Proteolysis-targeting chimeras (PROTACs) represent a promising class of drugs that can target disease-causing proteins more effectively than traditional small molecule inhibitors can, potentially revolutionizing drug discovery and treatment strategies. However, the links between in vitro and in vivo data are poorly understood, hindering a comprehensive understanding of the absorption, distribution, metabolism, and excretion (ADME) of PROTACs. In this work, ¹⁴C-labeled vepdegestrant (ARV-471), which is currently in phase III clinical trials for breast cancer, was synthesized as a model PROTAC to characterize its preclinical ADME properties and simulate its clinical pharmacokinetics (PK) by establishing a physiologically based pharmacokinetics (PBPK) model. For in vitro-in vivo extrapolation (IVIVE), hepatocyte clearance correlated more closely with in vivo rat PK data than liver microsomal clearance did. PBPK models, which were initially developed and validated in rats, accurately simulate ARV-471's PK across fed and fasted states, with parameters within 1.75-fold of the observed values. Human models, informed by in vitro ADME data, closely mirrored postoral dose plasma profiles at 30 mg. Furthermore, no human-specific metabolites were identified in vitro and the metabolic profile of rats could overlap that of humans. This work presents a roadmap for developing future PROTAC medications by elucidating the correlation between in vitro and in vivo characteristics.

Glycolytic metabolism enzymes have been implicated in the immunometabolism field through changes in metabolic status. PGK1 is a catalytic enzyme in the glycolytic pathway. Here, we set up a high-throughput screen platform to identify PGK1 inhibitors. DC-PGKI is an ATP-competitive inhibitor of PGK1 with an affinity of K _d = 99.08 nmol/L. DC-PGKI stabilizes PGK1 in vitro and in vivo, and suppresses both glycolytic activity and the kinase function of PGK1. In addition, DC-PGKI unveils that PGK1 regulates production of IL-1β and IL-6 in LPS-stimulated macrophages. Mechanistically, inhibition of PGK1 with DC-PGKI results in NRF2 (nuclear factor-erythroid factor 2-related factor 2, NFE2L2) accumulation, then NRF2 translocates to the nucleus and binds to the proximity region of Il-1β and Il-6 genes, and inhibits LPS-induced expression of these genes. DC-PGKI ameliorates colitis in the dextran sulfate sodium (DSS)-induced colitis mouse model. These data support PGK1 as a regulator of macrophages and suggest potential utility of PGK1 inhibitors in the treatment of inflammatory bowel disease.