Dong Zou

The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models

Purpose: Non–small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK+) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. Experimental Design: A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib–ALK co-structure was determined. Results: Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK+ cell lines, brigatinib inhibited native ALK (IC50, 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK+ tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Conclusions: Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK+, crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527–38. ©2016 AACR.

Discovery of 5-(arenethynyl) hetero-monocyclic derivatives as potent inhibitors of BCR-ABL including the T315I gatekeeper mutant.

Ponatinib (AP24534) was previously identified as a pan-BCR-ABL inhibitor that potently inhibits the T315I gatekeeper mutant, and has advanced into clinical development for the treatment of refractory or resistant CML. In this study, we explored a novel series of five and six membered monocycles as alternate hinge-binding templates to replace the 6,5-fused imidazopyridazine core of ponatinib. Like ponatinib, these monocycles are tethered to pendant toluanilides via an ethynyl linker. Several compounds in this series displayed excellent in vitro potency against both native BCR-ABL and the T315I mutant. Notably, a subset of inhibitors exhibited desirable PK and were orally active in a mouse model of T315I-driven CML.

Abstract 2671: Structural analysis of the inhibitory mechanism of AP24534, a pan-BCR-ABL inhibitor overriding the T315I gatekeeper mutation

Although durable response in many chronic myeloid leukemia (CML) patients can be achieved by imatinib treatment, point mutations in the kinase domain of the BCR-ABL fusion protein can lead to drug resistance and relapse. Nilotinib and dasatinib, the two second generation Abl kinase inhibitors, are active against most imatinib resistant mutants with a few exceptions, particularly the T315I gatekeeper residue mutation. Using a structure-based approach we have identified AP24534, a molecule that potently inhibits the T315I mutant and all other mutants tested, thus acting as a pan-BCR-ABL inhibitor. To elucidate the basis for this unique profile of AP24534, we have used a combination of X-ray crystallographic and structure-activity relationship analysis on AP24534 and its analogs. The crystal structure of the T315I mutant Abl in complex with the inhibitor clearly demonstrates that AP24534 binds to Abl in a DFG-out binding mode with the ethynyl chemical moiety skirting the mutated gate-keeper residue avoiding steric clashes. The X-ray co-structures of both the wild type and the T315I mutant Abl, in conjunction with the SAR analysis, further reveal that AP24534 retains potency against the T315I and other imatinib-resistant ABL mutants through optimized multiple points of contact, balancing and distributing the overall binding affinity so that mutation-based disruption of one facet of the binding network results in only a slight reduction in binding affinity. Our structural analysis enables us to predict and rationalize the different degree of sensitivity of ABL point mutants to AP24534, and to understand how AP24534 can completely abrogate resistance in cell-based mutagenesis screens at relatively low concentrations. AP24534 is currently being investigated in a Phase I clinical trial in patients with refractory and resistant CML and other hematological malignancies. 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 2671.