Inhibitors of the molecular chaperone heat shock protein 90 (Hsp90) are currently generating significant interest in clinical development as potential treatments for cancer. In a preceding publication (DOI: 10.1021/jm100059d ) we describe Astex's approach to screening fragments against Hsp90 and the subsequent optimization of two hits into leads with inhibitory activities in the low nanomolar range. This paper describes the structure guided optimization of the 2,4-dihydroxybenzamide lead molecule 1 and details some of the drug discovery strategies employed in the identification of AT13387 (35), which has progressed through preclinical development and is currently being tested in man.

Inhibitors of the chaperone Hsp90 are potentially useful as chemotherapeutic agents in cancer. This paper describes an application of fragment screening to Hsp90 using a combination of NMR and high throughput X-ray crystallography. The screening identified an aminopyrimidine with affinity in the high micromolar range and subsequent structure-based design allowed its optimization into a low nanomolar series with good ligand efficiency. A phenolic chemotype was also identified in fragment screening and was found to bind with affinity close to 1 mM. This fragment was optimized using structure based design into a resorcinol lead which has subnanomolar affinity for Hsp90, excellent cell potency, and good ligand efficiency. This fragment to lead campaign improved affinity for Hsp90 by over 1,000,000-fold with the addition of only six heavy atoms. The companion paper (DOI: 10.1021/jm100060b) describes how the resorcinol lead was optimized into a compound that is now in clinical trials for the treatment of cancer.

Here we report the discovery of a highly conserved novel binding site located at the interface between the protease and helicase domains of the Hepatitis C Virus (HCV) NS3 protein. Using a chemical lead, identified by fragment screening and structure-guided design, we demonstrate that this site has a regulatory function on the protease activity via an allosteric mechanism. We propose that compounds binding at this allosteric site inhibit the function of the NS3 protein by stabilising an inactive conformation and thus represent a new class of direct acting antiviral agents.

A ubiquitously expressed chaperone, heat shock protein 90 (HSP90) is of considerable interest as an oncology target because tumor cells and oncogenic proteins are acutely dependent on its activity. AT13387 (2,4-dihydroxy-5-isopropyl-phenyl)-[5-(4-methyl-piperazin-1-ylmethyl)-1,3-dihydro-isoindol-2-yl] methanone, L-lactic acid salt) a novel, high-affinity HSP90 inhibitor, which is currently being clinically tested, has shown activity against a wide array of tumor cell lines, including lung cancer cell lines. This inhibitor has induced the degradation of specific HSP90 client proteins for up to 7 days in tumor cell lines in vitro. The primary driver of cell growth (mutant epidermal growth factor receptors) was particularly sensitive to HSP90 inhibition. The long duration of client protein knockdown and suppression of phospho-signaling seen in vitro after treatment with AT13387 was also apparent in vivo, with client proteins and phospho-signaling suppressed for up to 72 h in xenograft tumors after treatment with a single dose of AT13387. Pharmacokinetic analyses indicated that while AT13387 was rapidly cleared from blood, its retention in tumor xenografts was markedly extended, and it was efficacious in a range of xenograft models. AT13387's long duration of action enabled, in particular, its efficacious once weekly administration in human lung carcinoma xenografts. The use of longer-acting HSP90 inhibitors, such as AT13387, on less frequent dosing regimens has the potential to maintain antitumor efficacy as well as minimize systemic exposure and unwanted effects on normal tissues. (Cancer Sci 2012; 103: 522-527) T he super-chaperone system is involved in the folding and maturation of newly synthesized proteins.(1,2) HSP90 in particular aids in the folding and maturation of a distinct subset of proteins, which includes kinases, cell surface receptors and transcription factors. (3,4) The N-terminal domain ATPase activity of HSP90 is essential for this function.(5) Inhibition of this domain induces remodeling of the HSP90 chaperone complex, resulting in the recruitment of ubiquitin ligases, polyubiquitination and subsequent proteasomal degradation of HSP90 client proteins.(6,7) Through this mechanism, the inhibition of a single target enzyme can have a wide effect on the stability and, hence, the function of a large set of client proteins. As many oncogenic proteins are HSP90 clients, HSP90 inhibition has been found to have broad antitumor effects.(8-10) In contrast to more recent targeted therapies, where the appearance of new driver mutations or resistance mutations result in a loss of efficacy, client protein mutation increases dependence on HSP90 chaperoning activity as these mutations tend to render the proteins less stable. (11)(12)(13) Previous studies have also demonstrated that the constitutively activated mutant forms of EGFR are particularly dependent on HSP90 both in vitro and in vivo, (14)(15)(16) indicating an HSP90 inhibitor may be particularly efficacious in mutant EGFR tumors.After HSP90 inhibiti...

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