The ubiquitously expressed protein tyrosine phosphatase SHP2 is required for signaling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 suppresses RAS/MAPK signaling and inhibit the proliferation of RTK-driven cancer cell lines. Here, we describe the first reported fragment-to-lead campaign against SHP2, where X-ray crystallography and biophysical techniques were used to identify fragments binding to multiple sites on SHP2. Structure-guided optimization, including several computational methods, led to the discovery of two structurally distinct series of SHP2 inhibitors binding to the previously reported allosteric tunnel binding site (Tunnel Site). One of these series was advanced to a low-nanomolar lead that inhibited tumor growth when dosed orally to mice bearing HCC827 xenografts. Furthermore, a third series of SHP2 inhibitors was discovered binding to a previously unreported site, lying at the interface of the C-terminal SH2 and catalytic domains.

Supplementary figures 1-7 show the effects of AST029 on ERK signalling in RAS-mutant cell lines (1) and tumour xenograft tissue (3), ASTX029 plasma PK linearity (2), ASTX029 activity in a cell line panel showing MAPK mutation status (4) the effects of ASTX029 on mouse body weight (5), PKPD effects of ASTX029 following b.i.d dosing to Colo205 tumour-bearing mice (6) and PD effects of ASTX029 following qd dosing to A375R tumour-bearing mice (7).

Table listing details of antibodies used

Supplementary tables 1-6 summarise the kinase panel screen data (1) mouse PK parameters (2) and further details of the anti-tumour activity (5) conferred by ASTX029, plus details of cell lines used in this study (3 and 4) and further details of the effects of ASTX029 and other MAPK inhibitors in models of MAPK resistance (6).

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

Abstract 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).

Background: SHP2 is a ubiquitously expressed protein tyrosine phosphatase required for signalling downstream of receptor tyrosine kinases (RTKs) and plays a role in regulating many cellular processes. Genetic knockdown and pharmacological inhibition of SHP2 inhibits proliferation of RTK-driven cancer cell lines and suppresses RAS/MAPK signalling. As SHP2 is a master regulator of RTK signalling and RTK deregulation often leads to a wide range of cancers, SHP2 inhibitors are therefore a promising therapeutic approach and several compounds are being tested in the clinic. Using our fragment-based screening approach, PyramidTM, we identified fragment hits binding to the tunnel region between the phosphatase domain and the C-SH2 domain of SHP2. Here we describe the optimisation of these mM fragment hits into potent SHP2 antagonists with in vitro and in vivo antitumor activity.

Abstract 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.

Background: The MAPK signaling pathway is commonly upregulated in human cancers due to oncogenic mutations of upstream components such as BRAF or KRAS. Targeting of the MAPK pathway has been clinically validated, with agents targeting BRAF and MEK approved. As the final node in the MAPK pathway, ERK is also an attractive therapeutic target for the treatment of MAPK-activated cancers. We have previously described the discovery of a chemical series targeting ERK using fragment-based drug design. ASTX029 is a highly potent and selective ERK inhibitor from this series, which is now being tested in a Phase 1/2 clinical trial (NCT03520075). Here, we describe the activity of ASTX029 in a number of MAPK-activated cell-based and in vivo models.

Abstract 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.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Abstract 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.

Lp-PLA2 has been explored as a target for a number of inflammation associated diseases, including cardiovascular disease and dementia. This article describes the discovery of a new fragment derived chemotype that interacts with the active site of Lp-PLA2. The starting fragment hit was discovered through an X-ray fragment screen and showed no activity in the bioassay (IC50 > 1 mM). The fragment hit was optimized using a variety of structure-based drug design techniques, including virtual screening, fragment merging, and improvement of shape complementarity. A novel series of Lp-PLA2 inhibitors was generated with low lipophilicity and a promising pharmacokinetic profile.

Elevated levels of human lipoprotein-associated phospholipase A2 (Lp-PLA2) are associated with cardiovascular disease and dementia. A fragment screen was conducted against Lp-PLA2 in order to identify novel inhibitors. Multiple fragment hits were observed in different regions of the active site, including some hits that bound in a pocket created by movement of a protein side chain (approximately 13 Å from the catalytic residue Ser273). Using structure guided design, we optimized a fragment that bound in this pocket to generate a novel low nanomolar chemotype, which did not interact with the catalytic residues.

The members of the NSD subfamily of lysine methyl transferases are compelling oncology targets due to the recent characterization of gain-of-function mutations and translocations in several hematological cancers. To date, these proteins have proven intractable to small molecule inhibition. Here, we present initial efforts to identify inhibitors of MMSET (aka NSD2 or WHSC1) using solution phase and crystal structural methods. On the basis of 2D NMR experiments comparing NSD1 and MMSET structural mobility, we designed an MMSET construct with five point mutations in the N-terminal helix of its SET domain for crystallization experiments and elucidated the structure of the mutant MMSET SET domain at 2.1 Å resolution. Both NSD1 and MMSET crystal systems proved resistant to soaking or cocrystallography with inhibitors. However, use of the close homologue SETD2 as a structural surrogate supported the design and characterization of N-alkyl sinefungin derivatives, which showed low micromolar inhibition against both SETD2 and MMSET.