 5′ ligand moiety from site 3. Compared with Hsp90:NECA, this movement likely accounts for the energetic penalty incurred when Hsp90 binds NPCA.
      Compared with Hsp90:NPCA, the structures of Hsp90:NEoCA and Hsp90:NEaCA reveal altered positioning of the terminal hydroxyl and amine groups. In Hsp90:NEoCA, the ∈ OH of NEoCA points away from the back wall of site 3 and makes hydrogen bond interactions with the carbonyl oxygen of Gly-135 and with a water molecule (Fig. 6D). NEaCA, on the other hand, contains a charged terminal amino group that adopts two conformations of equal occupancy. In both cases, the ∈ amino group makes extensive hydrogen-bonding interactions with the carbonyl oxygens of Asn-106, Thr-109, and either the carbonyl oxygen of Ile-110 or the OH of Tyr-139, depending on the conformation of the terminal amino group. The Kd of NEaCA binding to both Grp94 and Hsp90 is substantially worse than for NECA, NPCA, or NEoCA. This cannot be ascribed to unfavorable steric clashes because the poses of NEoCA and NEaCA are essentially identical when bound to Grp94, and the conformation of both Grp94 and Hsp90 is unaltered in the protein:NEaCA complexes. Rather, the reduced binding affinity likely reflects the energetic costs of burying a fully charged amino group deep in a protein cavity, perhaps displacing stabilizing water molecules in the process.

      Discussion

      Of the three side pockets identified near the ATP-binding cavity of hsp90 chaperones, sites 1 and 2 have been exploited previously for the design of paralog-selective inhibitors (
      • Patel P.D.
      • Yan P.
      • Seidler P.M.
      • Patel H.J.
      • Sun W.
      • Yang C.
      • Que N.S.
      • Taldone T.
      • Finotti P.
      • Stephani R.A.
      • Gewirth D.T.
      • Chiosis G.
      Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2.
      ,
      • Ernst J.T.
      • Liu M.
      • Zuccola H.
      • Neubert T.
      • Beaumont K.
      • Turnbull A.
      • Kallel A.
      • Vought B.
      • Stamos D.
      Correlation between chemotype-dependent binding conformations of HSP90α/β and isoform selectivity: implications for the structure-based design of HSP90α/β-selective inhibitors for treating neurodegenerative diseases.
      • Ernst J.T.
      • Neubert T.
      • Liu M.
      • Sperry S.
      • Zuccola H.
      • Turnbull A.
      • Fleck B.
      • Kargo W.
      • Woody L.
      • Chiang P.
      • Tran D.
      • Chen W.
      • Snyder P.
      • Alcacio T.
      • Nezami A.
      • et al.
      Identification of novel HSP90α/β isoform selective inhibitors using structure-based drug design. demonstration of potential utility in treating CNS disorders such as Huntington’s disease.
      ,
      • Patel H.J.
      • Patel P.D.
      • Ochiana S.O.
      • Yan P.
      • Sun W.
      • Patel M.R.
      • Shah S.K.
      • Tramentozzi E.
      • Brooks J.
      • Bolaender A.
      • Shrestha L.
      • Stephani R.
      • Finotti P.
      • Leifer C.
      • Li Z.
      • Gewirth D.T.
      • et al.
      Structure-activity relationship in a purine-scaffold compound series with selectivity for the endoplasmic reticulum Hsp90 paralog Grp94.
      • Que N.L.S.
      • Crowley V.M.
      • Duerfeldt A.S.
      • Zhao J.
      • Kent C.N.
      • Blagg B.S.J.
      • Gewirth D.T.
      Structure-based design of a Grp94-selective inhibitor: exploiting a key residue in Grp94 to optimize paralog-selective binding.
      ,
      • Ohkubo S.
      • Kodama Y.
      • Muraoka H.
      • Hitotsumachi H.
      • Yoshimura C.
      • Kitade M.
      • Hashimoto A.
      • Ito K.
      • Gomori A.
      • Takahashi K.
      • Shibata Y.
      • Kanoh A.
      • Yonekura K.
      TAS-116, a highly selective inhibitor of heat shock protein 90α and β, demonstrates potent antitumor activity and minimal ocular toxicity in preclinical models.
      ). Site 3, in contrast, has been relatively neglected, in part because site 3 is further away from the central ATP-binding cavity, requiring longer linkers between the central scaffold and its attached substituents and making targeting more difficult. The structural differences between site 3 in Grp94 and Hsp90 are also subtle, and access to site 3 is not governed by structural rearrangements to the N-terminal domain as they are for sites 1 and 2.
      Here we have shown that inhibitors based on the NECA scaffold all target site 3 and bind with a 5- to 9-fold preference for Grp94 over Hsp90. The origins of the preferential binding of these compounds to Grp94 appear to be due to the effect of Gly-135 in Hsp90 on the trajectory of the 5′ carboxamido substituent. Initial modeling studies (
      • Soldano K.L.
      • Jivan A.
      • Nicchitta C.V.
      • Gewirth D.T.
      Structure of the N-terminal domain of GRP94: basis for ligand specificity and regulation.
      ) indicated that NECA would not be able to access site 3 in yeast Hsp90 in the pose adopted when it bound to Grp94. The structural data presented here indicate that, although the adenine and ribose of NECA bind to the central pocket of Hsp90 in the same manner as Grp94, Hsp90-bound NECA redirects its carboxamido moiety toward the rear of site 3 by altering the amido dihedral angle to avoid a clash with the carbonyl oxygen of Gly-135. Although the structure of site 3 is not altered in Hsp90:NECA, the energetic strain imposed by the adoption of this alternate ligand binding pose correlates with the modestly lower binding affinity of NECA to Hsp90 compared with Grp94.
      In an attempt to improve ligand affinity and selectivity for Grp94, we hypothesized that compounds that insert deeper into site 3 would incur a binding advantage over NECA because of increased interactions between the ligand and the pocket. However, the binding data reported here show that NPCA, NEoCA, and NEaCA all bind to Grp94 and Hsp90 with weaker affinity than the parent compound, NECA. In the case of Grp94, examination of the structures of the Grp94:NPCA, Grp94:NEoCA, and Grp94:NEaCA complexes shows that the 5′ moieties all adopt similar poses that directs the 5′ moiety further toward the rear of site 3. This results in a gauche conformation at the terminal end of the 5′ substituents, which incurs a modest energetic penalty compared with the anti pose. In the case of NPCA and NEoCA, the energetic penalty for this conformation compared with NECA reduces the binding affinity by only 2- to 3-fold. Incorporation of the amino substituent, however, leads to a nearly 50-fold decrease in binding affinity, indicating that burial of a charged group in such a constrained pocket is strongly disfavored.
      A similar trend in the binding affinities for NPCA, NEoCA, and NEaCA is also observed for Hsp90. Incorporation of the additional methyl or hydroxyl group results in a 4- to 5-fold loss of binding affinity, whereas addition of the charged amino group lowers the affinity by 50-fold compared with the parent compound, NECA. Interestingly, the maximal selectivity between Grp94 and Hsp90 in this series is found with NPCA, which gains an additional factor of 2 in selectivity for Grp94 over Hsp90 compared with NECA. This can be rationalized by the structure of Hsp90:NPCA, which shows that the nonpolar ∈ methyl group is positioned facing the rear of the binding site, where it impinges on the position of Tyr-139, in turn displacing Tyr-139 deeper into the hydrophobic core of the Hsp90 N-terminal domain. The observed repositioning of Tyr-139 seen in Hsp90:NPCA is rare and has only been observed in Hsp90: ligand complexes where the ligand forms hydrogen-bonding interactions with the hydroxyl of the tyrosine side chain, which presumably stabilizes the altered rotamer. No such ligand-mediated stabilization is observed between NPCA and Tyr-139, leading to the conclusion that the distal rotamer is energetically disfavored.
      Because of the large sample requirements and low-throughput nature of ITC, most ligand binding assays for Hsp90 chaperones have made use of a fluorescence polarization competition binding assay (
      • Kim J.
      • Felts S.
      • Llauger L.
      • He H.
      • Huezo H.
      • Rosen N.
      • Chiosis G.
      Development of a fluorescence polarization assay for the molecular chaperone Hsp90.
      ,
      • Taldone T.
      • Patel P.D.
      • Patel M.
      • Patel H.J.
      • Evans C.E.
      • Rodina A.
      • Ochiana S.
      • Shah S.K.
      • Uddin M.
      • Gewirth D.
      • Chiosis G.
      Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series.
      ). We compared binding of NECA and its derivatives to Grp94 and Hsp90 using both ITC and FP and showed that the IC50 measurements resulting from the FP assay underestimates the binding affinity for Hsp90, leading to overestimation of the selectivity of Grp94-selective inhibitors. The difference between the two assays can be traced to the different affinities for Hsp90 and Grp94 of the fluorescent tracer ligand. When IC50 measurements are converted to Ki values, the FP and ITC measurements are in good agreement. This suggests that future studies of paralog-selective ligands may benefit from comparing Ki values instead of IC50 values.
      The binding data presented here agree with earlier studies showing that NECA binds selectively to Grp94 (
      • Hutchison K.A.
      • Fox I.H.
      Purification and characterization of the adenosine A2-like binding site from human placental membrane.
      ,
      • Hutchison K.A.
      • Nevins B.
      • Perini F.
      • Fox I.H.
      Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins.
      • Rosser M.F.
      • Nicchitta C.V.
      Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94: I: evidence for allosteric regulation of ligand binding.
      ). A recent report from Liu and Street (
      • Liu S.
      • Street T.O.
      5′-N-ethylcarboxamidoadenosine is not a paralog-specific Hsp90 inhibitor.
      ), however, suggests that NECA binds preferentially to Hsp90, not Grp94. The discrepancy between these conclusions likely reflects the assays used to measure selectivity. Liu and Street (
      • Liu S.
      • Street T.O.
      5′-N-ethylcarboxamidoadenosine is not a paralog-specific Hsp90 inhibitor.
      ) monitored ligand selectivity by interference with ATP hydrolysis, which relies on competition between NECA and ATP for the ATP-binding site on the chaperone. It has been established previously that, although the affinity of ATP for Grp94 is on par with that reported for NECA here, the affinity of ATP for human Hsp90 is nearly two orders of magnitude weaker than for Grp94 (
      • Taldone T.
      • Patel P.D.
      • Patel M.
      • Patel H.J.
      • Evans C.E.
      • Rodina A.
      • Ochiana S.
      • Shah S.K.
      • Uddin M.
      • Gewirth D.
      • Chiosis G.
      Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series.
      ,
      • Ge J.
      • Normant E.
      • Porter J.R.
      • Ali J.A.
      • Dembski M.S.
      • Gao Y.
      • Georges A.T.
      • Grenier L.
      • Pak R.H.
      • Patterson J.
      • Sydor J.R.
      • Tibbitts T.T.
      • Tong J.K.
      • Adams J.
      • Palombella V.J.
      Design, synthesis, and biological evaluation of hydroquinone derivatives of 17-amino-17-demethoxygeldanamycin as potent, water-soluble inhibitors of Hsp90.
      ). Competition between ATP and NECA for Grp94 and Hsp90 that does not account for the difference between the ATP affinities of the two paralogs would significantly underestimate the intrinsic selectivity of NECA in favor of Hsp90.
      Recently, several inhibitors have been reported that were designed to target site 3 in Grp94 (
      • Crowley V.M.
      • Huard D.J.E.
      • Lieberman R.L.
      • Blagg B.S.J.
      Second generation Grp94-selective inhibitors provide opportunities for the inhibition of metastatic cancer.
      ,
      • Crowley V.M.
      • Khandelwal A.
      • Mishra S.
      • Stothert A.R.
      • Huard D.J.
      • Zhao J.
      • Muth A.
      • Duerfeldt A.S.
      • Kizziah J.L.
      • Lieberman R.L.
      • Dickey C.A.
      • Blagg B.S.
      Development of glucose regulated protein 94-selective inhibitors based on the BnIm and Radamide scaffold.
      ). These compounds, which contain bisphenyl (PDB Chemical Component (CC) ID VC1, VC5) or furan-imidazole (CC ID 6C0) substituents off the core resorcinylic scaffold exhibit submicromolar affinity for Grp94 and have FP IC50 selectivities over Hsp90 of up to 70-fold. The evidence that site 3 is targeted by the resorcinylic substituents of these inhibitors comes from crystal structures (PDB codes 5IN9, 6AOL, and 6AOM) of complexes formed by soaking the compounds into preformed crystals of apoGrp94NΔ41. Although the structures are modeled with the furan imidazole and bisphenyl substituents inserted into site 3, the electron density for these pendant moieties is discontinuous and weak. This observation is inconsistent with the submicromolar IC50 values reported for these compounds and prompts reconsideration of their possible mode of binding. Notably, all of the compounds designed to target site 3 bear a strong resemblance to the resorcinylic benzyl imidazole compound BnIm (CC ID 9QY) (
      • Duerfeldt A.S.
      • Peterson L.B.
      • Maynard J.C.
      • Ng C.L.
      • Eletto D.
      • Ostrovsky O.
      • Shinogle H.E.
      • Moore D.S.
      • Argon Y.
      • Nicchitta C.V.
      • Blagg B.S.
      Development of a Grp94 inhibitor.
      ). A recent structure of BnIm bound to Grp94NΔ41 that was generated by cocrystallization of the ligand with the protein (PDB code 5WMT) showed that the benzyl imidazole moiety, whose placement is supported by strong electron density, occupies site 1 in the ATP pocket, not site 3. In addition, in Grp94NΔ41:BnIm, the resorcinylic scaffold is flipped about its pseudosymmetric resorcinylic–imidizole axis to insert the methyl ester moiety into an exposed site 2 (
      • Que N.L.S.
      • Crowley V.M.
      • Duerfeldt A.S.
      • Zhao J.
      • Kent C.N.
      • Blagg B.S.J.
      • Gewirth D.T.
      Structure-based design of a Grp94-selective inhibitor: exploiting a key residue in Grp94 to optimize paralog-selective binding.
      ). Unlike site 3, sites 1 and 2 are inaccessible in unliganded Grp94, and the structure of Grp94NΔ41:BnIm showed that significant conformational changes to the protein were required to open sites 1 and 2 for ligand occupancy. These conformational changes are not possible in apo Grp94N or Grp94NΔ41 crystals without disrupting the crystal lattice. Thus, soaking site 1 or site 2-binding ligands into preformed apoGrp94 crystals limits the occupancy possibilities for the resorcinylic substituents to either site 3 or the open mouth of the ATP-binding cavity, which is what is observed in the soaks. Taken together, the similarities between BnIm and VC1, VC5, and 6C0 and the lack of strong crystallographic support for the modeled conformations suggest that the later compounds target sites 1 and 2 of Grp94 but not site 3. This, in turn, suggests that, to date, the only compounds that are bona fide site 3 binders are Radamide and the NECA series described here.
      The rigidity of the NECA scaffold effectively directs the carboxamido substituent into site 3 of Grp94, and modest selectivity over Hsp90 suggests that site 3 has potential as a paralog-selective side pocket. However, functional and therapeutic exploitation of the NECA scaffold has been slowed by concerns about cross-reactivity with adenosine A2 receptors as well as by the modest affinity of NECA for Grp94. With regard to the later concern, NECA and the derivatives tested here exhibit high solubility in aqueous solutions (>200 mm). It is possible that next-generation NECA derivatives, designed with higher lipophilicity, might effectively drive the equilibrium in favor of Grp94 binding and improve the potency of these compounds.

      Experimental procedures

      Protein expression and purification

      The N-terminal domain of Grp94 (Grp94NΔ41) (residues 69–337; Δ41 refers to deletion of the charged linker, residues 287–327, which are replaced by four glycine residues) was overexpressed in Escherichia coli strain BL21 Star DE3 as GST fusions. Expression, purification, and removal of GST by thrombin proteolysis were done as described previously (
      • Soldano K.L.
      • Jivan A.
      • Nicchitta C.V.
      • Gewirth D.T.
      Structure of the N-terminal domain of GRP94: basis for ligand specificity and regulation.
      ). Near full-length Grp94 (residues 73–754 Δ41) was expressed in E. coli and purified as described previously (
      • Dollins D.E.
      • Warren J.J.
      • Immormino R.M.
      • Gewirth D.T.
      Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones.
      ). The N-terminal domain of human Hsp90α (residues 1–236) was expressed in E. coli as an N-terminal His tag fusion and purified as described previously (
      • Immormino R.M.
      • Kang Y.
      • Chiosis G.
      • Gewirth D.T.
      Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors.
      ).

      Reagents

      NECA was purchased from Sigma. NPCA, NEoCA, and NEaCA were provided by E. Toone (Duke University) and synthesized in a two-step process starting from 2′,3′-isopropyl-ideneadenosine and propylamine, isopropenyl chloroformate, or ethylenediamine (all from Aldrich) according to published protocols (
      • de Zwart M.
      • Kourounakis A.
      • Kooijman H.
      • Spek A.L.
      • Link R.
      • von Frijtag Drabbe Künzel J.K.
      • Ijzerman A.P.
      5′-N-substituted carboxamidoadenosines as agonists for adenosine receptors.
      ).

      Analytical HPLC-MS

      Mass spectra of NECA and its derivatives were obtained on an Advion Expression LCMS instrument with electrospray ionization. Analytical HPLC was performed on an Agilent Technologies 1260 Infinity Quaternary LC system. The purity and identity of each compound were verified by HPLC-MS using the following method: 12-min gradient of increasing concentrations of acetonitrile in water (5% → 100%) containing 0.1% formic acid with a flow rate of 0.4 ml/min and UV detection at λ = 218 and 260 nm on an Agilent Poroshell 120 EC-C18, 3.0 mm × 450 mm, 2.7-μm column. Compounds had a purity of 95% or more.

      Isothermal titration calorimetry

      Proteins were equilibrated into DTT-free assay buffer by repeated concentration and dilution using an ultrafiltration spin filter (Millipore). Concentrated ligand solutions in DMSO, typically 100 mm, were diluted into assay buffer. The standard ITC assay buffer contained 40 mm Hepes (pH 7.5), 100 mm NaCl, and 1% DMSO. Titrations were carried out at 25 °C using a VP-ITC calorimeter (Microcal, Inc.) with the ligand solution (500–1500 μm) loaded into the titration syringe and the protein (30–150 μm) into the cell. ITC titration against PU-H71, a well-characterized Hsp90 inhibitor, was used as a benchmark for both instrument and protein quality control.
      ITC assays consisted of 29 injections of 10 μl (2 μl for the first injection) each, with 5-min intervals between injections, and the stirring speed was set to 310 rpm. The first injection was discarded in all titrations. Reference power was set at 10 or 15 μCal s−1. Data were fit to a one-site model using Origin 7 Software. Two replicate titrations were carried out, and the reported parameters represent the average of the two replicates.
      The fluorescence polarization competition binding assay was carried out as described previously (
      • Taldone T.
      • Patel P.D.
      • Patel M.
      • Patel H.J.
      • Evans C.E.
      • Rodina A.
      • Ochiana S.
      • Shah S.K.
      • Uddin M.
      • Gewirth D.
      • Chiosis G.
      Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series.
      ). The protein concentration was 10 nm, and the geldanamycin-Cy3b concentration was 6 nm. Competition reactions were incubated for 24 h at 4 °C prior to analysis.

      Ki calculation

      Ki values for competition binding experiments were calculated from the FP IC50 values using the method of Nikolovska-Coleska et al. (
      • Nikolovska-Coleska Z.
      • Wang R.
      • Fang X.
      • Pan H.
      • Tomita Y.
      • Li P.
      • Roller P.P.
      • Krajewski K.
      • Saito N.G.
      • Stuckey J.A.
      • Wang S.
      Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization.
      ) using equations as described previously (
      • Huck J.D.
      • Que N.L.S.
      • Sharma S.
      • Taldone T.
      • Chiosis G.
      • Gewirth D.T.
      Structures of Hsp90α and Hsp90β bound to a purine-scaffold inhibitor reveal an exploitable residue for drug selectivity.
      ) and implemented in a spreadsheet calculator (
      • Nikolovska-Coleska Z.
      • Wang R.
      • Fang X.
      • Pan H.
      • Tomita Y.
      • Li P.
      • Roller P.P.
      • Krajewski K.
      • Saito N.G.
      • Stuckey J.A.
      • Wang S.
      Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization.
      ). Input data for the calculation were total protein concentration (10 nm), tracer ligand concentration (6 nm), tracer ligand Kd for the protein, and measured IC50 values. Tracer ligand Kd values for Grp94 (2.5 nm) and Hsp90α (0.7 nm) were taken from a previous report (
      • Taldone T.
      • Patel P.D.
      • Patel M.
      • Patel H.J.
      • Evans C.E.
      • Rodina A.
      • Ochiana S.
      • Shah S.K.
      • Uddin M.
      • Gewirth D.
      • Chiosis G.
      Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series.
      ).

      NECA inhibitor IC50 measurements

      ATP hydrolysis rates were measured using the PiPer Phosphate assay kit (Thermo Fisher Scientific) in 96-well fluorescent assay plates (Corning). Nearly full-length Grp94 (73–754Δ287–327) was buffer-exchanged into 1× ATPase buffer (40 mm HEPES-KOH (pH 7.4), 150 mm KCl, and 5 mm MgCl2), concentrated to 50 μm, and diluted prior to the experiment. Experimental setup included a 50 μl:50 μl mixture of PiPer reagent (100 μm Amplex Red reagent containing 4 units/ml maltose phosphorylase, 0.4 mm maltose, 2 units/ml glucose oxidase, and 0.4 units/ml horseradish peroxidase) and the ATPase reaction (5 μl of ATP solution, 5 μl of inhibitor solution, and 40 μl of protein or 40 μl of 1× ATPase buffer for ATP-only wells). Inhibitors were prepared from stocks in DMSO and serially diluted in 1× ATPase buffer before addition to the plate. The effects of NECA, NPCA, and NEoCA were tested in the range of 0.781–100 μm. NEaCA was tested in the range of 2.91–372 μm. The final concentration of protein in each well was between 2.2–2.7 μm depending on the plate, and the final ATP concentration was 15 μm. Plates were incubated at 37 °C for 4 h, and the reactions were quenched on ice.
      Fluorescence was measured at 544 nm/590 nm (excitation/emission) on a SpectraMax Gemini XS plate reader (Molecular Devices) with 30 readings/well. Data were background-corrected by subtracting the average of wells containing ATP only. Percent activity was calculated by dividing the measured fluorescence of inhibitor-treated wells by the average of wells containing protein, ATP, and DMSO (i.e. no inhibitor). DMSO had no effect on ATPase activity. Experiments are averages of six independent measurements. The data were plotted using Prism, and inhibitor concentration was transformed to logarithmic scale. The data were then fit using the dose–response inhibition equation: log(inhibitor) versus response (percent activity compared with DMSO). IC50 was determined by measuring the concentration of inhibitor required to inhibit 50% activity.

      Crystallization and structure solution

      Complexes between Grp94NΔ41 and NPCA, NEoCA, and NEaCA were formed by mixing concentrated protein at 30 mg/ml with 1–2.5 μl of concentrated ligand in DMSO to a final concentration of 5 mm and incubating the mixture on ice for 15 min. Crystals were formed by hanging drop vapor diffusion at 18 °C by mixing 2 μl of Grp94NΔ41:ligand with an equal volume of reservoir solution consisting of 100 mm Tris (pH 7.6), 25 mm MgCl2, and 30%–35% PEG 400. Crystals formed in 1–3 days. Crystals of apoHsp90N were formed by hanging drop vapor diffusion at 4 °C by mixing 2 μl of protein at 25 mg/ml with an equal volume of cold reservoir solution consisting of 100 mm BisTris propane (pH 6.4), l0–50 mm MgCl2, and 10–20% PEG 3350. Crystals formed in 1–3 days. Soaks were performed by adding 1 μl of a 9 mm ligand solution in reservoir solution to the crystal-containing drops. Crystals were harvested after 3–4 h of soaking.
      Grp94NΔ41 crystals were harvested directly from crystallization drops without further stabilization and flash-frozen in liquid nitrogen. Crystals of Hsp90N:ligand soaks were removed from the mother liquor in a loop, placed briefly in a drop containing reservoir solution, followed by transfer to a cryostabilization solution of 100 mm BisTris propane (pH 6.4), 25 mm MgCl2, and 30% PEG 3350. Crystals were removed after a few seconds and flash-frozen in liquid nitrogen.
      Structure solution was carried out by molecular replacement using PDB code 1QY5 as the search model for Grp94 and 1YER as the search model for Hsp90. Refinement was carried out in Phenix.

      Author contributions

      J. D. H., G. C., and D. T. G. formal analysis; J. D. H., N. L. S. Q., R. M. I., L. S., T. T., G. C., and D. T. G. investigation; J. D. H., N. L. S. Q., R. M. I., and L. S. writing-review and editing; R. M. I., L. S., G. C., and D. T. G. conceptualization; G. C. and D. T. G. data curation; G. C. and D. T. G. supervision; G. C. and D. T. G. funding acquisition; G. C. and D. T. G. validation; G. C. and D. T. G. methodology; G. C. and D. T. G. writing-original draft; G. C. and D. T. G. project administration.

      Acknowledgments

      We thank Dr. E. Toone (Duke University) for chemical synthesis of the NECA derivatives, and W. J. Aw and C. Campomizzi for assistance with crystal growth for PDB codes 6CYI, 6CYH, and 6CYG. X-ray data were collected at Advanced Photon Source beamlines 23ID-B, 22-BM, and 17-ID and SSRL beamline 12-2.

      Supplementary Material

      References

        • Marzec M.
        • Eletto D.
        • Argon Y.
        GRP94: An HSP90-like protein specialized for protein folding and quality control in the endoplasmic reticulum.
        Biochim. Biophys. Acta. 2012; 1823 (22079671): 774-787
        • Johnson J.L.
        Evolution and function of diverse Hsp90 homologs and cochaperone proteins.
        Biochim. Biophys. Acta. 2012; 1823 (22008467): 607-613
        • Taipale M.
        • Jarosz D.F.
        • Lindquist S.
        HSP90 at the hub of protein homeostasis: emerging mechanistic insights.
        Nat. Rev. Mol. Cell Biol. 2010; 11 (20531426): 515-528
        • Patel P.D.
        • Yan P.
        • Seidler P.M.
        • Patel H.J.
        • Sun W.
        • Yang C.
        • Que N.S.
        • Taldone T.
        • Finotti P.
        • Stephani R.A.
        • Gewirth D.T.
        • Chiosis G.
        Paralog-selective Hsp90 inhibitors define tumor-specific regulation of HER2.
        Nat. Chem. Biol. 2013; 9 (23995768): 677-684
        • Randow F.
        • Seed B.
        Endoplasmic reticulum chaperone gp96 is required for innate immunity but not cell viability.
        Nat. Cell Biol. 2001; 3 (11584270): 891-896
        • Liu B.
        • Staron M.
        • Hong F.
        • Wu B.X.
        • Sun S.
        • Morales C.
        • Crosson C.E.
        • Tomlinson S.
        • Kim I.
        • Wu D.
        • Li Z.
        Essential roles of grp94 in gut homeostasis via chaperoning canonical Wnt pathway.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110 (23572575): 6877-6882
        • Yang Y.
        • Liu B.
        • Dai J.
        • Srivastava P.K.
        • Zammit D.J.
        • Lefrançois L.
        • Li Z.
        Heat shock protein gp96 is a master chaperone for Toll-like receptors and is important in the innate function of macrophages.
        Immunity. 2007; 26 (17275357): 215-226
        • Wanderling S.
        • Simen B.B.
        • Ostrovsky O.
        • Ahmed N.T.
        • Vogen S.M.
        • Gidalevitz T.
        • Argon Y.
        GRP94 is essential for mesoderm induction and muscle development because it regulates insulin-like growth factor secretion.
        Mol. Biol. Cell. 2007; 18 (17634284): 3764-3775
        • Wu B.X.
        • Hong F.
        • Zhang Y.
        • Ansa-Addo E.
        • Li Z.
        GRP94/gp96 in Cancer: Biology, Structure, Immunology, and Drug Development.
        Adv Cancer Res. 2016; 129: 165-190
        • Ansa-Addo E.A.
        • Thaxton J.
        • Hong F.
        • Wu B.X.
        • Zhang Y.
        • Fugle C.W.
        • Metelli A.
        • Riesenberg B.
        • Williams K.
        • Gewirth D.T.
        • Chiosis G.
        • Liu B.
        • Li Z.
        Clients and oncogenic roles of molecular chaperone gp96/grp94.
        Curr. Top. Med. Chem. 2016; 16 (27072698): 2765-2778
        • Hua Y.
        • White-Gilbertson S.
        • Kellner J.
        • Rachidi S.
        • Usmani S.Z.
        • Chiosis G.
        • Depinho R.
        • Li Z.
        • Liu B.
        Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma.
        Clin. Cancer Res. 2013; 19 (24077352): 6242-6251
        • Jhaveri K.
        • Taldone T.
        • Modi S.
        • Chiosis G.
        Advances in the clinical development of heat shock protein 90 (Hsp90) inhibitors in cancers.
        Biochim. Biophys. Acta. 2012; 1823 (22062686): 742-755
        • Neckers L.
        • Workman P.
        Hsp90 molecular chaperone inhibitors: are we there yet?.
        Clin. Cancer Res. 2012; 18 (22215907): 64-76
        • Joshi S.
        • Wang T.
        • Araujo T.L.S.
        • Sharma S.
        • Brodsky J.L.
        • Chiosis G.
        Adapting to stress: chaperone networks in cancer.
        Nat. Rev. Cancer. 2018; 18 (29795326): 562-575
        • Rodina A.
        • Wang T.
        • Yan P.
        • Gomes E.D.
        • Dunphy M.P.
        • Pillarsetty N.
        • Koren J.
        • Gerecitano J.F.
        • Taldone T.
        • Zong H.
        • Caldas-Lopes E.
        • Alpaugh M.
        • Corben A.
        • Riolo M.
        • Beattie B.
        • et al.
        The epichaperome is an integrated chaperome network that facilitates tumour survival.
        Nature. 2016; 538 (27706135): 397-401
        • Taldone T.
        • Wang T.
        • Rodina A.
        • Pillarsetty N.V.K.
        • Digwal C.S.
        • Sharma S.
        • Yan P.
        • Joshi S.
        • Pagare P.P.
        • Bolaender A.
        • Roboz G.J.
        • Guzman M.L.
        • Chiosis G.
        A chemical biology approach to the chaperome in cancer-hsp90 and beyond.
        Cold Spring Harb. Perspect. Biol. 2019; (pii: a034116) (30936118)
        • Wang T.
        • Rodina A.
        • Dunphy M.P.
        • Corben A.
        • Modi S.
        • Guzman M.L.
        • Gewirth D.T.
        • Chiosis G.
        Chaperome heterogeneity and its implications for cancer study and treatment.
        J. Biol. Chem. 2019; 294 (30409908): 2162-2179
        • Gewirth D.T.
        paralog-specific Hsp90 inhibitors: a brief history and a bright future.
        Curr. Top. Med. Chem. 2016; 16 (27072700): 2779-2791
        • Ernst J.T.
        • Liu M.
        • Zuccola H.
        • Neubert T.
        • Beaumont K.
        • Turnbull A.
        • Kallel A.
        • Vought B.
        • Stamos D.
        Correlation between chemotype-dependent binding conformations of HSP90α/β and isoform selectivity: implications for the structure-based design of HSP90α/β-selective inhibitors for treating neurodegenerative diseases.
        Bioorg. Med. Chem. Lett. 2014; 24 (24332488): 204-208
        • Ernst J.T.
        • Neubert T.
        • Liu M.
        • Sperry S.
        • Zuccola H.
        • Turnbull A.
        • Fleck B.
        • Kargo W.
        • Woody L.
        • Chiang P.
        • Tran D.
        • Chen W.
        • Snyder P.
        • Alcacio T.
        • Nezami A.
        • et al.
        Identification of novel HSP90α/β isoform selective inhibitors using structure-based drug design. demonstration of potential utility in treating CNS disorders such as Huntington’s disease.
        J. Med. Chem. 2014; 57 (24673104): 3382-3400
        • Patel H.J.
        • Patel P.D.
        • Ochiana S.O.
        • Yan P.
        • Sun W.
        • Patel M.R.
        • Shah S.K.
        • Tramentozzi E.
        • Brooks J.
        • Bolaender A.
        • Shrestha L.
        • Stephani R.
        • Finotti P.
        • Leifer C.
        • Li Z.
        • Gewirth D.T.
        • et al.
        Structure-activity relationship in a purine-scaffold compound series with selectivity for the endoplasmic reticulum Hsp90 paralog Grp94.
        J. Med. Chem. 2015; 58 (25901531): 3922-3943
        • Que N.L.S.
        • Crowley V.M.
        • Duerfeldt A.S.
        • Zhao J.
        • Kent C.N.
        • Blagg B.S.J.
        • Gewirth D.T.
        Structure-based design of a Grp94-selective inhibitor: exploiting a key residue in Grp94 to optimize paralog-selective binding.
        J. Med. Chem. 2018; 61 (29528635): 2793-2805
        • Soldano K.L.
        • Jivan A.
        • Nicchitta C.V.
        • Gewirth D.T.
        Structure of the N-terminal domain of GRP94: basis for ligand specificity and regulation.
        J. Biol. Chem. 2003; 278 (12970348): 48330-48338
        • Hutchison K.A.
        • Fox I.H.
        Purification and characterization of the adenosine A2-like binding site from human placental membrane.
        J. Biol. Chem. 1989; 264 (2584200): 19898-19903
        • Hutchison K.A.
        • Nevins B.
        • Perini F.
        • Fox I.H.
        Soluble and membrane-associated human low-affinity adenosine binding protein (adenotin): properties and homology with mammalian and avian stress proteins.
        Biochemistry. 1990; 29 (2378869): 5138-5144
        • Rosser M.F.
        • Nicchitta C.V.
        Ligand interactions in the adenosine nucleotide-binding domain of the Hsp90 chaperone, GRP94: I: evidence for allosteric regulation of ligand binding.
        J. Biol. Chem. 2000; 275 (10816561): 22798-22805
        • Kim J.
        • Felts S.
        • Llauger L.
        • He H.
        • Huezo H.
        • Rosen N.
        • Chiosis G.
        Development of a fluorescence polarization assay for the molecular chaperone Hsp90.
        J. Biomol. Screen. 2004; 9 (15296636): 375-381
        • Taldone T.
        • Patel P.D.
        • Patel M.
        • Patel H.J.
        • Evans C.E.
        • Rodina A.
        • Ochiana S.
        • Shah S.K.
        • Uddin M.
        • Gewirth D.
        • Chiosis G.
        Experimental and structural testing module to analyze paralogue-specificity and affinity in the Hsp90 inhibitors series.
        J. Med. Chem. 2013; 56 (23965125): 6803-6818
        • Nikolovska-Coleska Z.
        • Wang R.
        • Fang X.
        • Pan H.
        • Tomita Y.
        • Li P.
        • Roller P.P.
        • Krajewski K.
        • Saito N.G.
        • Stuckey J.A.
        • Wang S.
        Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization.
        Anal. Biochem. 2004; 332 (15325294): 261-273
        • Dollins D.E.
        • Immormino R.M.
        • Gewirth D.T.
        Structure of unliganded GRP94, the endoplasmic reticulum Hsp90: basis for nucleotide-induced conformational change.
        J. Biol. Chem. 2005; 280 (15951571): 30438-30447
        • Vallée F.
        • Carrez C.
        • Pilorge F.
        • Dupuy A.
        • Parent A.
        • Bertin L.
        • Thompson F.
        • Ferrari P.
        • Fassy F.
        • Lamberton A.
        • Thomas A.
        • Arrebola R.
        • Guerif S.
        • Rohaut A.
        • Certal V.
        • et al.
        Tricyclic series of heat shock protein 90 (Hsp90) inhibitors part I: discovery of tricyclic imidazo[4,5-c]pyridines as potent inhibitors of the Hsp90 molecular chaperone.
        J. Med. Chem. 2011; 54 (21972823): 7206-7219
        • Ohkubo S.
        • Kodama Y.
        • Muraoka H.
        • Hitotsumachi H.
        • Yoshimura C.
        • Kitade M.
        • Hashimoto A.
        • Ito K.
        • Gomori A.
        • Takahashi K.
        • Shibata Y.
        • Kanoh A.
        • Yonekura K.
        TAS-116, a highly selective inhibitor of heat shock protein 90α and β, demonstrates potent antitumor activity and minimal ocular toxicity in preclinical models.
        Mol. Cancer Ther. 2015; 14 (25416789): 14-22
        • Liu S.
        • Street T.O.
        5′-N-ethylcarboxamidoadenosine is not a paralog-specific Hsp90 inhibitor.
        Protein Sci. 2016; 25 (27667530): 2209-2215
        • Ge J.
        • Normant E.
        • Porter J.R.
        • Ali J.A.
        • Dembski M.S.
        • Gao Y.
        • Georges A.T.
        • Grenier L.
        • Pak R.H.
        • Patterson J.
        • Sydor J.R.
        • Tibbitts T.T.
        • Tong J.K.
        • Adams J.
        • Palombella V.J.
        Design, synthesis, and biological evaluation of hydroquinone derivatives of 17-amino-17-demethoxygeldanamycin as potent, water-soluble inhibitors of Hsp90.
        J. Med. Chem. 2006; 49 (16854066): 4606-4615
        • Crowley V.M.
        • Huard D.J.E.
        • Lieberman R.L.
        • Blagg B.S.J.
        Second generation Grp94-selective inhibitors provide opportunities for the inhibition of metastatic cancer.
        Chemistry. 2017; 23 (28857290): 15775-15782
        • Crowley V.M.
        • Khandelwal A.
        • Mishra S.
        • Stothert A.R.
        • Huard D.J.
        • Zhao J.
        • Muth A.
        • Duerfeldt A.S.
        • Kizziah J.L.
        • Lieberman R.L.
        • Dickey C.A.
        • Blagg B.S.
        Development of glucose regulated protein 94-selective inhibitors based on the BnIm and Radamide scaffold.
        J. Med. Chem. 2016; 59 (27003516): 3471-3488
        • Duerfeldt A.S.
        • Peterson L.B.
        • Maynard J.C.
        • Ng C.L.
        • Eletto D.
        • Ostrovsky O.
        • Shinogle H.E.
        • Moore D.S.
        • Argon Y.
        • Nicchitta C.V.
        • Blagg B.S.
        Development of a Grp94 inhibitor.
        J. Am. Chem. Soc. 2012; 134 (22642269): 9796-9804
        • Dollins D.E.
        • Warren J.J.
        • Immormino R.M.
        • Gewirth D.T.
        Structures of GRP94-nucleotide complexes reveal mechanistic differences between the hsp90 chaperones.
        Mol. Cell. 2007; 28 (17936703): 41-56
        • Immormino R.M.
        • Kang Y.
        • Chiosis G.
        • Gewirth D.T.
        Structural and quantum chemical studies of 8-aryl-sulfanyl adenine class Hsp90 inhibitors.
        J. Med. Chem. 2006; 49 (16884307): 4953-4960
        • de Zwart M.
        • Kourounakis A.
        • Kooijman H.
        • Spek A.L.
        • Link R.
        • von Frijtag Drabbe Künzel J.K.
        • Ijzerman A.P.
        5′-N-substituted carboxamidoadenosines as agonists for adenosine receptors.
        J. Med. Chem. 1999; 42 (10212124): 1384-1392
        • Huck J.D.
        • Que N.L.S.
        • Sharma S.
        • Taldone T.
        • Chiosis G.
        • Gewirth D.T.
        Structures of Hsp90α and Hsp90β bound to a purine-scaffold inhibitor reveal an exploitable residue for drug selectivity.
        Proteins. 2019; 87 (31141217): 869-877