ASTX029, a Novel Dual-mechanism ERK Inhibitor, Modulates Both the Phosphorylation and Catalytic Activity of ERK

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).

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2

“…Potency was maintained in this 3,5disubstituted analogue, as well as observing improved CYP3A4 metabolic stability, with a half-life of 35 min (Table 1). The crystal structure of the 15-ERK2 complex 22 confirmed the designed binding mode, in which the methoxy group contacted the aryl side chain of Tyr64 and the fluoro group occupied a small space between methylenes in the side chains of Tyr64 and Arg67, forming a close contact (2.9 Å) with the hydroxyl side chain of Thr68 (Figure 5a). A wide range of additional monosubstituted and disubstituted analogues were also prepared, but generally showed less promising stability in the CYP3A4 assay, exemplified by the 3,5-difluoro analogue 16 with a half-life of less than 5 min.…”

“…The performance in the PD assays translated into efficacy in the Colo205 xenograft tumor growth models. Compound 15 elicited tumor growth inhibition dose dependently in the range 25–75 mg/kg (Figure c) . Analogues 17 , 20 , and 25 also elicited tumor growth inhibition at their highest doses in the 75–100 mg/kg range and with a lesser overall effect than 15 (Figure S2).…”

“…Given its promising performance in the Colo205 PD and efficacy models, compound 15 was selected for further characterization . For example, compound 15 was shown to have good in vivo efficacy against six other xenografted cell lines.…”

“…In this paper, we disclose our lead optimization strategy, focusing on enhancing the predicted pharmacokinetics in humans and culminating in the discovery of the clinical candidate ASTX029 (compound 15 ). Detailed pharmacological profiling of ASTX029 has been published elsewhere …”

“…Detailed pharmacological profiling of ASTX029 has been published elsewhere. 22 ■ RESULTS AND DISCUSSION Metabolic Studies on the Lead Compound. In the preceding paper, we characterized the lead 7 as a highly potent and selective inhibitor of ERK kinase activity and a modulator of ERK phosphorylation (pERK), with an IC 50 of 3.5 nM versus pRSK and 2.3 nM versus pERK in A375 cells.…”