Hsp90 molecular chaperones in eukaryotic cells play essential roles in the folding and activation of a range of client proteins involved in cell cycle regulation, steroid hormone responsiveness, and signal transduction. The biochemical mechanism of Hsp90 is poorly understood, and the involvement of ATP in particular is controversial. Crystal structures of complexes between the N-terminal domain of the yeast Hsp90 chaperone and ADP/ATP unambiguously identify a specific adenine nucleotide binding site homologous to the ATP-binding site of DNA gyrase B. This site is the same as that identified for the antitumor agent geldanamycin, suggesting that geldanamycin acts by blocking the binding of nucleotides to Hsp90 and not the binding of incompletely folded client polypeptides as previously suggested. These results finally resolve the question of the direct involvement of ATP in Hsp90 function.

Gastropod mollusks have been used for over 2500 years to produce the "Tyrian purple" dye made famous by the Phoenicians. This dye is constituted of mixed bromine-substituted indigo and indirubin isomers. Among these, the new natural product 6-bromoindirubin and its synthetic, cell-permeable derivative, 6-bromoindirubin-3'-oxime (BIO), display remarkable selective inhibition of glycogen synthase kinase-3 (GSK-3). Cocrystal structure of GSK-3beta/BIO and CDK5/p25/indirubin-3'-oxime were resolved, providing a detailed view of indirubins' interactions within the ATP binding pocket of these kinases. BIO but not 1-methyl-BIO, its kinase inactive analog, also inhibited the phosphorylation on Tyr276/216, a GSK-3alpha/beta activation site. BIO but not 1-methyl-BIO reduced beta-catenin phosphorylation on a GSK-3-specific site in cellular models. BIO but not 1-methyl-BIO closely mimicked Wnt signaling in Xenopus embryos. 6-bromoindirubins thus provide a new scaffold for the development of selective and potent pharmacological inhibitors of GSK-3.

Activation of client proteins by the Hsp90 molecular chaperone is dependent on binding and hydrolysis of ATP, which drives a molecular clamp via transient dimerization of the N-terminal domains. The crystal structure of the middle segment of yeast Hsp90 reveals considerable evolutionary divergence from the equivalent regions of other GHKL protein family members such as MutL and GyrB, including an additional domain of new fold. Using the known structure of the N-terminal nucleotide binding domain, a model for the Hsp90 dimer has been constructed. From this structure, residues implicated in the ATPase-coupled conformational cycle and in interactions with client proteins and the activating cochaperone Aha1 have been identified, and their roles functionally characterized in vitro and in vivo.

Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.

The DNA damage response (DDR) is essential for maintaining the genomic integrity of the cell and its disruption is one of the 'Hallmarks of Cancer'. Classically, defects in the DDR have been exploited therapeutically in the treatment of cancer by radiation therapies or by genotoxic chemotherapies. More recently, protein components of the DDR systems are being identified as promising avenues for targeted cancer therapeutics. Here we present an in-depth analysis of the function, disease role and therapeutic potential of ~450 expert-curated human DDR genes. We discuss the current state of DDR drugs both FDA approved or under clinical investigation. We examine large-scale genomic and expression data in 15 cancers to identify deregulated components of the DDR in these tumours, and we apply systematic computational analysis to identify DDR proteins amenable to modulation by small molecules, highlighting potential novel therapeutic targets. 3The DNA Damage Response (DDR) evolved in response to the exposure of the genome to exogenous and endogenous genotoxins. Unless repaired in an error-free process, DNA damage can result in mutations and altered cellular behavior. Consequently, cells deploy a diverse repertoire of mechanisms to maintain genetic integrity 1 (see TABLE 1). These mechanisms involve the DNA repair processes themselves, the systems that regulate and organize them, and the systems that integrate DNA damage repair with the cell cycle 2 .Disruption of the DDR is observed in many cancers [3][4][5] , and underlies the genomic instability that accompanies tumourigenesis and progression. However, in the majority of cases, the specific underlying defects are poorly characterised 6,7 . Conversely, there are well-described cancers where disruption of a DDR mechanism is directly causal. In addition to these licensed drugs, there are a number of compounds currently under clinical evaluation that target DDR pathways directly. These targets include the protein kinases involved in cell cycle DNA checkpoint for DNA damage and/or replicative stress (eg CHEK1, WEE1), and individual enzymes involved in base excision repair (BER; APEX1), direct repair (MGMT), non-homologous DNA double strand break repair (NHEJ; PRKDC / DNA-PK) and telomere maintenance (TM; TERT).The initial rationale for development of DDR enzyme-targeted drugs focused on their use as potentiators, inhibiting repair of damage caused by radiotherapy and/or conventional genotoxins 11 .However, this approach has been extended to stand-alone use, targeting DNA repair pathways critical to tumour survival by exploiting synthetic sensitivity/lethality 16 (SSL). SSL arises when a combination of loss-of-function in two or more genes leads to cell death, while loss-of-function in only one of them does not. The therapeutic aim is to exploit genetic defects essential to a tumour's survival by combining the defect in an affected pathway with a pharmacologically induced defect in a compensating pathway 17 . 4The best example to date is the pharmaceutical inhibition...