Enzyme-mediated drug-drug interactions: a review of in vivo and in vitro methodologies, regulatory guidance, and translation to the clinic.

Enzyme-mediated pharmacokinetic drug-drug interactions can be caused by altered activity of drug metabolizing enzymes in the presence of a perpetrator drug, mostly via inhibition or induction. We identified a gap in the literature for a state-of-the art detailed overview assessing this type of DDI risk in the context of drug development. This manuscript discusses in vitro and in vivo methodologies employed during the drug discovery and development process to predict clinical enzyme-mediated DDIs, including the determination of clearance pathways, metabolic enzyme contribution, and the mechanisms and kinetics of enzyme inhibition and induction. We discuss regulatory guidance and highlight the utility of in silico physiologically-based pharmacokinetic modeling, an approach that continues to gain application and traction in support of regulatory filings. Looking to the future, we consider DDI risk assessment for targeted protein degraders, an emerging small molecule modality, which does not have recommended guidelines for DDI evaluation. Our goal in writing this report was to provide early-career researchers with a comprehensive view of the enzyme-mediated pharmacokinetic DDI landscape to aid their drug development efforts.

Transporter-mediated drug-drug interactions: regulatory guidelines, in vitro and in vivo methodologies and translation, special populations, and the blood-brain barrier.

This review, part of a special issue on drug-drug interactions (DDIs) spearheaded by the International Society for the Study of Xenobiotics (ISSX) New Investigators, explores the critical role of drug transporters in absorption, disposition, and clearance in the context of DDIs. Over the past two decades, significant advances have been made in understanding the clinical relevance of these transporters. Current knowledge on key uptake and efflux transporters that affect drug disposition and development is summarized. Regulatory guidelines from the FDA, EMA, and PMDA that inform the evaluation of potential transporter-mediated DDIs are discussed in detail. Methodologies for preclinical and clinical testing to assess potential DDIs are reviewed, with an emphasis on the utility of physiologically based pharmacokinetic (PBPK) modeling. This includes the application of relative abundance and expression factors to predict human pharmacokinetics (PK) using preclinical data, integrating the latest regulatory guidelines. Considerations for assessing transporter-mediated DDIs in special populations, including pediatric, hepatic, and renal impairment groups, are provided. Additionally, the impact of transporters at the blood-brain barrier (BBB) on the disposition of CNS-related drugs is explored. Enhancing the understanding of drug transporters and their role in drug disposition and toxicity can improve efficacy and reduce adverse effects. Continued research is essential to bridge remaining gaps in knowledge, particularly in comparison with cytochrome P450 (CYP) enzymes.

METTL7A (TMT1A) and METTL7B (TMT1B) Are Responsible for Alkyl S-Thiol Methyl Transferase Activity in Liver.

S-methylation of drugs containing thiol-moieties often alters their activity and results in detoxification. Historically, scientists attributed methylation of exogenous aliphatic and phenolic thiols to a putative S-adenosyl-L-methionine (SAM)-dependent membrane-associated enzyme referred to as thiol methyltransferase (TMT). This putative TMT appeared to have a broad substrate specificity and methylated the thiol metabolite of spironolactone, mertansine, ziprasidone, captopril, and the active metabolites of the thienopyridine prodrugs, clopidogrel, and prasugrel. Despite TMT's role in the S-methylation of clinically relevant drugs, the enzyme(s) responsible for this activity remained unknown. We recently identified methyltransferase-like protein 7B (METTL7B) as an alkyl thiol methyltransferase. METTL7B is an endoplasmic reticulum-associated protein with similar biochemical properties and substrate specificity to the putative TMT. Yet, the historic TMT inhibitor 2,3-dichloro-α-methylbenzylamine (DCMB) did not inhibit METTL7B, indicating that multiple enzymes contribute to TMT activity. Here we report that methyltransferase-like protein 7A (METTL7A), an uncharacterized member of the METTL7 family, is also a SAM-dependent thiol methyltransferase. METTL7A exhibits similar biochemical properties to METTL7B and putative TMT, including inhibition by DCMB (IC50 = 1.17 µM). Applying quantitative proteomics to human liver microsomes and gene modulation experiments in HepG2 and HeLa cells, we determined that TMT activity correlates closely with METTL7A and METTL7B protein levels. Furthermore, purification of a novel His-GST-tagged recombinant protein and subsequent activity experiments prove that METTL7A can selectively methylate exogenous thiol-containing substrates, including 7α-thiospironolactone, dithiothreitol, 4-chlorothiophenol, and mertansine. We conclude that the METTL7 family encodes for two enzymes, METTL7A and METTL7B, which are now renamed thiol methyltransferase 1A (TMT1A) and thiol methyltransferase 1B (TMT1B), respectively, that are responsible for thiol methylation activity in human liver microsomes. SIGNIFICANCE STATEMENT: We identified methyltransferase-like protein 7A (thiol methyltransferase 1A) and methyltransferase-like protein 7B (thiol methyltransferase 1B) as the enzymes responsible for the microsomal alkyl thiol methyltransferase (TMT) activity. These are the first two enzymes directly associated with microsomal TMT activity. S-methylation of commonly prescribed thiol-containing drugs alters their pharmacological activity and/or toxicity, and identifying the enzymes responsible for this activity will improve our understanding of the drug metabolism and pharmacokinetic (DMPK) properties of alkyl- or phenolic thiol-containing therapeutics.

Epigenetics in drug disposition & drug therapy: symposium report of the 24th North American meeting of the International Society for the Study of Xenobiotics (ISSX).

The 24th North American International Society for the Study of Xenobiotics (ISSX) meeting, held virtually from September 13 to 17, 2021, embraced the theme of "Broadening Our Horizons." This reinforces a key mission of ISSX: striving to share innovative science related to drug discovery and development. Session speakers and the ISSX New Investigators Group, which supports the scientific and professional development of student and early career ISSX members, elected to highlight the scientific content presented during the captivating session titled, "Epigenetics in Drug Disposition & Drug Therapy." The impact genetic variation has on drug response is well established; however, this session underscored the importance of investigating the role of epigenetics in drug disposition and drug discovery. Session speakers, Drs. Ning, McClay, and Lazarus, detailed mechanisms by which epigenetic players including long non-coding RNA (lncRNAs), microRNA (miRNAs), DNA methylation, and histone acetylation can alter the expression of genes involved in pharmacokinetics, pharmacodynamics, and toxicity. Dr. Ning detailed current knowledge about miRNAs and lncRNAs and the mechanisms by which they can affect the expression of drug metabolizing enzymes (DMEs) and nuclear receptors. Dr. Lazarus discussed the potential role of miRNAs on UDP-glucuronosyltransferase (UGT) expression and activity. Dr. McClay provided evidence that aging alters methylation and acetylation of DMEs in the liver, affecting gene expression and activity. These topics, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are herein discussed, along with exciting future perspectives for epigenetics in drug disposition and drug discovery research.

Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers.

RAS-driven cancers comprise up to 30% of human cancers. RMC-6236 is a RAS(ON) multi-selective noncovalent inhibitor of the active, GTP-bound state of both mutant and wild-type variants of canonical RAS isoforms with broad therapeutic potential for the aforementioned unmet medical need. RMC-6236 exhibited potent anticancer activity across RAS-addicted cell lines, particularly those harboring mutations at codon 12 of KRAS. Notably, oral administration of RMC-6236 was tolerated in vivo and drove profound tumor regressions across multiple tumor types in a mouse clinical trial with KRASG12X xenograft models. Translational PK/efficacy and PK/PD modeling predicted that daily doses of 100 mg and 300 mg would achieve tumor control and objective responses, respectively, in patients with RAS-driven tumors. Consistent with this, we describe here objective responses in two patients (at 300 mg daily) with advanced KRASG12X lung and pancreatic adenocarcinoma, respectively, demonstrating the initial activity of RMC-6236 in an ongoing phase I/Ib clinical trial (NCT05379985). SIGNIFICANCE: The discovery of RMC-6236 enables the first-ever therapeutic evaluation of targeted and concurrent inhibition of canonical mutant and wild-type RAS-GTP in RAS-driven cancers. We demonstrate that broad-spectrum RAS-GTP inhibition is tolerable at exposures that induce profound tumor regressions in preclinical models of, and in patients with, such tumors. This article is featured in Selected Articles from This Issue, p. 897.

Human METTL7B is an alkyl thiol methyltransferase that metabolizes hydrogen sulfide and captopril.

Methylation of alkyl thiols is a biotransformation pathway designed to reduce thiol reactivity and potential toxicity, yet the gene and protein responsible for human alkyl thiol methyltransferase (TMT) activity remain unknown. Here we demonstrate with a range of experimental approaches using cell lines, in vitro systems, and recombinantly expressed enzyme, that human methyltransferase-like protein 7B (METTL7B) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to hydrogen sulfide (H2S) and other exogenous thiol small molecules. METTL7B gene modulation experiments, including knockdown in HepG2 cells and overexpression in HeLa cells, directly alter the methylation of the drug captopril, a historic probe substrate for TMT activity. Furthermore, recombinantly expressed and purified wild-type METTL7B methylates several thiol compounds, including H2S, 7α-thiospironolactone, L-penicillamine, and captopril, in a time- and concentration-dependent manner. Typical for AdoMet-dependent small molecule methyltransferases, S-adenosyl-L-homocysteine (AdoHcy) inhibited METTL7B activity in a competitive fashion. Similarly, mutating a conserved aspartate residue, proposed to anchor AdoMet into the active site, to an alanine (D98A) abolished methylation activity. Endogenous thiols such as glutathione and cysteine, or classic substrates for other known small molecule S-, N-, and O-methyltransferases, were not substrates for METTL7B. Our results confirm, for the first time, that METTL7B, a gene implicated in multiple disease states including rheumatoid arthritis and breast cancer, encodes a protein that methylates small molecule alkyl thiols. Identifying the catalytic function of METTL7B will enable future pharmacological research in disease pathophysiology where altered METTL7B expression and, potentially H2S levels, can disrupt cell growth and redox state.