Search

Epidermal growth factor receptor (EGFR) inhibitors have clinical utility in the treatment of non-small cell lung cancer (NSCLC) patients. Despite encouraging clinical efficacy with these agents, many patients develop resistance due to sensitizing (or activating) mutations ultimately leading to disease progression. In the majority of the cases, this resistance is due to the T790M mutation and frequently coexisting L858R. In addition, EGFR wild type receptor inhibition can lead to on target related dose limiting toxicities such as rash and diarrhea. We describe herein the identification of a mutant selective lead compound 12, an irreversible covalent inhibitor of EGFR T790M/L858R resistance mutations with selectivity over the wild type form. Significant tumor growth inhibition in preclinical models was observed with this lead.

Bruton tyrosine kinase (BTK) is an important target in oncology and (auto)immunity. Various BTK inhibitors have been approved or are currently in clinical development. A novel BTK inhibitor series was developed starting with a quinazoline core. Moving from a quinazoline to a quinoline core provided a handle for selectivity for BTK over EGFR and resulted in the identification of potent and selective BTK inhibitors with good potency in human whole blood assay. Furthermore, proof of concept of this series for BTK inhibition was shown in an in vivo mouse model using one of the compounds identified.

Several small-molecule Bruton tyrosine kinase (BTK) inhibitors are in development for B cell malignancies and autoimmune disorders, each characterized by distinct potency and selectivity patterns. Herein we describe the pharmacologic characterization of BTK inhibitor acalabrutinib [compound 1, ACP-196 (4-[8-amino-3-[(2S)-1-but-2-ynoylpyrrolidin-2-yl]imidazo[1,5-a]pyrazin-1-yl]-N-(2-pyridyl)benzamide)]. Acalabrutinib possesses a reactive butynamide group that binds covalently to Cys481 in BTK. Relative to the other BTK inhibitors described here, the reduced intrinsic reactivity of acalabrutinib helps to limit inhibition of off-target kinases having cysteine-mediated covalent binding potential. Acalabrutinib demonstrated higher biochemical and cellular selectivity than ibrutinib and spebrutinib (compounds 2 and 3, respectively). Importantly, off-target kinases, such as epidermal growth factor receptor (EGFR) and interleukin 2-inducible T cell kinase (ITK), were not inhibited. Determination of the inhibitory potential of anti-immunoglobulin M-induced CD69 expression in human peripheral blood mononuclear cells and whole blood demonstrated that acalabrutinib is a potent functional BTK inhibitor. In vivo evaluation in mice revealed that acalabrutinib is more potent than ibrutinib and spebrutinib. Preclinical and clinical studies showed that the level and duration of BTK occupancy correlates with in vivo efficacy. Evaluation of the pharmacokinetic properties of acalabrutinib in healthy adult volunteers demonstrated rapid absorption and fast elimination. In these healthy individuals, a single oral dose of 100 mg showed approximately 99% median target coverage at 3 and 12 hours and around 90% at 24 hours in peripheral B cells. In conclusion, acalabrutinib is a BTK inhibitor with key pharmacologic differentiators versus ibrutinib and spebrutinib and is currently being evaluated in clinical trials.

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity. Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z'-LYTE) with IC50 determination at a fixed time point. On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB). Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells. Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells. Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates. Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested. Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively. No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. Disclosures Kaptein: Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Item does not contain fulltext MK2 kinase is a promising drug discovery target for the treatment of inflammatory diseases. Here, we describe the discovery of novel MK2 inhibitors using X-ray crystallography and structure-based drug design. The lead has in vivo efficacy in a short-term preclinical model.