Covalent EGFR inhibitor analysis reveals importance of reversible interactions to potency and mechanisms of drug resistance.

Covalent inhibition is a reemerging paradigm in kinase drug design, but the roles of inhibitor binding affinity and chemical reactivity in overall potency are not well-understood. To characterize the underlying molecular processes at a microscopic level and determine the appropriate kinetic constants, specialized experimental design and advanced numerical integration of differential equations are developed. Previously uncharacterized investigational covalent drugs reported here are shown to be extremely effective epidermal growth factor receptor (EGFR) inhibitors (kinact/Ki in the range 10(5)-10(7) M(-1)s(-1)), despite their low specific reactivity (kinact ≤ 2.1 × 10(-3) s(-1)), which is compensated for by high binding affinities (Ki < 1 nM). For inhibitors relying on reactivity to achieve potency, noncovalent enzyme-inhibitor complex partitioning between inhibitor dissociation and bond formation is central. Interestingly, reversible binding affinity of EGFR covalent inhibitors is highly correlated with antitumor cell potency. Furthermore, cellular potency for a subset of covalent inhibitors can be accounted for solely through reversible interactions. One reversible interaction is between EGFR-Cys797 nucleophile and the inhibitor's reactive group, which may also contribute to drug resistance. Because covalent inhibitors target a cysteine residue, the effects of its oxidation on enzyme catalysis and inhibitor pharmacology are characterized. Oxidation of the EGFR cysteine nucleophile does not alter catalysis but has widely varied effects on inhibitor potency depending on the EGFR context (e.g., oncogenic mutations), type of oxidation (sulfinylation or glutathiolation), and inhibitor architecture. These methods, parameters, and insights provide a rational framework for assessing and designing effective covalent inhibitors.

Tumor P-Glycoprotein Correlates with Efficacy of PF-3758309 in in vitro and in vivo Models of Colorectal Cancer.

P-glycoprotein (P-gp), a member of the ATP-binding cassette transporter family, is overexpressed in a number of different cancers and some studies show that P-gp overexpression can be correlated to poor prognosis or therapeutic resistance. Here we sought to elucidate if PF-3758309 (PF-309), a novel p-21 activated kinase inhibitor, efficacy was influenced by tumor P-gp. Based on in vitro proliferation data, a panel of colorectal cancer cell lines were ranked as sensitive or resistant and ABCB1 (P-gp) expression was evaluated by microarray for these cell lines. P-gp expression was determined by western blot and activity determined by rhodamine efflux assay. Knock down of P-gp and pharmacologic inhibition of P-gp to restore PF-309 activity was performed in vitro. PF-309 activity was evaluated in vivo in cell line xenograft models and in primary patient derived tumor xenografts (PDTX). Mice were treated with 25 mg/kg PF-309 orally, twice daily. On the last day of treatment, tumor and plasma were collected for PF-309 analysis. Here we show that ABCB1 gene expression correlates with resistance to PF-309 treatment in vitro and the expression and activity of P-gp was verified in a panel of resistant cells. Furthermore, inhibition of P-gp increased the sensitivity of resistant cells, resulting in a 4-100-fold decrease in the IC50s. Eleven cell line xenografts and 12 PDTX models were treated with PF-309. From the cell line xenografts, we found a significant correlation between ABCB1 gene expression profiles and tumor response. We evaluated tumor and plasma concentrations for eight tumor models (three cell line xenografts and five PDTX models) and a significant correlation was found between tumor concentration and response. Additionally, we show that tumor concentration is approximately fourfold lower in tumors that express P-gp, verified by western blot. Our in vitro and in vivo data strongly suggests that PF-309 efficacy is influenced by the expression of tumor P-gp.

Molecular conformations, interactions, and properties associated with drug efficiency and clinical performance among VEGFR TK inhibitors.

Analyses of compounds in clinical development have shown that ligand efficient-molecules with privileged physical properties and low dose are less likely to fail in the various stages of clinical testing, have fewer postapproval withdrawals, and are less likely to receive black box safety warnings. However, detailed side-by-side examination of molecular interactions and properties within single drug classes are lacking. As a class, VEGF receptor tyrosine kinase inhibitors (VEGFR TKIs) have changed the landscape of how cancer is treated, particularly in clear cell renal cell carcinoma, which is molecularly linked to the VEGF signaling axis. Despite the clear role of the molecular target, member molecules of this validated drug class exhibit distinct clinical efficacy and safety profiles in comparable renal cell carcinoma clinical studies. The first head-to-head randomized phase III comparative study between active VEGFR TKIs has confirmed significant differences in clinical performance [Rini BI, et al. (2011) Lancet 378:193-1939]. To elucidate how fundamental drug potency-efficiency is achieved and impacts differentiation within the VEGFR TKI class, we determined potencies, time dependence, selectivities, and X-ray structures of the drug-kinase complexes using a VEGFR2 TK construct inclusive of the important juxtamembrane domain. Collectively, the studies elucidate unique drug-kinase interactions that are dependent on distinct juxtamembrane domain conformations, resulting in significant potency and ligand efficiency differences. The identified structural trends are consistent with in vitro measurements, which translate well to clinical performance, underscoring a principle that may be broadly applicable to prospective drug design for optimal in vivo performance.