Hefti1 FF, Rosenthal1 A, Walicke1 PA, Wyatt S, Vergara1 G, Shelton DL, Davies AM. become an important pharmacological target for cancer as well as pain. TrkB is usually involved in calcium signaling2 and membrane depolarization2 and TrkC is usually important for sensory ganglia5 and cardiac health.5 More specifically, the TrkA receptor has been extensively implicated in driving tumor pathology,6C10 inflammatory and neuropathic pain,11C15 as well as chemosensitization.16 Therefore, the pharmacological modulation of TrkA represents a novel approach for the treatment of cancer as well as cancer associated pain and chemotherapy resistance. Efforts to inhibit the TrkA kinase have been made in the medical center,17 yet an FDA approved TrkA inhibitor has not yet been developed. Inhibitor progress has focused on creating novel chemotypes that are active on Trk receptors and validating and Trk biology.18 The generation of TrkA inhibitors has TAK-715 been conducted by diminishing the activity for other closely related receptor tyrosine kinases (RTKs) and enhancing TrkA activity.19 Additionally to generate TrkA inhibitors, modification of staurosporine has been completed.20 In another approach, TrkA inhibitors have been developed through the screening of a kinase library.21 Although some progress has been made on Trk inhibition, additional RTK selective inhibitors are still needed. In an orthogonal approach to develop TrkA inhibitors, we utilized a computational screening assay.22 A kinase-directed virtual library was screened against the Trk kinase crystal structure and identified compound 1 as a novel active CD4 (Fig. 1). We decided to progress forward with 1 because of the novelty of the pyrazine moiety. The majority of all pyrazine-based inhibitors are aminopyrazines either directed at non-receptor tyrosine kinases23 or serine/threonine kinases.24 Therefore, the investigation of pyrazine-based inhibitors for TrkA could produce compounds with interesting potency and/or selectivity profiles. Open in a separate windows Fig. 1 Trk computational hit from a TAK-715 kinase-directed virtual library. Results and conversation To validate the computational screen, compound 1 was synthesized utilizing a developed synthetic protocol (Plan 1). 2,6-Dichloropyrazine was reacted with 3-aminophenol to generate intermediate 1a. Triphosgene was then utilized to form the isocyanate, which was subjected to nucleophilic addition from 3-trifluoromethyl aniline to generate computational hit 1. Compound 1 was screened against TrkA and was found active (TrkA IC50 = 3.5 M) (Table 1). The inhibitor was modeled to identify possible points for optimization (Fig. 2). Compound 1 was found to hydrogen bond at the kinase hinge through a weak hydrogen bond (~4 ?) with M620. Additionally, 1 was predicted to form hydrogen bonds with D697 from the DFG (aspartic acid, phenylalanine, glycine) motif and E588 from the C-helix, and enter the DFG-out allosteric pocket. Compound 1 was not predicted to interact with the gatekeeper residue, F617. Open in a separate window Fig. 2 Computational binding mode of 1 1 in TrkC.18 PDB accession number 3V5Q. Predicted ligand/receptor hydrogen bonds are denoted with black dotted lines. 1 is not predicted to piCpi stack with F617. Open in a separate window Scheme 1 (a) values. Compounds that achieve less than ?10 kcal mol?1 affinity are progressed to an inhibitor-candidate stage. At this stage, predicted binding modes are investigated and compounds that contain prototypical drug-like properties are synthesized and evaluated biochemically. Procedure for computational modeling studies Computational modeling studies were completed using Auto-Dock Vina,22 AutoDock Tools, TAK-715 and Discovery Studio 3.5. Using AutoDock Tools, the Trk model was prepared as follows: (1) all water was removed, (2) all.