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 ...

Isoform-specific signaling of Akt, a major signaling hub and a prominent therapeutic target, remained poorly defined until recently. Subcellular distribution, tissue-specific expression, substrate specificity, and posttranslational modifications are believed to underlie isoform-specific signaling of Akt. The studies reported here show inhibition of Akt2 activity under physiologically relevant conditions of oxidation created by PDGF-induced reactive oxygen species. Combined MS and functional assays identified Cys124 located in the linker region between the N-terminal pleckstrin homology domain and the catalytic kinase domain as one of the unique regulatory redox sites in Akt2 with functional consequence on PDGF-stimulated glucose uptake. A model is proposed describing the consequence of increased endogenous oxidation induced by extracellular cues such as PDGF on Akt2 activity.disulfide | receptor tyrosine kinase | DCF | DCP-Bio1 P KB/Akt is a major signaling hub between cytokine, growth factor, and integrin signaling pathways of consequence to many biological processes. Energy storage, protein synthesis, cell survival and growth, cell cycle progression, and cell death are differentially regulated by the three known isoforms of Akt kinase: Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ (1). Although the molecular features underlying isoform-specific functional predominance are largely unknown, hypotheses to explain Akt isoform specificity include selective interactions with substrates and/ or binding partners, tissue specificity, subcellular location, and temporal changes in activation profiles of Akt isoforms (2). Posttranslational modifications may contribute to this isoform-specific signaling, but reports of modifications are lacking.Reactive oxygen species (ROS) are integral to cytokine and growth factor signaling; ROS generation in response to these extracellular cues is well documented (3). Earlier reports, in particular from Sundaresan et al. (4), showed ROS generation in response to PDGF stimulation of vascular smooth muscle cells and suggested that H 2 O 2 can relay redox signals to regulate physiological signaling in response to growth factors. More recently, it has been shown that PrxI phosphorylation at Y194 by Src family tyrosine kinases inhibited PrxI and resulted in H 2 O 2 accumulation at the cellular membrane where receptor tyrosine kinase activation occurs (5). Unanswered questions include which proteins are oxidized by receptor tyrosine kinase-induced ROS, which specific cysteine site(s) undergo oxidation, and the consequence of oxidation on activity of these proteins and propagation of receptor tyrosine kinase signaling. Recently, the synthesis of several dimedone-based chemoselective reagents capable of specific labeling of sulfenic acid oxidized proteins was reported; these reagents allow for specific enrichment and identification of oxidized proteins (6-10). In this study, we used a biotin-tagged 1,3-cyclohexadione derivative, DCP-Bio1 (Fig. 1A), to investigate isoform-specific effects of PDGF-induced ...

β-ketoesters are robust probes for labeling sulfenic acid (−SOH) proteins allowing quantitative cleavage of the tag for improved analysis of the labeled peptides by MS.

The rSCC-61/SCC-61 model provides the opportunity for future investigations of the redox-regulated mechanisms of response to combined radiation and Erlotinib in a preclinical setting.

The perception of reactive oxygen species has evolved over the past decade from agents of cellular damage to secondary messengers which modify signaling proteins in physiology and the disease state (e.g., cancer). New protein targets of specific oxidation are rapidly being identified. One emerging class of redox modification occurs to the thiol side chain of cysteine residues which can produce multiple chemically distinct alterations to the protein (e.g., sulfenic/sulfinic/sulfonic acid, disulfides). These post-translational modifications (PTM) are shown to affect the protein structure and function. Because redox-sensitive proteins can traffic between subcellular compartments that have different redox environments, cysteine oxidation enables a spatio-temporal control to signaling. Understanding ramifications of these oxidative modifications to the functions of signaling proteins is crucial for understanding cellular regulation as well as for informed-drug discovery process. The effects of EGFR oxidation of Cys797 on inhibitor pharmacology are presented to illustrate the principle. Taken together, cysteine redox PTM can impact both cell biology and drug pharmacology.