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The discovery and development of heat shock protein 70 (Hsp70) inhibitors is currently a hot topic in cancer. In the preceding paper in this issue ( 10.1021/jm401551n ), we have described structure-activity relationship studies in the first Hsp70 inhibitor class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein. These ligands contained an acrylamide to take advantage of an active cysteine embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. Here, we perform chemical modifications around the irreversible inhibitor scaffold to demonstrate that covalent modification is not a requirement for activity within this class of compounds. The study identifies derivative 27c, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. Collectively, the back-to-back manuscripts describe the first pharmacophores that favorably and selectively interact with a never explored pocket in Hsp70 and provide a novel blueprint for a cancer-oriented development of Hsp70-directed ligands.

Heat shock protein 70 (Hsp70) is an important emerging cancer target whose inhibition may affect multiple cancer-associated signaling pathways and, moreover, result in significant cancer cell apoptosis. Despite considerable interest from both academia and pharmaceutical companies in the discovery and development of druglike Hsp70 inhibitors, little success has been reported so far. Here we describe structure-activity relationship studies in the first rationally designed Hsp70 inhibitor class that binds to a novel allosteric pocket located in the N-terminal domain of the protein. These 2,5'-thiodipyrimidine and 5-(phenylthio)pyrimidine acrylamides take advantage of an active cysteine embedded in the allosteric pocket to act as covalent protein modifiers upon binding. The study identifies derivatives 17a and 20a, which selectively bind to Hsp70 in cancer cells. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. In summary, the described scaffolds represent a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics.

In this report we describe a simple synthesis of mercaptobenzonitriles from the reaction of fluorobenzonitriles with Na2S in DMF at room temperature and following direct treatment with Zn/HCl. Significantly, 2- and 4-fluorobenzonitriles substituted with chlorine or bromine, but not iodine, undergo selective substitution of fluorine at room temperature to yield synthetically useful halo-substituted mercaptobenzonitriles.

GPR40, a G-protein-coupled receptor has been well established to play a crucial role in regulating blood glucose levels. Hence, GPR40 is a potential target for future antidiabetic agents. The present 3D QSAR study is aimed at delineating structural parameters governing GPR40 agonistic activity. To meet this objective, a comparative molecular similarity indices analysis for 63 different GPR40 agonists was performed using two methods; a ligand-based 3D QSAR model employing the atom fit alignment method and a receptor-based 3D QSAR model that was derived from the predicted binding conformations obtained by docking all the GPR40 agonists at the active site of GPR40. The results of these studies showed the ligand-based model to be superior (r(cv)(2) value of 0.610) to the receptor-based model (r(cv)(2) value of 0.519) in terms of statistical data. The predictive ability of these models was evaluated using a test set of 15 compounds not included in the preliminary training set of 48 compounds. The predictive r(2) values for the ligand- and the receptor-based models were found to be 0.863 and 0.599, respectively. Further, interpretation of the comparative molecular similarity indices analysis contour maps with reference to the active site of GPR40 provided an insight into GPR40-agonist interactions.

Preparation of oxirane 3 was accomplished in two steps. 1H-1,2,4-triazole was reacted with 2,4-difluoro-α-chloroacetophenone 1 in presence of K2CO3 in refluxing toluene to provide compound 2. Compound 2 was treated with trimethylsulfoxonium iodide (TMSI) in aq. NaOH and toluene to provide oxirane 3. Oxirane 3, previously isolated as an oil, was crystallized from (DCM/MeOH) and characterized by X-ray crystallography: triclinic, space group P − 1, a = 7.3225 (15), b = 7.5833 (15), c = 9.856 (2) A, α = 91.908 (12), β = 100.824 (11), γ = 103.800 (11)°, V = 520.28 (18) A3, Z = 2. The molecule has a stepped conformation with nearly parallel triazole and phenyl rings. Lack of classical hydrogen-bond donors leads to packing dominated by weaker interactions, including C–H···N, C–H···F and F···F contacts. This important intermediate, normally an oil, has been crystallized, and its crystal structure includes a stepped conformation with nearly parallel triazole and phenyl rings. Lack of classical hydrogen-bond donors leads to packing dominated by weaker interactions, including C–H···N, C–H···F and F···F contacts.

A variety of substrates undergoes SNAr reaction of fluorine with Na2S to produce the corresponding mercaptobenzonitriles.

A number of compounds from different chemical classes are known to bind competitively to the ATP-pocket of Hsp90 and inhibit its chaperone function. The natural product geldanamycin was the first reported inhibitor of Hsp90 and since then synthetic inhibitors from purine, isoxazole and indazol-4-one chemical classes have been discovered and are currently or soon to be in clinical trials for the treatment of cancer. In spite of a similar binding mode to Hsp90, distinct biological profiles were demonstrated amongst these molecules, both in vitro and in vivo. To better understand the molecular basis for these dissimilarities, we report here the synthesis of chemical tools for three Hsp90 inhibitor classes. These agents will be useful for probing tumor-by-tumor the Hsp90 complexes isolated by specific inhibitors. Such information will lead to better understanding of tumor specific molecular markers to aid in their clinical development. It will also help to elucidate the molecular basis for the biological differences observed among Hsp90 inhibitors.

Benzotriazoles (III), (IV), (VI), and (VII) are prepared by ring opening of the crystalline key intermediate (II) in the presence of potassium carbonate.

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target., Author Summary Helminth diseases of humans remain a major problem in many parts of the tropics. Treatment of these parasitic infections is restricted to a limited number of drugs and few new compounds are in development. One of the major obstacles to the development of new therapeutics is the lack of high-throughput screens that can be adapted to parasitic species for the identification of small molecule inhibitors. Here we present a simple, inexpensive assay for the identification of inhibitors of Hsp90 in filarial worms. The assay, first described for the identification of Hsp90 inhibitors in tumor cells, does not require recombinant protein but relies upon the ability of a fluorescently labelled drug to bind to Hsp90 in the context of a soluble fraction of worm homogenate. We validated the assay using known inhibitors of Hsp90, including derivatives of the synthetic purine-scaffold series of Hsp90 inhibitors and were able to show a differential sensitivity to these compounds between human and Brugia Hsp90.

In an effort to find inhibitors that are effective against both Candida and Aspergillus spp., a series of 5(6)-(un)substituted benzotriazole analogs, represented by compounds 3a-3h and 3b'-3f', were prepared using a crystalline oxirane intermediate 1 previously synthesized in our laboratory. All the compounds were evaluated for inhibitory activity against various species of Candida and Aspergillus. Compounds 3b' (5,6-dimethylbenzotriazol-2-yl derivative), 3d (5-chlorobenzotriazol-1-yl derivative) and 3e' (6-methylbenzotriazol-1-yl derivative) exhibited potent antifungal activity, with the MICs for Candida spp. and Aspergillus niger, ranging from 1.6 microg/mL to 25 microg/mL and 12.5 microg/mL to 25 microg/mL, respectively. The present work describes the design, synthesis, regioisomer characterization (through COSY and NOESY 2D-NMR spectroscopy and single molecule X-ray crystallography), antifungal evaluation, molecular docking, and structure-activity relationships of the various 5(6)-(un)substituted benzotriazole analogs.

The urgent need for novel HCV antiviral agents has provided an impetus for understanding the structural requisites of NS5B polymerase inhibitors at the molecular level. Toward this objective, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) of 67 HCV NS5B polymerase inhibitors were performed using two methods. First, ligand-based 3D QSAR studies were performed based on the lowest energy conformations employing the atom fit alignment method. Second, receptor-based 3D QSAR models were derived from the predicted binding conformations obtained by docking all NS5B inhibitors at the allosteric binding site of NS5B (PDB ID: 2dxs). Results generated from the ligand-based model were found superior (r2cv values of 0.630 for CoMFA and 0.668 for CoMSIA) to those obtained by the receptor-based model (r2cv values of 0.536 and 0.561 for CoMFA and CoMSIA, respectively). The predictive ability of the models was validated using a structurally diversified test set of 22 compounds that had not been included in a preliminary training set of 45 compounds. The predictive r2 values for the ligand-based CoMFA and CoMSIA models were 0.734 and 0.800, respectively, while the corresponding predictive r2 values for the receptor-based CoMFA and CoMSIA models were 0.538 and 0.639, respectively. The greater potency of the tryptophan derivatives over that of the tyrosine derivatives was interpreted based on CoMFA steric and electrostatic contour maps. The CoMSIA results revealed that for a NS5B inhibitor to have appreciable inhibitory activity it requires hydrogen bond donor and acceptor groups at the 5-position of the indole ring and an R substituent at the chiral carbon, respectively. Interpretation of the CoMFA and CoMSIA contour maps in context of the topology of the allosteric binding site of NS5B provided insight into NS5B-inhibitor interactions. Taken together, the present 3D QSAR models were found to accurately predict the HCV NS5B polymerase inhibitory activity of structurally diverse test set compounds and to yield reliable clues for further optimization of the benzimidazole derivatives in the data set.

The urgent need for novel HCV antiviral agents has provided an impetus for understanding the structural requisites of NS5B polymerase inhibitors at the molecular level. Toward this objective, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) of 67 HCV NS5B polymerase inhibitors were performed using two methods. First, ligand-based 3D QSAR studies were performed based on the lowest energy conformations employing the atom fit alignment method. Second, receptor-based 3D QSAR models were derived from the predicted binding conformations obtained by docking all NS5B inhibitors at the allosteric binding site of NS5B (PDB ID: 2dxs). Results generated from the ligand-based model were found superior (r2cv values of 0.630 for CoMFA and 0.668 for CoMSIA) to those obtained by the receptor-based model (r2cv values of 0.536 and 0.561 for CoMFA and CoMSIA, respectively). The predictive ability of the models was validated using a structurally diversified test set of 22 compounds that had not been included in a preliminary training set of 45 compounds. The predictive r2 values for the ligand-based CoMFA and CoMSIA models were 0.734 and 0.800, respectively, while the corresponding predictive r2 values for the receptor-based CoMFA and CoMSIA models were 0.538 and 0.639, respectively. The greater potency of the tryptophan derivatives over that of the tyrosine derivatives was interpreted based on CoMFA steric and electrostatic contour maps. The CoMSIA results revealed that for a NS5B inhibitor to have appreciable inhibitory activity it requires hydrogen bond donor and acceptor groups at the 5-position of the indole ring and an R substituent at the chiral carbon, respectively. Interpretation of the CoMFA and CoMSIA contour maps in context of the topology of the allosteric binding site of NS5B provided insight into NS5B-inhibitor interactions. Taken together, the present 3D QSAR models were found to accurately predict the HCV NS5B polymerase inhibitory activity of structurally diverse test set compounds and to yield reliable clues for further optimization of the benzimidazole derivatives in the data set.

Glucose Regulated Protein 94 (Grp94) is an Hsp90 paralog localized in the lumen of the endoplasmic reticulum (ER) of higher eukaryotes. While the mechanisms associated with Grp94-pathogenic expression are still actively studied, the focus thus far of most cancer related studies has been primarily on the immunogenic activity of Grp94/peptide complexes and the involvement of this protein in the secretion of IGF-I and IGF-II and the regulation of Toll-like receptors and integrins. This status quo has recently changed when work from our lab has shown that in certain breast cancers the maintenance of a high density HER2 species at the plasma membrane and its associated aberrant signaling also requires Grp94, rendering these tumors highly sensitive to Grp94 inhibition. The discovery of selective and potent Grp94 inhibitors has been hindered due to the high similarity of the ATP-binding regulatory pocket of the Hsp90 paralogs. However, recent work from our lab has shown that this apparent roadblock can be overcome by using library screening and structural and computational analysis to discover purine-based molecules that show 100-fold selectivity for the Grp94 isoform. Herein, we detail for the first time the structure activity relationship of the selective purine derived Grp94 molecules. This work provides insights on how to overcome the high structural similarity of Hsp90s in the ligand binding pocket, and also reports selective and therapeutically relevant Grp94 inhibitors based on a purine scaffold. These initial inhibitors have potent activity against cancer cells providing an important platform for the development of Grp94 inhibitors with drug-like features and potential for clinical translation. Citation Format: Stefan O. Ochiana, Tony Taldone, Hardik J. Patel, Pallav Patel, Yan Pengrong, Weilin Sun, Anna Rodina, Smit Shah, Daniel T. Gewirth, Gabriela Chiosis. Development of selective GRP94 inhibitors for the treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2831. doi:10.1158/1538-7445.AM2015-2831