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The MAPK signaling pathway is commonly upregulated in human cancers. As the primary downstream effector of the MAPK pathway, ERK is an attractive therapeutic target for the treatment of MAPK-activated cancers and for overcoming resistance to upstream inhibition. ASTX029 is a highly potent and selective dual-mechanism ERK inhibitor, discovered using fragment-based drug design. Because of its distinctive ERK-binding mode, ASTX029 inhibits both ERK catalytic activity and the phosphorylation of ERK itself by MEK, despite not directly inhibiting MEK activity. This dual mechanism was demonstrated in cell-free systems, as well as cell lines and xenograft tumor tissue, where the phosphorylation of both ERK and its substrate, ribosomal S6 kinase (RSK), were modulated on treatment with ASTX029. Markers of sensitivity were highlighted in a large cell panel, where ASTX029 preferentially inhibited the proliferation of MAPK-activated cell lines, including those with BRAF or RAS mutations. In vivo, significant antitumor activity was observed in MAPK-activated tumor xenograft models following oral treatment. ASTX029 also demonstrated activity in both in vitro and in vivo models of acquired resistance to MAPK pathway inhibitors. Overall, these findings highlight the therapeutic potential of a dual-mechanism ERK inhibitor such as ASTX029 for the treatment of MAPK-activated cancers, including those which have acquired resistance to inhibitors of upstream components of the MAPK pathway. ASTX029 is currently being evaluated in a first in human phase I–II clinical trial in patients with advanced solid tumors (NCT03520075).

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.

We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role. Mol Cancer Ther; 10(9); 1542–52. ©2011 AACR.

The RAS-RAF-MEK-ERK signalling cascade is activated through mutations in RAS or RAF in over 30% of cancers. The successful development of inhibitors of BRAF and MEK kinases has led to effective treatment particularly of melanomas whose tumor growth is driven by activating mutations in BRAF such as V600E. Despite these successes, resistance emerges after several months, leading to increased signaling through ERK1/2. This has prompted the development of direct inhibitors of ERK1/2, several of which are in early clinical trials. The majority of clinical ERK1/2 inhibitors are ATP competitive, blocking ERK1/2 catalytic phosphorylation of downstream substrates such as RSK, but do not modulate phosphorylation of ERK1/2 by MEK. Crystal structural studies performed by us and others on the pERK1/2 modulating inhibitor SCH772984 suggested that it induces a conformational change in the glycine-rich loop of ERK2, which leads to Tyr36 becoming tucked under the loop and creating a new binding pocket. We hypothesized that this binding mode might underlie the ability of SCH772984 to block the phosphorylation of ERK1/2, and initiated a fragment-based approach to develop novel, orally bioavailable inhibitors that elicit a similar conformational change and also modulate the phosphorylation of ERK1/2. Using screening methods including high-throughput X-ray crystallography and biophysical assays, we identified fragments binding to both the hinge and the inducible pocket of ERK2. Progressive rounds of structure-guided fragment optimization and growing led to an understanding of inhibitor structure determinants required to induce the conformational change in ERK2. These efforts, together with iterative optimization in a screening cascade including measurement of pRSK and pERK levels and antiproliferative activity in RAS and BRAF mutant cells, led to the discovery of a novel series of pERK modulating ERK1/2 inhibitors. The lead compound shows low nanomolar potency in biochemical ERK1/2 assays and an excellent kinome selectivity profile. In BRAF and RAS mutant cell lines, the lead shows low nanomolar cell proliferation IC50 values, while sparing cell lines not driven by the MAPK pathway. The lead exhibits robust antitumor activity upon oral dosing in a range of subcutaneous xenograft models including the mutant BRAF colorectal line Colo205, providing a promising basis for further optimization towards clinical pERK1/2 modulating ERK1/2 inhibitors. Citation Format: Tom D. Heightman, Valerio Berdini, Hannah Braithwaite, Ildiko Buck, Megan Cassidy, Juan Castro, Aurélie Courtin, James Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte Griffiths-Jones, John Lyons, Vanessa Martins, Sandra Muench, Joanne Munck, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, Mike Reader, David Rees, Sharna Rich, Caroline Richardson, Harpreet Saini, Neil Thompson, Nicola Wallis, Hugh Walton, Nicola Wilsher, Alison Woolford, Chris Murray. Fragment-based discovery of a highly potent, orally bioavailable ERK1/2 inhibitor that modulates the phosphorylation and catalytic activity of ERK1/2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B161.

The MAPK pathway is commonly hyper-activated in human cancers due to the occurrence of oncogenic mutations in RAF, RAS and the upregulation of RTKs. The therapeutic potential of MAPK pathway inhibition has been demonstrated by the clinical efficacy of RAF and MEK1/2 (MEK) inhibitors in the treatment of BRAF-mutant melanoma. However, response to such agents is short-lived due to the onset of resistance mechanisms, which in the majority of cases result in the reactivation of ERK1/2 (ERK) signalling. Therefore, the direct targeting of ERK is an attractive therapeutic approach to overcoming the limitations of RAF or MEK inhibitors. Here, we describe a novel, potent, and selective ERK inhibitor, which inhibits both ERK catalytic activity and also the phosphorylation of ERK by MEK. Using fragment-based drug discovery we have developed a selective ERK inhibitor, which inhibits in vitro ERK catalytic activity with a low nM IC50 value. This lead compound has strong antiproliferative effects in a wide range of MAPK-activated cell lines, including the BRAF-mutant cell lines A375 (melanoma) and Colo205 (colorectal), the KRAS-mutant cell lines HCT116 (colorectal), Calu6 (lung) and Panc05.04 (pancreatic), and the NRAS-mutant cell line Ma-mel-27 (melanoma). The lead compound potently inhibits ERK cell signalling. The potent (nM) inhibition of RSK phosphorylation (a direct ERK substrate) was confirmed in A375 (BRAF-mutant melanoma) cells, using MSD analysis. In addition to inhibiting downstream ERK signalling, we demonstrated by ELISA and Western blotting that the lead compound confers a decrease in phospho-ERK levels in both BRAF-mutant and KRAS-mutant cell lines. We investigated the biochemical mechanism of the modulation of ERK phosphorylation in vitro and demonstrated that the compound prevents the phosphorylation of ERK by MEK (at key ERK activation loop residues, T202/Y204), without directly inhibiting MEK activity. The compound was profiled in a range of subcutaneous xenograft models including A375 (BRAF-mutant melanoma) and Calu-6 (KRAS-mutant lung). Once-daily oral dosing of the lead compound conferred significant antitumor activity in a range of in vivo efficacy studies. The compound potently inhibited the phosphorylation of downstream ERK substrates (including RSK) in tumor xenograft tissue. There was a clear relationship between in vivo compound concentrations and the modulation of ERK substrate phosphorylation. Furthermore, as was demonstrated in vitro, we confirmed that in addition to inhibiting ERK catalytic activity the compound potently inhibited the phosphorylation of ERK itself, in both KRAS and BRAF-mutant tumor xenografts. Here, we characterize a novel, highly potent, selective ERK inhibitor, which inhibits both ERK catalytic activity and also the upstream phosphorylation of ERK by MEK. These data support the further optimization of this series of compounds for clinical development. Citation Format: Joanne M. Munck, Valerio Berdini, Luke D. Bevan, Hannah Braithwaite, Ildiko M. Buck, Megan Cassidy, Juan Castro, Aurelie Courtin, James E. Day, Charlotte East, Lynsey Fazal, Brent Graham, Charlotte M. Griffiths-Jones, Tom D. Heightman, Chris J. Hindley, Birikiti Kidane, Justyna Kucia-Tran, John F. Lyons, Vanessa Martins, Sandra Muench, Chris W. Murray, David Norton, Marc O'Reilly, Nick Palmer, Puja Pathuri, Mike Reader, David C. Rees, Sharna J. Rich, Caroline J. Richardson, Harpreet K. Saini, Alpesh Shah, Lukas Stanczuk, Neil T. Thompson, Hugh Walton, Nicola E. Wilsher, Alison J. Woolford, Nicola G. Wallis. Characterization of a novel ERK1/2 inhibitor, which modulates the phosphorylation and catalytic activity of ERK1/2 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B154.

Fibroblast growth factors (FGFs) and their receptors (FGFR1 through 4) regulate a variety of key cellular processes, including proliferation, migration, survival, and differentiationa. Aberrant activation of FGF/FGFR is strongly implicated in oncogenic signalling in many tumor types. This has stimulated the development of a number of FGFR inhibitors, with diverse kinase inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We conducted a fragment screening campaign and this resulted in identification of a 6-aminoquinoxalinyl fragment with a binding affinity in the micromolar range. Structure-guided medicinal chemistry led to the identification of a novel quinoxaline-based chemical series with nanomolar affinity for FGFR1, 2, 3, and 4, activity in cells, and selectivity with respect to VEGFR-2. Further optimisation resulted in the generation of JNJ-42756493, a compound with favourable drug-like properties that demonstrated strong anti-tumoral activity in a FGFR2-dependent SNU-16 human gastric carcinoma xenograft model. This report represents the first disclosure of the structure-activity relationships as well as the chemical synthesis pathway of the JNJ-42756493 series and illustrates how a fragment-based drug discovery approach has been efficiently used to discover FGFR1-4 inhibitors with nanomolar affinity. aTurner, N. and Grose, R. Nat. Rev. Cancer, 2010, 10, 116-129. Citation Format: Patrick R. Angibaud, Laurence Mevellec, Gordon Saxty, Christophe Adelinet, Rhalid Akkari, Valerio Berdini, Pascal Bonnet, Marine Bourgeois, Xavier Bourdrez, Anne Cleasby, Helene Colombel, Imre Csoka, Werner Embrechts, Eddy Freyne, Ronaldus Gilissen, Eleonora Jovcheva, Peter King, Jean Lacrampe, Delphine Lardeau, Yannick Ligny, Steve Mcclue, Lieven Meerpoel, David R. Newell, Martin Page, Alexandra Papanikos, Elisabeth Pasquier, Isabelle Pilatte, Virginie Poncelet, Olivier Querolle, David C. Rees, Sharna Rich, Bruno Roux, Elodie Sement, Yvan Simonnet, Matthew Squires, Virginie Tronel, Tinne Verhulst, Jorge Vialard, Marc Willems, Steven J. Woodhead, Berthold Wroblowski, Christopher W. Murray, Timothy Perera. Discovery of JNJ-42756493, a potent fibroblast growth factor receptor (FGFR) inhibitor using a fragment based approach. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4748. doi:10.1158/1538-7445.AM2014-4748

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. A fragment screening campaign was conducted against the tyrosine kinase domain of FGFR1 to detect low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment inhibitors (molecular weight The poster will focus on the description of previously undescribed compounds bearing an imidazo[1,2-a]pyridine core scaffold where selectivity versus other protein kinases, for example FLT3, is obtained using the X-ray crystal structure and structure-based design. In summary we will illustrate how X-ray crystallography and fragment-based drug design (FBDD) can be used to discover compounds with activity in an FGFR driven xenograft model when dosed by the oral route. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1361. doi:10.1158/1538-7445.AM2011-1361

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFR) signalling as being key to the molecular pathology of cancer. FGFR is a receptor tyrosine kinase which activates the extracellular signal-regulated kinase / mitogen-activated protein kinase and the protein kinase B / Akt pathways which promote cell growth and survival. Amplification, over-expression or activating mutations of fibroblast growth factor receptors have been associated with bladder tumours, multiple myeloma, hormone-refractory prostate cancer and breast cancer. Multiple lead series of FGFR inhibitors were developed using Astex's fragment based medicinal chemistry approach, Pyramid™, linked to high throughput X-ray Crystallography. We describe here the characterisation of some examples of these lead molecules. In particular we detail the pharmacological profile of a compound from one of these lead series that demonstrated activity against FGFR 1-4 with an IC50 10 fold lower in cell lines lacking FGFR expression. We demonstrate inhibition of FGFR 2 and 3 phosphorylation in gastric and multiple myeloma cell lines respectively with associated inhibition of downstream signalling pathways. This lead molecule has an excellent pharmacokinetic profile and high oral bioavailibility in mice and rats. In xenograft models in mice where aberrant FGF signalling underlies tumour pathology, tumour growth inhibition is observed at doses of 100mg/kg /day orally for 21 days. This xenograft efficacy was observed in several models, with significantly lower activity in models where aberrant FGF signalling is not involved in tumour pathology. This suggests that the mechanism of action is consistent with FGFR inhibition. The pharmacological profile in these models is also distinct from other broader spectrum receptor tyrosine kinase inhibitors. The pre-clinical data shown here suggests that such compounds warrant further investigation pre-clinically and may benefit patients whose disease is driven by FGFR activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3626.

Recent data in a number of tumour types has implicated Fibroblast Growth Factor (FGF) and Fibroblast Growth Factor receptor (FGFr) signalling as being key to the molecular pathology of cancer. This poster will describe fragment based drug discovery using biophysical screening to identify initial fragments. Subsequently, in the fragments-to-leads stage a detailed structural understanding of the binding interactions between the fragment and its target protein utilised X-ray crystallography and NMR. Starting with different fragments allows several lead series to be identified, often by synthesizing only small numbers of compounds. A fragment screening campaign was conducted against the FGFr-1 to detect very low molecular weight compounds that bound to the hinge region of the kinase. The screening produced several fragment molecules (Molecular Weight This poster represents first disclosure of the structure of the lead series and illustrates how a fragment-based drug discovery approach can be efficiently used to discover compounds advanced nanomolar compounds with oral bioavailability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5778.