First generation EGFR TKIs (gefitinib, erlotinib) provide significant clinical benefit for NSCLC cancer patients with oncogenic EGFR mutations. Ultimately, these patients' disease progresses, often driven by a second-site mutation in the EGFR kinase domain (T790M). Another liability of the first generation drugs is severe adverse events driven by inhibition of WT EGFR. As such, our goal was to develop a highly potent irreversible inhibitor with the largest selectivity ratio between the drug-resistant double mutants (L858R/T790M, Del/T790M) and WT EGFR. A unique approach to develop covalent inhibitors, optimization of reversible binding affinity, served as a cornerstone of this effort. PF-06459988 was discovered as a novel, third generation irreversible inhibitor, which demonstrates (i) high potency and specificity to the T790M-containing double mutant EGFRs, (ii) minimal intrinsic chemical reactivity of the electrophilic warhead, (iii) greatly reduced proteome reactivity relative to earlier irreversible EGFR inhibitors, and (iv) minimal activity against WT EGFR.

Checkpoint kinase 1 (CHK1) is a key element in the DNA damage response pathway and plays a crucial role in the S-G(2)-phase checkpoint. Inhibiting CHK1 is a therapeutic strategy involving abrogation of the G2/M mitotic checkpoint defense of tumor cells toward lethal damage induced by DNA-directed chemotherapeutic agents. To date, most CHK1 inhibition approaches have involved targeting the ATP site of this kinase. In this study, we provide crystallographic and kinetic characterization of two small molecule inhibitors that bind to an allosteric site in the proximity of the CHK1 substrate site. Analysis of kinetic and biophysical data has led to the conclusion that these small molecule allosteric site inhibitors of CHK1 are reversible and are neither ATP- nor peptide substrate-competitive. K(i) values of 1.89 and 0.15 microM, respectively, have been determined for these compounds using a mixed inhibitor kinetic analysis. Cocrystal structures of the inhibitors bound to CHK1 reveal an allosteric site, unique to CHK1, located in the C-terminal domain and consisting of a shallow groove linked to a small hydrophobic pocket. The pocket displays induced fit characteristics in the presence of the two inhibitors. These findings establish the potential for the development of highly selective CHK1 inhibitors.

Rapid mutations of proteins that are targeted in cancer therapy often lead to drug resistance. Often, the mutation directly affects a drug's binding site, effectively blocking binding of the drug, but these mutations can have other effects such as changing the protein turnover half-life. Utilizing SILAC MS, we measured the cellular turnover rates of an important non-small cell lung cancer target, epidermal growth factor receptor (EGFR). Wild-type (WT) EGFR, EGFR with a single activating mutant (Del 746–750 or L858R), and the drug-resistant double mutant (L858R/T790M) EGFR were analyzed. In non-small cell lung cancer cell lines, EGFR turnover rates ranged from 28 hours in A431 cells (WT) to 7.5 hours in the PC-9 cells (Del 746–750 mutant). The measurement of EGFR turnover rate in PC-9 cells dosed with irreversible inhibitors has additional complexity due to inhibitor effects on cell viability and results were reported as a range. Finally, essential amino acid recycling (K and R) was measured in different cell lines. The recycling was different in each cell line, but the overall inclusion of the effect of amino acid recycling on calculating EGFR turnover rates resulted in a 10–20% reduction in rates.

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The African clawed frog Xenopus laevis is an important model organism for studies in developmental and cell biology, including cell-signaling. However, our knowledge of X. laevis protein posttranslational modifications remains scarce. Here, we used a mass spectrometry-based approach to survey the phosphoproteome of this species, compiling a list of 3225 phosphosites. We used this resource to study the conservation between the phosphoproteomes of X. laevis and 13 other species. We found that the degree of conservation of phosphorylation across species is predictive of sites with known molecular function, kinase interactions and functionally relevant phospho-regulatory interactions. In addition, using comparative protein structure models, we find that phosphosites within structured domains tend to be located at positions with high conformational flexibility. A fraction of sites appear to occur in inaccessible positions and have the potential to regulate protein conformation.