Design, synthesis, and evaluation of small molecule Hsp90 probes

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Abstract

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 among 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.

Introduction

Heat shock protein 90 (Hsp90) is a molecular chaperone with key roles in folding and maintaining the conformational integrity of its client proteins through its ATPase activity.1, 2 Because many of these proteins (i.e., Her2, Raf-1, Akt, Cdk4, Polo-1 kinase, cMet, mutant B-Raf, mutant p53, AR, ER, Bcr–Abl, HIF-1 alpha, hTERT) are associated with signaling pathways involved in cell regulation, it plays an important role in maintaining the transformed phenotype.3 As such, Hsp90 has become one of the highly pursued molecular targets in cancer therapy, with efforts in the development of Hsp90-inhibitory agents in neurodegenerative diseases and pathogenic resistance (i.e., viral, fungal, bacterial) closely following behind.3, 4 A search on esp@cenet patent database returned over 700 hits in September 2010, with several applications claiming distinct compositions of matter that interact with Hsp90.

At least five distinct Hsp90 inhibitor chemotypes are currently in clinical studies, with many others following in late-stage IND evaluation or different stages of pre-clinical evaluation.4, 5 While the interest in Hsp90 inhibitors has spread fast, knowledge on the target has however, lagged behind. It is known that while all current Hsp90 inhibitors in advanced stage (clinical or late-stage IND) inhibit Hsp90 by binding to its N-terminal site regulatory pocket, evidence suggests important distinctions in the in vitro and in vivo behavior of these agents.6, 7, 8, 9 Among most critical differences are the kinetics of client protein modulation and in vivo pharmacodynamic profiles which both can have a determining effect on the clinical efficacy and the therapeutic window of the Hsp90-agents.6, 7, 8, 9

In addition, with the advancement of Hsp90 inhibitors into several cancers, and potentially other diseases, it will be important to identify tumor- and disease-specific Hsp90 clients and implement these in the clinic as potential molecular markers to provide proof that the target, Hsp90, is inhibited. Identification of proper clinical biomarkers has become of utmost importance. The cost of successful anticancer drug development to the stage of approval has escalated recently to more than $800 million, and having a properly chosen pharmacodynamic disease biomarker can increase the efficacy of the process. In particular, it can be a useful indicator of drug activity and accelerate the ability to make go-no go decisions early in the clinical process.10

Most molecules currently known to inhibit Hsp90 function (Fig. 1) mimic or adopt scaffolds based on those of geldanamycin11 (GM, ansamycin class), PU-H7112 (purine class), NVP-AUY9227 (isoxazole class) and SNX-211213 (indazol-4-one class). Geldanamycin was the first identified Hsp90 inhibitor and as such has served a central role in the study of Hsp90 biology. Originally, it was believed to inhibit Src kinase directly, but by using geldanamycin covalently bound to Affi-Gel® 10 solid support, it was later shown to bind to Hsp90 and inhibit heterocomplex formation with Src.11 Biotinylated geldanamycin was also synthesized.14

Here, we report on the design and synthesis of molecules based on purine, isoxazole and indazol-4-one chemical classes attached to Affi-Gel® 10 beads and on the synthesis of a biotinylated purine compound. These are chemical tools to investigate and understand the molecular basis for the distinct behavior of Hsp90 inhibitors. They can be also used to better understand Hsp90 tumor biology by examining bound client proteins and co-chaperones. Understanding the tumor specific clients of Hsp90 most likely to be modulated by each Hsp90 inhibitor could lead to a better choice of pharmacodynamic markers, and thus a better clinical design. Not lastly, understanding the molecular differences among these Hsp90 inhibitors could result in identifying characteristics that could lead to the design of an Hsp90 inhibitor with most favorable clinical profile.

Section snippets

Design of Hsp90 probes and precursor evaluation

The attachment of small molecules to a solid support is a very useful method to probe their target and the target’s interacting partners. Indeed, as mentioned above, geldanamycin attached to solid support enabled for the identification of Hsp90 as its target.11 Perhaps the most crucial aspects in designing such chemical probes are determining the appropriate site for attachment of the small molecule ligand, and designing an appropriate linker between the molecule and the solid support. Our

Chemistry

Synthesis of PU-H71 beads (6) is shown in Scheme 1 and commences with the 9-alkylation of 8-arylsulfanylpurine (1)12 with 1,3-dibromopropane to afford 2 in 35% yield. The low yield obtained in the formation of 2 can be primarily attributed to unavoidable competing 3-alkylation. Five equivalents of 1,3-dibromopropane were used to ensure complete reaction of 1 and to limit other undesirable side-reactions, such as dimerization, which may also contribute to the low yield. 2 was reacted with tert

Biological evaluation

Next, we investigated whether the synthesized beads retained interaction with tumor Hsp90. Agarose beads covalently attached to either of PU-H71, NVP-AUY922, SNX-2112 or 2-methoxyethylamine (PU-, NVP-, SNX-, control-beads, respectively), were incubated with K562 chronic myeloid leukemia (CML) or MDA-MB-468 breast cancer cell extracts. As seen in Figure 3A, the Hsp90 inhibitor, but not the control-beads, efficiently isolated Hsp90 in the cancer cell lysates. Control-beads contain an Hsp90

Conclusions

In conclusion, we have prepared useful chemical tools based on three different Hsp90 inhibitors, each of a different chemotype. These were prepared either by attachment onto solid support, such as PU-H71 (purine), NVP-AUY922 (isoxazole) and SNX-2112 (indazol-4-one)-beads, or by biotinylation (PU-H71-biotin). The utility of these probes was demonstrated by their ability to efficiently isolate Hsp90 and, in the case of PU-H71 beads (6), isolate Hsp90 onco-protein containing complexes from cancer

General

1H and 13C NMR spectra were recorded on a Bruker 500 MHz instrument. Chemical shifts were reported in δ values in ppm downfield from TMS as the internal standard. 1H data were reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, br = broad, m = multiplet), coupling constant (Hz), integration. 13C chemical shifts were reported in δ values in ppm downfield from TMS as the internal standard. Low resolution mass spectra were obtained on a Waters Acquity Ultra

Acknowledgements

This work was supported in part by W.H. Goodwin and A. Goodwin and the Commonwealth Cancer Foundation for Research, The Experimental Therapeutics Center of Memorial Sloan-Kettering Cancer Center (MSKCC), the Translational and Integrative Medicine Research Fund of MSKCC, SPORE Pilot Award and Research & Therapeutics Program in Prostate Cancer, 1R01CA155226-01, U01AG032969-01A1, R21AG028811, 3P30CA008748-44S2, Leukemia and Lymphoma Society LLS#6114-10, the Byrne Fund, the Geoffrey Beene Cancer

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