Discovery of Selective Tertiary Amide Inhibitors of Cyclin-Dependent Kinase 2 (CDK2).

Cyclin-dependent kinases (CDKs) are key regulators of the cell cycle and are frequently altered in cancer cells, thereby leading to uncontrolled proliferation. In this context, CDK2 has emerged as an appealing target for anticancer drug development. Herein, we describe the discovery of a series of selective small molecule inhibitors of CDK2 beginning with historical compounds from our ERK2 program (e.g., compound 6). Structure-based drug design led to the potent and selective tool compound 32, where excellent selectivity against ERK2 and CDK4 was achieved by filling the lipophilic DFG-1 pocket and targeting interactions with CDK2-specific lower hinge binding residues, respectively. Compound 32 demonstrated 112% tumor growth inhibition in mice bearing OVCAR3 tumors with 50 mg/kg bis in die (BID) oral dosing.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a Pyridopyridazinone pan-RAF Kinase Inhibitor.

Structure-based optimization of a set of aryl urea RAF inhibitors has led to the identification of Type II pan-RAF inhibitor GNE-9815 (7), which features a unique pyrido[2,3-d]pyridazin-8(7H)-one hinge-binding motif. With minimal polar hinge contacts, the pyridopyridazinone hinge binder moiety affords exquisite kinase selectivity in a lipophilic efficient manner. The improved physicochemical properties of GNE-9815 provided a path for oral dosing without enabling formulations. In vivo evaluation of GNE-9815 in combination with the MEK inhibitor cobimetinib demonstrated synergistic MAPK pathway modulation in an HCT116 xenograft mouse model. To the best of our knowledge, GNE-9815 is among the most highly kinase-selective RAF inhibitors reported to date.

Targeting KRAS Mutant Cancers via Combination Treatment: Discovery of a 5-Fluoro-4-(3H)-quinazolinone Aryl Urea pan-RAF Kinase Inhibitor.

Optimization of a series of aryl urea RAF inhibitors led to the identification of type II pan-RAF inhibitor GNE-0749 (7), which features a fluoroquinazolinone hinge-binding motif. By minimizing reliance on common polar hinge contacts, this hinge binder allows for a greater contribution of RAF-specific residue interactions, resulting in exquisite kinase selectivity. Strategic substitution of fluorine at the C5 position efficiently masked the adjacent polar NH functionality and increased solubility by impeding a solid-state conformation associated with stronger crystal packing of the molecule. The resulting improvements in permeability and solubility enabled oral dosing of 7. In vivo evaluation of 7 in combination with the MEK inhibitor cobimetinib demonstrated synergistic pathway inhibition and significant tumor growth inhibition in a KRAS mutant xenograft mouse model.

Physiologically-Based Pharmacokinetic Model-Informed Drug Development for Fenebrutinib: Understanding Complex Drug-Drug Interactions.

Fenebrutinib is a CYP3A substrate and time-dependent inhibitor, as well as a BCRP and OATP1B transporter inhibitor in vitro. Physiologically-based pharmacokinetic (PBPK) modeling strategies with the ultimate goal of understanding complex drug-drug interactions (DDIs) and proposing doses for untested scenarios were developed. The consistency in the results of two independent approaches, PBPK simulation and endogenous biomarker measurement, supported that the observed transporter DDI is primarily due to fenebrutinib inhibition of intestinal BCRP, rather than hepatic OATP1B. A mechanistic-absorption model accounting for the effects of excipient complexation with fenebrutinib was used to rationalize the unexpected observation of itraconazole-fenebrutinib DDI (maximum plasma concentration (Cmax ) decreased, and area under the curve (AUC) increased). The totality of the evidence from sensitivity analysis and clinical and nonclinical data suggested that fenebrutinib is likely a sensitive CYP3A substrate. This advanced PBPK application allowed the use of model-informed approach to facilitate the development of concomitant medication recommendations for fenebrutinib without requiring additional clinical DDI studies.

Complex DDI by Fenebrutinib and the Use of Transporter Endogenous Biomarkers to Elucidate the Mechanism of DDI.

Mechanistic understanding of complex clinical drug-drug interactions (DDIs) with potential involvement of multiple elimination pathways has been challenging, especially given the general lack of specific probe substrates for transporters. Here, we conducted a clinical DDI study to evaluate the interaction potential of fenebrutinib using midazolam (MDZ; CYP3A), simvastatin (CYP3A and OATP1B), and rosuvastatin (BCRP and OATP1B) as probe substrates. Fenebrutinib (200 mg) increased the area under the curve (AUC) of these probe substrates twofold to threefold. To evaluate the mechanism of the observed DDIs, we measured the concentration of coproporphyrin I (CP-I) and coproporphyrin III (CP-III), endogenous biomarkers of OATP1B. There was no change in CP-I or CP-III levels with fenebrutinib, suggesting that the observed DDIs were caused by inhibition of CYP3A and BCRP rather than OATP1B, likely due to increased bioavailability. This is the first published account using an endogenous transporter biomarker to understand the mechanism of complex DDIs involving multiple elimination pathways.

Pharmacological Induction of RAS-GTP Confers RAF Inhibitor Sensitivity in KRAS Mutant Tumors.

Targeting KRAS mutant tumors through inhibition of individual downstream pathways has had limited clinical success. Here we report that RAF inhibitors exhibit little efficacy in KRAS mutant tumors. In combination drug screens, MEK and PI3K inhibitors synergized with pan-RAF inhibitors through an RAS-GTP-dependent mechanism. Broad cell line profiling with RAF/MEK inhibitor combinations revealed synergistic efficacy in KRAS mutant and wild-type tumors, with KRASG13D mutants exhibiting greater synergy versus KRASG12 mutant tumors. Mechanistic studies demonstrate that MEK inhibition induced RAS-GTP levels, RAF dimerization and RAF kinase activity resulting in MEK phosphorylation in synergistic tumor lines regardless of KRAS status. Taken together, our studies uncover a strategy to rewire KRAS mutant tumors to confer sensitivity to RAF kinase inhibition.