The occurrence of idiosyncratic adverse drug reactions during late clinical trials or after a drug has been released can lead to a severe restriction in its use and even in its withdrawal. Metabolic activation of relatively inert functional groups to reactive electrophilic intermediates is considered to be an obligatory event in the etiology of many drug-induced adverse reactions. Therefore, a thorough examination of the biochemical reactivity of functional groups/structural motifs in all new drug candidates is essential from a safety standpoint. A major theme attempted in this review is the comprehensive cataloging of all of the known bioactivation pathways of functional groups or structural motifs commonly utilized in drug design efforts. Potential strategies in the detection of reactive intermediates in biochemical systems are also discussed. The intention of this review is not to "black list" functional groups or to immediately discard compounds based on their potential to form reactive metabolites, but rather to serve as a resource describing the structural diversity of these functionalities as well as experimental approaches that could be taken to evaluate whether a "structural alert" in a new drug candidate undergoes bioactivation to reactive metabolites.
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 (k inact /K i in the range 10), despite their low specific reactivity (k inact ≤ 2.1 × 10), which is compensated for by high binding affinities (K i < 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 EGFRCys 797 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.cysteine oxidation | protein kinase | signaling | capture period | warhead interactions R eceptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR) tyrosine kinase, catalyze protein phosphorylation reactions to trigger signaling networks. Oncogenic activating mutations of EGFR lead to aberrant signaling for a subpopulation (10-30%) of nonsmall cell lung cancer patients (1). These mutations reside primarily in two regions of the EGFR catalytic domain [namely, the in-frame deletion mutations (e.g., Del746-750) preceding the N-terminal Cα-helix (exon 19) and the C-terminal activation loop L858R mutation (exon 21)] (2). Patients harboring these activating mutations usually respond to reversible ATP competitive drugs (e.g., erlotinib and gefitinib), but their effectiveness is limited by the emergence of drug resistance, in part, through an additional active site mutation (T790M and gatekeeper residue) in 50% of the responsive patients (3).A second generation of drug discovery dating back to the 1990s resulted in inhibitors that incorporate a chemically reactive Michael Acceptor (MA) electrophile (warhead) to target a cysteine nucleophile (EGFR-Cys 797 ) in the hinge region of the ATP binding cleft (4). The ...
Inhibition of acetyl-CoA carboxylase (ACC), with its resultant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation, has the potential to favorably affect the multitude of cardiovascular risk factors associated with the metabolic syndrome. To achieve maximal effectiveness, an ACC inhibitor should inhibit both the lipogenic tissue isozyme (ACC1) and the oxidative tissue isozyme (ACC2). Herein, we describe the biochemical and acute physiological properties of CP-610431, an isozyme-nonselective ACC inhibitor identified through high throughput inhibition screening, and CP-640186, an analog with improved metabolic stability. CP-610431 inhibited ACC1 and ACC2 with IC 50 s of ϳ50 nM. Inhibition was reversible, uncompetitive with respect to ATP, and non-competitive with respect to bicarbonate, acetyl-CoA, and citrate, indicating interaction with the enzymatic carboxyl transfer reaction. CP-610431 also inhibited fatty acid synthesis, triglyceride (TG) synthesis, TG secretion, and apolipoprotein B secretion in HepG2 cells (ACC1) with EC 50 s of 1.6, 1.8, 3.0, and 5.7 M, without affecting either cholesterol synthesis or apolipoprotein CIII secretion. CP-640186, also inhibited both isozymes with IC 50 s of ϳ55 nM but was 2-3 times more potent than CP-610431 in inhibiting HepG2 cell fatty acid and TG synthesis. CP-640186 also stimulated fatty acid oxidation in C2C12 cells (ACC2) and in rat epitrochlearis muscle strips with EC 50 s of 57 nM and 1.3 M. In rats, CP-640186 lowered hepatic, soleus muscle, quadriceps muscle, and cardiac muscle malonyl-CoA with ED 50 s of 55, 6, 15, and 8 mg/kg. Consequently, CP-640186 inhibited fatty acid synthesis in rats, CD1 mice, and ob/ob mice with ED 50 s of 13, 11, and 4 mg/kg, and stimulated rat whole body fatty acid oxidation with an ED 50 of ϳ30 mg/kg. Taken together, These observations indicate that isozyme-nonselective ACC inhibition has the potential to favorably affect risk factors associated with the metabolic syndrome.
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