TY - JOUR
T1 - Metabolic Activation of the Acrylamide Michael Acceptor Warhead in Futibatinib to an Epoxide Intermediate Engenders Covalent Inactivation of CYP3A
AU - Tang, Lloyd Wei Tat
AU - Fu, Jiaxin
AU - Koh, Siew Kwan
AU - Wu, Guoyi
AU - Zhou, Lei
AU - Chan, Eric Chun Yong
N1 - Funding Information:
L.W.T.T is supported by the National University of Singapore (NUS) President’s Graduate Fellowship (PGF). This work was supported by the Singapore Ministry of Education Tier 1 Academic Research Funding [Grant A-0008501-00-00] (to E.C.Y.C.). The authors declare that they have no conflicts of interest with the contents of this article. dx.doi.org/10.1124/dmd.122.000895. S This article has supplemental material available at dmd.aspetjournals.org.
Publisher Copyright:
Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics
PY - 2022/7/1
Y1 - 2022/7/1
N2 - Futibatinib (FUT) is a potent inhibitor of fibroblast growth factor receptor (FGFR) 1–4 that is currently under clinical investigation for intrahepatic cholangiocarcinoma. Unlike its predecessors, FUT possesses an acrylamide warhead, which enables it to bind covalently to a free cysteine residue in the FGFR kinase domain. However, it remains uninterrogated if this electrophilic a,b-unsaturated carbonyl scaffold could also directly or indirectly engender off-target covalent binding to nucleophilic centers on other cellular proteins. Here, we discovered that FUT inactivated both CYP3A isoforms with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratios of 12.5 and 51.4 lM, 0.25 and 0.06 minutes21, and ~52 and ~58 for CYP3A4 and CYP3A5, respectively. Along with its time-, concentration-, and cofactor-dependent inhibitory profiles, FUT also exhibited several cardinal features that were consistent with mechanism-based inactivation. Moreover, the nature of inactivation was unlikely to be pseudo-irreversible and instead arose from the covalent modification of the cytochrome P450 apoprotein and/or its heme moiety due to the lack of substantial enzyme activity recovery following dialysis and chemical oxidation, as well as the absence of the diagnostic Soret peak in spectral analyses. Finally, utilizing glutathione (GSH) trapping and high-resolution mass spectrometry, we illuminated that while the acrylamide moiety in FUT could nonenzymatically conjugate to GSH via Michael addition, it was not implicated in the covalent inactivation of CYP3A. Rather, we surmised that it likely stemmed from the metabolic activation of its acrylamide covalent warhead to a highly electrophilic epoxide intermediate that could covalently modify CYP3A and culminate in its catalytic inactivation. SIGNIFICANCE STATEMENT In this study, we reported for the first time the inactivation of CYP3A by futibatinib (FUT). Furthermore, using FUT as an exemplary targeted covalent inhibitor, our study revealed the propensity for its acrylamide Michael acceptor moiety to be metabolically activated to a highly electrophilic epoxide. Due to the growing resurgence of covalent inhibitors and the well-established toxicological ramifications associated with epoxides, we advocate that closer scrutiny be adopted when profiling the reactive metabolites of compounds possessing an a,b-unsaturated carbonyl scaffold.
AB - Futibatinib (FUT) is a potent inhibitor of fibroblast growth factor receptor (FGFR) 1–4 that is currently under clinical investigation for intrahepatic cholangiocarcinoma. Unlike its predecessors, FUT possesses an acrylamide warhead, which enables it to bind covalently to a free cysteine residue in the FGFR kinase domain. However, it remains uninterrogated if this electrophilic a,b-unsaturated carbonyl scaffold could also directly or indirectly engender off-target covalent binding to nucleophilic centers on other cellular proteins. Here, we discovered that FUT inactivated both CYP3A isoforms with inactivator concentration at half-maximum inactivation rate constant, maximum inactivation rate constant, and partition ratios of 12.5 and 51.4 lM, 0.25 and 0.06 minutes21, and ~52 and ~58 for CYP3A4 and CYP3A5, respectively. Along with its time-, concentration-, and cofactor-dependent inhibitory profiles, FUT also exhibited several cardinal features that were consistent with mechanism-based inactivation. Moreover, the nature of inactivation was unlikely to be pseudo-irreversible and instead arose from the covalent modification of the cytochrome P450 apoprotein and/or its heme moiety due to the lack of substantial enzyme activity recovery following dialysis and chemical oxidation, as well as the absence of the diagnostic Soret peak in spectral analyses. Finally, utilizing glutathione (GSH) trapping and high-resolution mass spectrometry, we illuminated that while the acrylamide moiety in FUT could nonenzymatically conjugate to GSH via Michael addition, it was not implicated in the covalent inactivation of CYP3A. Rather, we surmised that it likely stemmed from the metabolic activation of its acrylamide covalent warhead to a highly electrophilic epoxide intermediate that could covalently modify CYP3A and culminate in its catalytic inactivation. SIGNIFICANCE STATEMENT In this study, we reported for the first time the inactivation of CYP3A by futibatinib (FUT). Furthermore, using FUT as an exemplary targeted covalent inhibitor, our study revealed the propensity for its acrylamide Michael acceptor moiety to be metabolically activated to a highly electrophilic epoxide. Due to the growing resurgence of covalent inhibitors and the well-established toxicological ramifications associated with epoxides, we advocate that closer scrutiny be adopted when profiling the reactive metabolites of compounds possessing an a,b-unsaturated carbonyl scaffold.
UR - http://www.scopus.com/inward/record.url?scp=85133215055&partnerID=8YFLogxK
U2 - 10.1124/dmd.122.000895
DO - 10.1124/dmd.122.000895
M3 - Journal article
C2 - 35512804
AN - SCOPUS:85133215055
SN - 0090-9556
VL - 50
SP - 931
EP - 941
JO - Drug Metabolism and Disposition
JF - Drug Metabolism and Disposition
IS - 7
ER -