Niresh Hariparsad

TEAD proteins are the transcriptional effectors of the Hippo signaling pathway that bind coactivators YAP/TAZ to activate gene expression programs involved in cell survival, proliferation, and drug resistance. TEAD-dependent transcription is elevated in many tumor types through a variety of mechanisms including loss-of-function mutations in Hippo pathway tumor suppressors and as an adaptive resistance mechanism in response to EGFR tyrosine kinase inhibitor and KRAS inhibition. Therefore, targeting the YAP/TAZ–TEAD signaling axis has the potential to provide clinical benefit either as a monotherapy or combination strategy across diverse indications. In this study, we characterize the activity of AZ′4331, a TEAD palmitoylation inhibitor which displays antitumor efficacy in multiple preclinical models of Hippo pathway–dysregulated cancers. Metabolic labeling studies show that TEAD proteins rapidly cycle between apo and palmitoylated states and that AZ′4331 inhibits the palmitoylation of TEAD1 to 4 paralogs. Functionally, AZ′4331 reduces YAP–TEAD binding and TEAD-dependent transcription, leading to reduced cell-cycle progression and proliferation in Hippo pathway–altered NCI-H226 cells in vitro. AZ′4331 elicits robust monotherapy activity across mesothelioma and head and neck squamous cell carcinoma xenografts and significantly enhances the efficacy of osimertinib in EGFR-mutant non–small cell lung cancer in vitro. However, this combination benefit only translates in vivo when osimertinib is administered at doses below clinically relevant levels, suggesting that TEAD inhibition enhances the efficacy of osimertinib in settings where EGFR signaling is incompletely suppressed. Collectively, these findings highlight the promise of TEAD inhibition as a therapeutic strategy for the treatment of Hippo pathway–driven cancers.

NADPH-P450 oxidoreductase (POR) is the accepted redox-partner for drug-metabolising cytochrome P450s (CYPs) over NADH-cytochrome b ₅ reductase (Cyt b ₅ R). Accordingly, POR-centric NADPH-supplemented human liver microsomes (HLM) and recombinant CYP-POR systems complement human hepatocytes (HH) as drug-metabolism models. However, HH may exhibit inexplicably lower CYP activities relative to NADPH-HLM, particularly for CYP3A4-substrates. Here, we show the phenomenon can manifest as disparate CYP phenotyping in which NADPH-HLM and recombinant CYP-POR systems incorrectly identify CYP3A4 while HH accurately assign the main-metabolising CYP exemplified by CYP1A2 for savolitinib. Mechanistically, we serendipitously discover that HH CYP3A4-mediated midazolam-metabolism is increased by gefitinib and find that this can only be recapitulated in non-canonical Cyt b ₅ R-dependent NADH-HLM and recombinant CYP3A4-Cyt b ₅ R. We conclude that Cyt b ₅ R is important for HH-CYP3A4 and show HH-consistent CYP3A4-activities in NADH-HLM. Imaging NADH/NADPH in hepatocytes shows equivalent concentrations suggesting CYP redox-partnerships are cofactor-independent and likely influenced by protein-protein interactions as mimicking the dense-intracellular protein using albumin recapitulates HH savolitinib-metabolism in HLM.

Background: Accurate assessment of CYP2C induction-mediated drug–drug interactions (DDIs) remains a challenge, despite the importance of CYP2C enzymes in drug metabolism. Limitations in available models and scarce clinical induction data have hampered quantitative preclinical DDI risk evaluation. Methods: In this study, the authors utilized an all-human hepatocyte triculture system to capture CYP2C induction using the perpetrators rifampicin, efavirenz, carbamazepine, and apalutamide. In vitro induction parameters were quantified by measuring changes in both mRNA and enzyme activities for CYP2C8, CYP2C9, and CYP2C19. These induction parameters, along with CYP-specific intrinsic clearance (CL_int) for the victim compounds, were incorporated into a physiologically based pharmacokinetic (PBPK) model, and pharmacokinetics (PK) of known CYP2C substrates were predicted with and without co-administration of perpetrator compounds using clinical dosing regimens. The results were quantitatively compared with the currently utilized mechanistic static modeling (MSM) approach and the reported clinical DDI outcomes. Results: By incorporating the measured f_m of CYP2C substrates into PBPK modeling, we observed a lower propensity to over- or underpredict the exposure of these substrates as victims of CYP2C induction-based DDIs when co-administered with known perpetrators, which resulted in an excellent correlation to observed clinical outcomes. The MSM approach predicted the CYP3A4 induction-based DDI risk accurately but could not capture CYP2C induction with similar precision. Conclusions: Overall, this is the first study that demonstrates the utility of PBPK modeling as a complementary approach to MSM for CYP2C induction-based DDI risk assessment.

Accurate prediction of oral drug absorption in humans is essential for early drug development; however, physiologically relevant human models are lacking. This study aims to comprehensively assess the EpiIntestinal™, a human primary intestinal 3D model, for its ability to predict oral absorption ( F _a ), intestinal availability ( F _g ), CYP3A4 induction, and drug–drug interactions (DDIs). The model showed clinically relevant expression of a key drug‐metabolizing enzymes, transporters, and a nuclear receptor, pregnane X receptor (PXR). The model demonstrated a moderate improvement over Caco‐2 in correlating permeability coefficients with human absorption data for a set of 18 drugs. However, PBPK modeling, using EpiIntestinal™ permeability data, accurately predicted the clinical maximum plasma concentration ( C _max ) of the P‐gp substrates digoxin and dabigatran etexilate, unlike the significant underpredictions from Caco‐2 data. PBPK modeling using intrinsic clearance and permeability data from EpiIntestinal™ accurately predicted human F _g for CYP3A4/5 substrate drugs (except buspirone). Furthermore, the model demonstrated the induction of CYP3A4 and P‐gp (threefold) by a strong PXR inducer, rifampicin. Combining induction parameters of rifampicin from EpiIntestinal™ with those from the TruVivo (human hepatic model) into PBPK modeling accurately captured DDI effects on midazolam, a sensitive CYP3A4/5 substrate. Additionally, the model accurately predicted clinical outcomes of P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) mediated DDIs for ARV‐471. These data underscore the potential of EpiIntestinal™ in predicting human F _a and F _g , and in quantitatively assessing CYP3A4 induction and transporter‐based DDIs.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of T-cell signaling. Inhibition of HPK1 with small molecules has been shown to reinvigorate the immune system toward fighting tumours in preclinical models, thus it holds promise as a therapeutic strategy in cancer immunotherapy. Herein we report a series of pyrazine carboxamide pyrazoles as selective inhibitors of HPK1. Key to our approach was the development of late-stage functionalisation chemistry which allowed for rapid SAR generation. Through these efforts, we discovered difluoroethyl pyrazole 16a, an in vivo tool which elicited the desired pharmacodynamic response in mice. Further, we describe the optimization of synthetic chemistry which could support preclinical studies of a member of this series of substituted pyrazoles.

Hematopoietic progenitor kinase 1 (HPK1) is a negative regulator of the T cell receptor signaling pathway and is therefore a target of interest for immunooncology. Nonselective HPK1 inhibitors may affect other kinase components of T cell activation, blunting the beneficial impact of enhanced T cell activity that results from HPK1 inhibition itself. Here, we report the discovery of pyrazine carboxamide HPK1 inhibitors and their optimization through structure-based drug design to afford a highly selective HPK1 inhibitor, compound 24 (AZ3246). This compound induces IL-2 secretion in T cells with an EC₅₀ of 90 nM without inhibiting antagonistic kinases, exhibits pharmacokinetic properties consistent with oral dosing, and demonstrates antitumor activity in the EMT6 syngeneic mouse model.