Starting from the MLPCN probe compound ML300, a structure-based optimization campaign was initiated against the recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CL pro ). X-ray structures of SARS-CoV-1 and SARS-CoV-2 3CL pro enzymes in complex with multiple ML300-based inhibitors, including the original probe ML300, were obtained and proved instrumental in guiding chemistry toward probe compound 41 (CCF0058981). The disclosed inhibitors utilize a noncovalent mode of action and complex in a noncanonical binding mode not observed by peptidic 3CL pro inhibitors. In vitro DMPK profiling highlights key areas where further optimization in the series is required to obtain useful in vivo probes. Antiviral activity was established using a SARS-CoV-2-infected Vero E6 cell viability assay and a plaque formation assay. Compound 41 demonstrates nanomolar activity in these respective assays, comparable in potency to remdesivir. These findings have implications for antiviral development to combat current and future SARS-like zoonotic coronavirus outbreaks.

Lapatinib is a dual tyrosine kinase inhibitor associated with rare but potentially severe idiosyncratic hepatotoxicity. We have previously shown that cytochromes P450 CYP3A4 and CYP3A5 quantitatively contribute to lapatinib bioactivation, leading to formation of a reactive, potentially toxic quinone imine. CYP3A5 is highly polymorphic; however, the impact of CYP3A5 polymorphism on lapatinib metabolism has not been fully established. The goal of this study was to determine the effect of CYP3A5 genotype and individual variation in CYP3A activity on the metabolic activation of lapatinib using human-relevant in vitro systems. Lapatinib metabolism was examined using CYP3A5-genotyped human liver microsomes and cryopreserved human hepatocytes. CYP3A and CYP3A5-selective activities were measured in liver tissues using probe substrates midazolam and T-5 (T-1032), respectively, to evaluate the correlation between enzymatic activity and lapatinib metabolite formation. Drug metabolites were measured by high-performance liquid chromatographytandem mass spectrometry. Further, the relative contributions of CYP3A4 and CYP3A5 to lapatinib O-debenzylation were estimated using selective chemical inhibitors of CYP3A. The results from this study demonstrated that lapatinib O-debenzylation and quinone imine-GSH conjugate formation were highly correlated with hepatic CYP3A activity, as measured by midazolam 19-hydroxylation. CYP3A4 played a dominant role in lapatinib bioactivation in all liver tissues evaluated. The CYP3A5 contribution to lapatinib bioactivation varied by individual donor and was dependent on CYP3A5 genotype and activity. CYP3A5 contributed approximately 20%-42% to lapatinib O-debenzylation in livers from CYP3A5 expressers. These findings indicate that individual CYP3A activity, not CYP3A5 genotype alone, is a key determinant of lapatinib bioactivation and likely influences exposure to reactive metabolites. SIGNIFICANCE STATEMENT This study is the first to examine the effect of CYP3A5 genotype, total CYP3A activity, and CYP3A5-selective activity on lapatinib bioactivation in individual human liver tissues. The results of this investigation indicate that lapatinib bioactivation via oxidative O-debenzylation is highly correlated with total hepatic CYP3A activity, and not CYP3A5 genotype alone. These findings provide insight into the individual factors, namely, CYP3A activity, that may affect individual exposure to reactive, potentially toxic metabolites of lapatinib.

1. Failure to predict human pharmacokinetics of aldehyde oxidase (AO) substrates using traditional allometry has been attributed to species differences in AO metabolism. 2. To identify appropriate species for predicting human in vivo clearance by single-species scaling (SSS) or multispecies allometry (MA), we scaled in vitro intrinsic clearance (CL) of five AO substrates obtained from hepatic S9 of mouse, rat, guinea pig, monkey and minipig to human in vitro CL. 3. When predicting human in vitro CL, average absolute fold-error was ≤2.0 by SSS with monkey, minipig and guinea pig (rat/mouse >3.0) and was <3.0 by most MA species combinations (including rat/mouse combinations). 4. Interspecies variables, including fraction metabolized by AO (F) and hepatic extraction ratios (E) were estimated in vitro. SSS prediction fold-errors correlated with the animal:human ratio of E (r = 0.6488), but not F (r = 0.0051). 5. Using plasma clearance (CL) from the literature, SSS with monkey was superior to rat or mouse at predicting human CL of BIBX1382 and zoniporide, consistent with in vitro SSS assessments. 6. Evaluation of in vitro allometry, F and E may prove useful to guide selection of suitable species for traditional allometry and prediction of human pharmacokinetics of AO substrates.