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XIAP antagonism measurements in HEK293-XIAP-Caspase-9 xenografts

XIAP antagonism in A375 cells in the presence or absence of TNF-α

Preparation of 2-chloro-1-{6-[(4-fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}ethan-1-one

Fold induction in secreted cytokine concentrations in PBMC supernatants after IAP antagonist treatment

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.

Comparison of XIAP activity, cIAP1 activity and selectivity of clinical IAP antagonists

IncuCyte images from neutralization of TNF-α activity with anti-TNF-α experiment in MDA-MB-231 cells

Additional data from ASTX669 in vivo xenograft studies

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.

The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signalling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Recent advances have shown that genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signalling and inhibits proliferation of RTK-driven cancer cell lines. SHP2 is now understood to act upstream of RAS and plays a role in KRAS-driven cancers, an area of research which is rapidly growing. Considering that RTK deregulation often leads to a wide range of cancers and the newly appreciated role of SHP2 in KRAS-driven cancers, SHP2 inhibitors are therefore a promising therapeutic approach. SHP2 contains two N-terminal tandem SH2 domains (N-SH2, C-SH2), a catalytic phosphatase domain and a C-terminal tail. SHP2 switches between “open” active and “closed” inactive forms due to autoinhibitory interactions between the N-SH2 domain and the phosphatase domain. Historically, phosphatases were deemed undruggable as there had been no advancements with active site inhibitors. We hypothesised that fragment screening would be highly applicable and amenable to this target to enable alternative means of inhibition through identification of allosteric binding sites. Here we describe the first reported fragment screen against SHP2. Using our fragment-based PyramidTM approach, screening was carried out on two constructs of SHP2; a closed autoinhibited C-terminal truncated form (phosphatase and both SH2 domains), as well as the phosphatase-only domain. A combination of screening methods such as X-ray crystallography and NMR were employed to identify fragment hits at multiple sites on SHP2, including the tunnel-like allosteric site reported by Chen et al, 2016. Initial fragment hits had affinities for SHP2 in the range of 1mM as measured by ITC. Binding of these hits was improved using structure-guided design to generate compounds which inhibit SHP2 phosphatase activity and are promising starting points for further optimization. Citation Format: Philip J. Day, Valerio Berdini, Juan Castro, Gianni Chessari, Thomas G. Davies, James E. Day, Satoshi Fukaya, Chris Hamlett, Keisha Hearn, Steve Hiscock, Rhian Holvey, Satoru Ito, Yasuo Kodama, Kenichi Matsuo, Yoko Nakatsuru, Nick Palmer, Amanda Price, Tadashi Shimamura, Jeffrey D.St. Denis, Nicola G. Wallis, Glyn Williams, Christopher N. Johnson. Fragment-based drug discovery to identify small molecule allosteric inhibitors of SHP2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1039.

In response to cellular stress, the p53 tumor suppressor is activated to modulate cell cycle progression, DNA repair, and cell death. The activity of p53 is tightly regulated by MDM2, an E3 ubiquitin ligase that targets p53 for proteasomal degradation. Inhibition of the MDM2-p53 interaction in tumors carrying wild-type p53 can therefore reactivate p53 and elicit an anti-cancer effect. Small molecule inhibitors of the MDM2-p53 interaction remains a promising strategy for cancer therapy and a number of these compounds are in clinical development. An isoindolinone series, identified by the Northern Institute for Cancer Research (NICR), has been used as a starting point for the development of potent MDM2-p53 inhibitors. Structure based drug design was applied during lead optimisation to gain potency whilst also focusing on stabilizing the main metabolically labile position and reducing lipophilicity. This approach led to potent compounds with EC50 Citation Format: Lynsey Fazal, Maria Ahn, Luke Bevan, Ildiko Buck, Juan Castro, Gianni Chessari, Ben Cons, Keisha Hearn, Steven Howard, Chris Johnson, Judith Reeks, Emiliano Tamanini, Neil Thompson, Hugh Walton, Pamela Williams, Ruth H. Bawn, Tim J. Blackburn, Celine Cano, Sarah J. Cully, Bernard Golding, Roger Griffin, Karen Haggerty, Ian Hardcastle, Herbie Newell, Martin Noble, Huw Thomas, Elaine Willmore, Yan Zhao, Steve Wedge. Development of a potent class of small molecule inhibitors of the MDM2-p53 protein-protein interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1652.

We investigated the cellular response to two MDM2-p53 antagonists (RG7388 and Cpd 1) in a panel of 19 tumor cell lines and in patient-derived chronic lymphocytic leukemia (CLL) cells. RG7388 (Idasanutlin) is an MDM2-p53 antagonist developed by Roche and Cpd1 a novel potent and selective MDM2-p53 antagonist developed as part of an ongoing Alliance between Newcastle University, Cancer Research UK and Astex Pharmaceuticals. RG7388 and Cpd1 potently inhibited the proliferation of many cell lines, with MDM2-amplified SJSA-1 osteosarcoma cells and cells from haematological malignancies being the most sensitive (e.g. GI50 values in the acute myeloid leukemia cell line MOLM13 were 33 + 16 nM for RG7388 and 8 + 1 nM for Cpd 1). We also examined induction of apoptosis by measuring caspase 3/7 activation (24 h treatment). SJSA-1 cells showed a 20-fold increase in caspase activation and Molm13 cells a 6-fold increase (measured at 300nM with RG7388). However, cell lines from solid tumours such as the colorectal carcinoma HCT116 or the hepatocellular carcinoma HepG2 did not show any induction of apoptosis, even at concentrations up to 1µM RG3788. In primary CLL cells, Cpd 1 reduced cell viability (48 h) with an average LC50 of 131 + 46 nM (n = 4), compared to 400 + 55 nM for RG7388 (n =23). We also quantified mRNA levels in 25 primary CLL samples and found that RG7388 induced a predominantly pro-apoptotic gene signature: following 6h treatment, 1µM RG7388 resulted in an average 8-fold induction of PUMA and 3.5-fold increase in BAX compared to a 1.6-fold induction of CDKN1A (p21). Our data confirm that targeting the MDM2-p53 interaction is an effective strategy to inhibit growth and viability of tumor cells, and that some cells, including those expressing high levels of MDM2 or those derived from hematological malignancies, display a striking apoptotic response. Citation Format: Elaine Willmore, Yan Zhao, Carmela Ciardullo, Laura Woodhouse, Huw D. Thomas, Maria Ahn, Lynsey Fazal, Martin E. Noble, Ian Hardcastle, Steven Howard, Gianni Chessari, John Lunec, Steve Wedge. The anti-proliferative and pro-apoptotic effect of MDM2-p53 antagonists evaluated in human tumor cells lines and chronic lymphocytic leukemia patient samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1870.

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.

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

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 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

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.

Heat Shock Protein 90 (HSP90) is a member of a family of molecular chaperone proteins which directs the folding of polypeptides into functional configurations affecting stabilisation and activation. Many of these proteins are oncogenes regulating tumor cell growth, survival and apoptosis. This poster will focus on the screening and medicinal chemistry work that led to the identification of AT13387, a high affinity HSP90 inhibitor that is currently in clinical trials for the treatment of cancer. A fragment screening campaign was conducted against the N-terminal domain of HSP90 to detect very low molecular weight compounds (Molecular Weight Subsequent lead optimisation focussed on the improvement of in vivo distribution properties via the addition of basic moieties to the lead molecule. These compounds showed encouraging in vivo pharmacology and biological profiles, and further medicinal chemistry work led to the discovery of AT13387, an inhibitor with sub-nanomolar affinity, prolonged duration of action and excellent in vivo anti-tumor efficacy. This poster represents first disclosure of the structure of AT13387 and illustrates how a fragment-based drug discovery approach can be efficiently used to discover compounds suitable for clinical testing in oncology. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A211.