Sulfoximine-substituted trifluoromethylpyrimidine analogs as inhibitors of proline-rich tyrosine kinase 2 (PYK2) show reduced hERG activity.

The synthesis, in vitro properties, and in vivo pharmacokinetics for a series of sulfoximine-substituted trifluoromethylpyrimidines as inhibitors of proline-rich tyrosine kinase, a target for the possible treatment of osteoporosis, are described. These compounds were prepared as surrogates of the corresponding sulfone compound 1. Sulfone 1 was an attractive PYK2 lead compound; however, subsequent studies determined this compound possessed high dofetilide binding, which is an early indicator of cardiovascular safety. Surprisingly, the corresponding sulfoximine analogs displayed significantly lower dofetilide binding, which, for N-methylsulfoximine (S)-14a, translated to lower activity in a patch clamp hERG K(+) ion channel screen. In addition, compound (S)-14a shows good oral exposure in a rat pharmacokinetic model.

Trifluoromethylpyrimidine-based inhibitors of proline-rich tyrosine kinase 2 (PYK2): structure-activity relationships and strategies for the elimination of reactive metabolite formation.

The synthesis and SAR for a series of diaminopyrimidines as PYK2 inhibitors are described. Using a combination of library and traditional medicinal chemistry techniques, a FAK-selective chemical series was transformed into compounds possessing good PYK2 potency and 10- to 20-fold selectivity against FAK. Subsequent studies found that the majority of the compounds were positive in a reactive metabolite assay, an indicator for potential toxicological liabilities. Based on the proposed mechanism for bioactivation, as well as a combination of structure-based drug design and traditional medicinal chemistry techniques, a follow-up series of PYK2 inhibitors was identified that maintained PYK2 potency, FAK selectivity and HLM stability, yet were negative in the RM assay.

The contribution of E2F-regulated transcription to Drosophila PCNA gene function.

E2F proteins control cell cycle progression by predominantly acting as either activators or repressors of transcription. How the antagonizing activities of different E2Fs are integrated by cis-acting control regions into a final transcriptional output in an intact animal is not well understood. E2F function is required for normal development in many species, but it is not completely clear for which genes E2F-regulated transcription provides an essential biological function. To address these questions, we have characterized the control region of the Drosophila PCNA gene. A single E2F binding site within a 100-bp enhancer is necessary and sufficient to direct the correct spatiotemporal program of G1-S-regulated PCNA expression during development. This dynamic program requires both E2F-mediated transcriptional activation and repression, which, in Drosophila, are thought to be carried out by two distinct E2F proteins. Our data suggest that functional antagonism between these different E2F proteins can occur in vivo by competition for the same binding site. An engineered PCNA gene with mutated E2F binding sites supports a low level of expression that can partially rescue the lethality of PCNA null mutants. Thus, E2F regulation of PCNA is dispensable for viability, but is nonetheless important for normal Drosophila development.

Phosphoproteomic characterization of PYK2 signaling pathways involved in osteogenesis.

The PYK2 tyrosine kinase is a negative regulator of bone formation, but aside from the requirement for PYK2 kinase activity there has been little progress toward understanding of the molecular mechanism involved in this function. To gain insight into the signaling pathways modulated by PYK2 we sought to identify PYK2 substrates. Challenges inherent to a quantitative phosphoproteomic analysis for non-receptor tyrosine kinases were overcome by employing an inducible PYK2 overexpression system in NIH3T3 cells in combination with a selective PYK2 inhibitor. The identification of a number of known PYK2 substrates and interacting partners validated the methodology. Results of the inducible cell system were extended to a cell model of osteogenesis, examining the effect of the PYK2 inhibitor on the phosphorylation state of targets identified in the phosphoproteomic study. Consistent with phosphoproteomic analysis, increased osteogenesis associated with a selective PYK2 inhibitor was accompanied by reduced phosphorylation of paxillin, Gab1 and p130(Cas), along with reduction of phosphorylation levels of the Met activation loop. These results further confirmed the utility of the methodology and point to a previously unknown bi-directional activation pathway between PYK2 and Met.

Structural characterization of proline-rich tyrosine kinase 2 (PYK2) reveals a unique (DFG-out) conformation and enables inhibitor design.

Proline-rich tyrosine kinase 2 (PYK2) is a cytoplasmic, non-receptor tyrosine kinase implicated in multiple signaling pathways. It is a negative regulator of osteogenesis and considered a viable drug target for osteoporosis treatment. The high-resolution structures of the human PYK2 kinase domain with different inhibitor complexes establish the conventional bilobal kinase architecture and show the conformational variability of the DFG loop. The basis for the lack of selectivity for the classical kinase inhibitor, PF-431396, within the FAK family is explained by our structural analyses. Importantly, the novel DFG-out conformation with two diarylurea inhibitors (BIRB796, PF-4618433) reveals a distinct subclass of non-receptor tyrosine kinases identifiable by the gatekeeper Met-502 and the unique hinge loop conformation of Leu-504. This is the first example of a leucine residue in the hinge loop that blocks the ATP binding site in the DFG-out conformation. Our structural, biophysical, and pharmacological studies suggest that the unique features of the DFG motif, including Leu-504 hinge-loop variability, can be exploited for the development of selective protein kinase inhibitors.

Proline-rich tyrosine kinase 2 regulates osteoprogenitor cells and bone formation, and offers an anabolic treatment approach for osteoporosis.

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.

Cell-autonomous roles of the ecdysoneless gene in Drosophila development and oogenesis.

Steroid signaling underlies developmental processes in animals. Mutations that impair steroidogenesis in the fruit fly Drosophila melanogaster provide tools to dissect steroid hormone action genetically. The widely used temperature-sensitive mutation ecdysoneless(1) (ecd(1)) disrupts production of the steroid hormone ecdysone, and causes developmental and reproductive defects. These defects cannot be satisfactorily interpreted without analysis of the ecd gene. Here, we show that ecd encodes an as yet functionally undescribed protein that is conserved throughout eukaryotes. The ecd(1) conditional allele contains an amino acid substitution, whereas three non-conditional larval lethal mutations result in truncated Ecd proteins. Consistent with its role in steroid synthesis, Ecd is expressed in the ecdysone-producing larval ring gland. However, development of ecd-null early larval lethal mutants cannot be advanced by Ecd expression targeted to the ring gland or by hormone feeding. Cell-autonomous ecd function, suggested by these experiments, is evidenced by the inability of ecd(-) clones to survive within developing imaginal discs. Ecd is also expressed in the ovary, and is required in both the follicle cells and the germline for oocyte development. These defects, induced by the loss of ecd, provide the first direct evidence for a cell-autonomous function of this evolutionarily conserved protein.