Pamela A. Williams

Discovery of an Allosteric Mechanism for the Regulation of Hcv Ns3 Protein Function.

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.

Structure of the BTB Domain of Keap1 and Its Interaction with the Triterpenoid Antagonist CDDO

The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.

Fragment-based discovery of 6-azaindazoles as inhibitors of bacterial DNA ligase.

Herein we describe the application of fragment-based drug design to bacterial DNA ligase. X-ray crystallography was used to guide structure-based optimization of a fragment-screening hit to give novel, nanomolar, AMP-competitive inhibitors. The lead compound 13 showed antibacterial activity across a range of pathogens. Data to demonstrate mode of action was provided using a strain of S. aureus, engineered to overexpress DNA ligase.

Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-Linked Inhibitor of Apoptosis Protein (XIAP).

XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.

Abstract 2944: AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models.

Melanoma is a highly aggressive malignancy with an exceptional ability to develop resistance and no curative therapy is available for patients with metastatic disease. Inhibitor of apoptosis proteins (IAP) play a key role in preventing cell death by apoptosis. In normal cell, IAPs are highly regulated by endogenous antagonists (e.g. SMAC) but in melanoma cell lines and in patient samples expression levels of IAPs are generally high and depleting IAPs by siRNA tended to reduce cell viability, with XIAP reduction being the most efficient [1]. Small molecule IAP antagonists have the ability to switch IAP-controlled pro-survival pathways towards apoptosis and cell death. Recent evidence suggests that a true dual antagonist of both cIAP1 and XIAP will promote an effective apoptotic response through generation of death-inducing ripoptosome complexes, with resultant caspase activation [2, 3]. We have used our fragment-based drug discovery technology PyramidTM to derive a non-peptidomimetic IAP antagonist, AT-IAP, which does not have an alanine warhead and has nanomolar cellular potency for both XIAP and cIAP1. Initial pharmacokinetic and pharmacodynamic modeling of AT-IAP in mice bearing the MDA-MB-231 cell line indicated that daily oral dosing of AT-IAP at 30 mg/kg ensures high concentrations of compound in tumor and plasma over a 24 h period with resultant inhibition of both XIAP and cIAP1 and induction of apoptosis markers (cleaved PARP and cleaved caspase-3). In this paper, we describe the characterization of AT-IAP in melanoma models. An in vitro cell line proliferation screen demonstrated that 36% of melanoma cell lines exhibited enhanced sensitivity to AT-IAP, which was improved on addition of exogenous 1 ng/ml TNF-α (92% of cell lines were sensitive to AT-IAP + TNF-α). Sensitivity of melanoma cells to AT-IAP has also been confirmed in a panel of 20 primary melanoma tumors in colony formation assays set up in the presence and absence of added TNF-α. Finally, a set of biomarkers has been identified and used to predict single agent activity of AT-IAP in a range of melanoma cell line and patient derived xenograft models. [1] Engesaeter et al., Cancer Biology & Therapy, 2011, 12 (1), 47 [2] Ndubaku et al., ACS Chem Biol., 2009, 4 (7), 557 [3] Meier, P., Nat Rev. Cancer, 2010, 10 (8), 561 Citation Format: Gianni Chessari, Ahn Maria, Ildiko Buck, Elisabetta Chiarparin, Joe Coyle, James Day, Martyn Frederickson, Charlotte Griffiths-Jones, Keisha Hearn, Steven Howard, Tom Heightman, Petra Hillmann, Aman Iqbal, Christopher N. Johnson, Jon Lewis, Vanessa Martins, Joanne Munck, Mike Reader, Lee Page, Anna Hopkins, Alessia Millemaggi, Caroline Richardson, Gordon Saxty, Tomoko Smyth, Emiliano Tamanini, Neil Thompson, George Ward, Glyn Williams, Pamela Williams, Nicola Wilsher, Alison Woolford. AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2944. doi:10.1158/1538-7445.AM2013-2944

ASTX660, a Novel Non-peptidomimetic Antagonist of cIAP1/2 and XIAP, Potently Induces TNFα-Dependent Apoptosis in Cancer Cell Lines and Inhibits Tumor Growth

Because of their roles in the evasion of apoptosis, inhibitor of apoptosis proteins (IAP) are considered attractive targets for anticancer therapy. Antagonists of these proteins have the potential to switch prosurvival signaling pathways in cancer cells toward cell death. Various SMAC-peptidomimetics with inherent cIAP selectivity have been tested clinically and demonstrated minimal single-agent efficacy. ASTX660 is a potent, non-peptidomimetic antagonist of cIAP1/2 and XIAP, discovered using fragment-based drug design. The antagonism of XIAP and cIAP1 by ASTX660 was demonstrated on purified proteins, cells, and in vivo in xenograft models. The compound binds to the isolated BIR3 domains of both XIAP and cIAP1 with nanomolar potencies. In cells and xenograft tissue, direct antagonism of XIAP was demonstrated by measuring its displacement from caspase-9 or SMAC. Compound-induced proteasomal degradation of cIAP1 and 2, resulting in downstream effects of NIK stabilization and activation of noncanonical NF-κB signaling, demonstrated cIAP1/2 antagonism. Treatment with ASTX660 led to TNFα-dependent induction of apoptosis in various cancer cell lines in vitro, whereas dosing in mice bearing breast and melanoma tumor xenografts inhibited tumor growth. ASTX660 is currently being tested in a phase I–II clinical trial (NCT02503423), and we propose that its antagonism of cIAP1/2 and XIAP may offer improved efficacy over first-generation antagonists that are more cIAP1/2 selective. Mol Cancer Ther; 17(7); 1381–91. ©2018 AACR.

Abstract A55: Potent, dual cIAP1/XIAP antagonists induce apoptosis in a melanoma stem cell population.

The inhibitor of apoptosis proteins (IAP) are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. IAP antagonists activate the E3 ligase function of cIAP1 and stimulate rapid autoubiquitylation and proteosomal degradation of both cIAP1 and cIAP2. Elimination of these proteins leads to a switch in TNFα signalling from being pro-survival to being pro-apoptotic. However, a strong pro-apoptotic effect from cIAP loss cannot be achieved without sustained antagonism of XIAP-mediated caspase inhibition. Therefore, a best in class profile for IAP antagonists requires potent dual antagonism of cIAP1 and XIAP. Astex has used fragment based-drug discovery to develop a second generation of IAP antagonists, which are non-peptidomimetic and do not contain an alanine as a warhead. This series has the ability not only to efficiently degrade cIAP1 but also to potently antagonize XIAP, delivering a dual cIAP1/XIAP inhibitory profile which is not apparent in the first generation of IAP antagonists based on an alanine warhead. Here, we report the structural understanding of the unique molecular profile of the series together with the enhanced activity of these compounds in melanoma cancer stem cells (CSC). CSC populations are more resistant to apoptosis than the bulk cell population and they have been associated with resistance to cancer therapy, relapse and cancer progressions. Blockade of the apoptotic pathway by up-regulation of anti-apoptotic factors has been implicated in conferring resistance in CSC fractions and increased XIAP expression has also been reported in these cells. We have analysed the CD133+ population of three melanoma cell lines (SK-MEL-2, SK-MEL-5 and SK-MEL-28) and measured activation of caspase-3 (NucView™ cell staining) after treatment with IAP antagonists in presence of TNFα. Our potent dual cIAP1/XIAP antagonists (XIAP EC50 35 nM in cells). The enhanced XIAP potency of our compounds is overriding the resistance in CSC subpopulations, highlighting the importance of dual antagonism in promoting efficient induction of apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A55. Citation Format: Gianni Chessari, Maria Ahn, Keisha Hearn, Christopher N. Johnson, Jon Lewis, Neil Thompson, George Ward, Pamela Williams. Potent, dual cIAP1/XIAP antagonists induce apoptosis in a melanoma stem cell population. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A55.

Abstract 2018: Discovery of potent dual inhibitors of both XIAP and cIAP1 using fragment based drug discovery

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL XIAP and cIAP1 are members of the inhibitor of apoptosis (IAP) protein family. Both proteins have the ability to attenuate apoptosis induced through intrinsic and extrinsic stimuli via inhibition of caspase-3, -7, -8 and -9. The defining feature of both XIAP and cIAP1 is the presence in their protein sequence of 3 Baculoviral IAP Repeat (BIR) domains, which are necessary for their antiapoptotic activity. The mitochondrial protein SMAC uses its N-terminal region (AVPI) to interact with BIR domains and deactivate the antiapoptotic function of IAPs. Several companies and academic groups have active programs developing SMAC peptidomimetic compounds based on the AVPI motif. In general, those compounds have the tendency to be cIAP1 selective like their tetrapeptide progenitor (AVPI IC50 values for XIAP-BIR3 and cIAP1-BIR3 are 0.3 uM and 0.016 uM respectively). Using our fragment-based screening approach, PyramidTM, we identified a non-peptidomimetic chemotype which binds with similar potency to the BIR3 domain of both XIAP and cIAP1. Hit optimisation using a structure based approach led to the discovery of potent true dual XIAP and cIAP1 antagonists with good in vivo physico-chemical profile and no P450 or hERG liabilities. Dual XIAP/cIAP1 inhibitors have potential for more effective apoptosis and less toxicity associated with cytokine production. Compounds were initially characterised in fluorescence polarisation binding assays using XIAP-BIR3 or cIAP1-BIR3 domains. Robust induction of apoptosis was observed in two sensitive breast cancer cell lines (EC50s well below 0.1 uM in EVSA-T and MDA-MB-231); whilst HCT116 cells (colon cancer) were insensitive (unless exogenous TNF-α was added). This in vitro cell line killing was demonstrated to correlate closely with cIAP1 antagonism and hence a parallel cell assay was established to measure XIAP antagonism. An engineered HEK293 cell line was stably co-transfected with full length FLAG-tagged human XIAP cDNA and full length (untagged) human caspase-9 cDNA. Inhibition of caspase-9 binding to XIAP was measured in immunoprecipitation assays. This gave us a sensitive read-out for XIAP antagonism in cells which could be plotted against the most sensitive cell killing read-out (from the EVSA-T cell line) to establish relative XIAP vs cIAP1 selectivities and to select dual antagonists of both IAPs. Potent compounds (HEK293-EC50 <0.01 uM and EVSA-T-EC50 <0.01 uM) were further characterised in PKPD studies in mice bearing MDA-MB-231 xenografts. Compounds with good oral exposure achieved high concentration in tumor over 24h periods which ensured excellent inhibition of both XIAP and cIAP1 with consequent reduction of cIAP1 levels and induction of apoptosis markers (PARP, Caspase-3). Finally, dual XIAP/cIAP1 inhibitors have been investigated in xenograft models (melanoma, breast and colorectal cancer) and have achieved significant efficacy at tolerated doses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2018. doi:1538-7445.AM2012-2018

Abstract A1: The physiological form of MetAP2 can be inhibited through binding to either of the two active‐site metals

Methionine aminopeptidases (MetAP) are metalloenzymes that remove the N‐terminal initiator methionine from newly synthesized polypeptides allowing essential post‐translational modifications such as acetylation and myristoylation to take place. MetAP2, one of the two eukaryotic forms of the enzyme, was identified as the target of fumagillin, a natural product with anti‐angiogenic properties that inhibits the proliferation of endothelial cells. Clinical activity has been seen for a semi‐synthetic analogue of fumagillin, TNP470, suggesting MetAP2 is a good target for inhibiting angiogenesis. In vitro, MetAP2 appears to have sites for two divalent metal ions within its active site but there has been much discussion around the identity and number of metal ions actually present in the physiological states of the various MetAPs. An understanding of the physiologically relevant metalloform of the enzyme is essential for designing inhibitors that are active in cells. We have used tool compounds that bind the active site metals in diverse ways to investigate the relevance of the two potential metal binding sites in MetAP2. Using our fragment‐based screening approach, Pyramid™, we screened the manganese‐form of the MetAP2 enzyme. We identified multiple low-molecular weight fragment hits and confirmed their modes of binding to the two metals in the active site of MetAP2 by X‐ray crystallography. Three hit series, which bound metal 1 only, metal 2 only or both metals 1 and 2 were chosen for further optimization using structure‐based drug design. Optimized lead compounds had potent inhibitory activity against the in vitro MetAP2 enzyme (∼100 nM) and in HUVEC proliferation assays, whilst also showing greater than 1000‐fold selectivity for MetAP2 over MetAP1. Examples from each series, representing different active site metal binding modes, were used as tool compounds to investigate the mechanism of action in cells. The levels of the MetAP2 substrate, 14‐3‐3, were monitored by western blot in HUVECs treated with these compounds. Levels of methionylated 14‐3‐3 increased upon treatment with compounds from each of our series indicating the substrate was not being processed and that in each case the compound was inhibiting MetAP2 in these cells. These data indicate that the physiological form of MetAP2 can be inhibited by compounds which bind solely to either of the two active‐site metals, suggesting that both metals must be present in the intra‐cellular form of MetAP2 and allowing multiple approaches to inhibiting this key angiogenic target. The lead series identified here provide chemically diverse scaffolds for further optimization of drug like properties. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A1.