Appropriate control of cell death is a fundamental biological process which is frequently dysregulated during tumor development and therapeutic resistance. Apoptosis is a form of regulated cell death initiated by either the extracellular environment (extrinsic) or following internal cellular damage (intrinsic). It is controlled by the BCL-2 family which includes anti-apoptotic regulators like BCL-2, BCL-XL and MCL-1 that bind and sequester various pro-apoptotic BH3-only proteins (BIM, BAD, BID, NOXA, PUMA, etc.), and the pro-apoptotic effectors (BAK, BAX, etc.) responsible for mitochondrial pore formation and MOMP (mitochondrial outer membrane permeabilization). MOMP results in intermembrane space protein release, leading to caspase activation in an irreversible path to programmed cell death. Of the anti-apoptotic regulators, MCL-1 is one of the most frequently and highly amplified genes in human cancers such as myeloid leukemia making it a compelling therapeutic target. Since BCL-2 proteins interact through protein-protein interactions, they have long been elusive targets. The success of selective BCL-2 protein inhibitor Venetoclax in the treatment of various hematological cancers, however spurred interest in MCL-1 as an oncology target. Using structure-based drug design, major breakthroughs were made in the development of MCL-1 inhibitors, with several candidates entering clinical studies in the past five years. JNJ-4355, a highly potent 1,4-indolyl macrocycle (MCL-1 Ki = 18 pM, Cell (MOLP8) AC50 = 8.7 nM) was optimized to address shortcomings from first generation MCL-1 inhibitors: it has improved physicochemical properties (CHI LogD7.4 = 2.35, EPSA = 151 Å2), resulting in greatly improved equilibrium solubility (3.14 mM in buffer pH 7) and reduced protein binding (99.93%). JNJ-4355 showed promising in vitro potency data in cancer cell lines and AML patient-derived samples (cell killing AC50 0.29-75 nM in 25/27 evaluable samples). In vivo MCL-1:BAK complex disruption was confirmed in a mouse MOLM13 (AML) xenograft. Efficacy was demonstrated in a mouse MOLP8 (multiple myeloma) xenograft resulting in complete tumor regression after a single IV dose of JNJ-4355. Citation Format: Frederik J. Rombouts, Lento William, Ingrid Velter, Ann Vos, Aldo Peschiulli, Reuillon Tristan, Maria Dominguez Blanco, Matthieu Jouffroy, Lisa McQueen, Helena Steyvers, Mariette Bekkers, Cristina Altrocchi, Beth Pietrak, Seong Joo Koo, Lawrence Szewczuk, David Walker, Kathryn Packman, Ruud Bueters, Petra Vinken, Amy Johnson, Ricardo Attar, Ulrike Philippar. In pursuit of MCL-1 inhibitors with improved therapeutic window for the treatment of hematological malignancies: Discovery of JNJ-4355 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2133.
Aims Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have proven valuable for studies in drug discovery and safety, although limitations regarding their structural and electrophysiological characteristics persist. In this study, we investigated the electrophysiological properties of Pluricyte® CMs, a commercially available hiPSC-CMs line with a ventricular phenotype, and assessed arrhythmia incidence by IKr block at the single-cell and 2D monolayer level. Methods and results Action potentials were measured at different pacing frequencies, using dynamic clamp. Through voltage-clamp experiments, we determined the properties of INa, IKr, and ICaL. Intracellular Ca2+ measurements included Ca2+-transients at baseline and during caffeine perfusion. Effects of IKr block were assessed in single hiPSC-CMs and 2D monolayers (multi-electrode arrays). Action-potential duration (APD) and its rate dependence in Pluricyte® CMs were comparable to those reported for native human CMs. INa, IKr, and ICaL revealed amplitudes, kinetics, and voltage dependence of activation/inactivation similar to other hiPSC-CM lines and, to some extent, to native CMs. Near-physiological Ca2+-induced Ca2+ release, response to caffeine and excitation–contraction coupling gain characterized the cellular Ca2+-handling. Dofetilide prolonged the APD and field-potential duration, and induced early afterdepolarizations. Beat-to-beat variability of repolarization duration increased significantly before the first arrhythmic events in single Pluricyte® CMs and 2D monolayers, and predicted pending arrhythmias better than action-potential prolongation. Conclusion Taking their ion-current characteristics and Ca2+ handling into account, Pluricyte® CMs are suitable for in vitro studies on action potentials and field potentials. Beat-to-beat variability of repolarization duration proved useful to evaluate the dynamics of repolarization instability and demonstrated its significance as proarrhythmic marker in hiPSC-CMs during IKr block.
Myeloid cell leukemia-1 (MCL-1) is a member of the antiapoptotic BCL-2 proteins family and a key regulator of mitochondrial homeostasis. Overexpression of MCL-1 is found in many cancer cells and contributes to tumor progression, which makes it an attractive therapeutic target. Pursuing our previous study of macrocyclic indoles for the inhibition of MCL-1, we report herein the impact of both pyrazole and indole isomerism on the potency and overall properties of this family of compounds. We demonstrated that the incorporation of a fluorine atom on the naphthalene moiety was a necessary step to improve cellular potency and that, combined with the introduction of various side chains on the pyrazole, it enhanced solubility significantly. This exploration culminated in the discovery of compounds (R a)-10 and (R a)-15, possessing remarkable cellular potency and properties
Introduction: Cardiotoxicity is one of the leading causes of compound attrition during drug development. Most in vitro screening platforms aim at detecting acute cardio-electrophysiological changes and drug-induced chronic functional alterations are often not studied in the early stage of drug development. Therefore, we developed an assay using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) that evaluates both drug-induced acute and delayed electrophysiological and cytotoxic effects of reference compounds with clinically known cardiac outcomes.Methods: hiPSC-CMs were seeded in 48-well multielectrode array (MEA) plates and were treated with four doses of reference compounds (covering and exceeding clinical free plasma peak concentrations -fCmax values) and MEA recordings were conducted for 4 days. Functional-electrophysiological (field-potentials) and viability (impedance) parameters were recorded with a MEA machine.Results: To assess this platform, we tested tyrosine-kinase inhibitors with high-cardiac risk profile (sunitinib, vandetanib and nilotinib) and low-cardiac risk (erlotinib), as well as known classic cardiac toxic drugs (doxorubicin and BMS-986094), ion-channel trafficking inhibitors (pentamidine, probucol and arsenic trioxide) and compounds without known clinical cardiotoxicity (amoxicillin, cetirizine, captopril and aspirin). By evaluating the effects of these compounds on MEA parameters, the assay was mostly able to recapitulate different drug-induced cardiotoxicities, represented by a prolongation of the field potential, changes in beating rate and presence of arrhythmic events in acute (<2 h) or delayed phase ≥24 h, and/or reduction of impedance during the delayed phase (≥24 h). Furthermore, a few reference compounds were tested in hiPSC-CMs using fluorescence- and luminescence-based plate reader assays, confirming the presence or absence of cytotoxic effects, linked to changes of the impedance parameters measured in the MEA assay. Of note, some cardiotoxic effects could not be identified at acute time points (<2 h) but were clearly detected after 24 h, reinforcing the importance of chronic drug evaluation.Discussion: In conclusion, the evaluation of chronic drug-induced cardiotoxicity using a hiPSC-CMs in vitro assay can contribute to the early de-risking of compounds and help optimize the drug development process.
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