This approach provided both ATP-competitive active site, and noncompetitive, presumably allosteric site inhibitors. For example, CID755673 and kb-NB142-70 inhibited PKD1 in vitro in the low nanomolar range and suppressed PKD1 autophosphorylation at Ser916 in LNCaP prostate cancer cells in the low micromolar range. CID1893668, CID2011756, and CID5389142 also inhibited phorbol ester-induced endogenous PKD1 activation in LNCaP prostate cancer cells in a concentration-dependent manner. Using a small, targeted library of diverse VX-661 biological activity kinase inhibitors, we have now identified twenty-eight new ATP-competitive inhibitors of PKD. Among these, eight displayed excellent selectivity towards PKD with little or no inhibitory activity for CAMK or PKC, two structurally and functionally closely related kinases. Additionally, we have developed a homology model of PKD and investigated at the molecular level the interactions of these PKD inhibitors in the active site of the kinase. The newly discovered PKD inhibitors hold promise for the further development of clinically effective PKD-specific inhibitors. The specificity of the newly identified PKD1 inhibitors was assessed using in vitro kinase assays against PKC and CAMK, two families of kinases functionally and structurally related to PKD. PKC, like PKD, is a DAG/phorbol ester receptor and a direct activator of PKD. The PKC/PKD pathway is a key signaling pathway that accounts for PKD-mediated cellular responses. The kinase domain of PKD bears high sequence homology to the CAMK family of kinases. Functionally, CAMK also partially overlaps with PKD in regulation of certain substrates and signaling events; for example, both kinases phosphorylate class IIa HDACs and have been implicated in cardiac hypertrophy. Thus, selectivity against these two related kinase families is a highly desirable feature of a specific PKD NAN-190 (hydrobromide) inhibitor. In this study, we counter-screened the twenty-eight PKD1 inhibitory agents for inhibition of PKCa, PKCd and CAMKIIa in order to get an initial profile for the potential PKD selectivity, since these are the functionally most closely related kinases. The compounds were examined at concentration. To further explore the mechanism of action of these active PKD1 compounds, molecular modeling technologies were utilized to investigate the putative binding modes using our reported protocols. The three-dimensional structure of PKD1 and the catalytic domain which consists of two lobes and an intervening linker was built based on high-resolution crystal structures of homologues. The sequence of the PKD1 kinase domain, which extends from Glu587 to Ser835, was submitted to the I-TASSER server for 3D structure prediction. Protein structures activated serine-threonine kinase were chosen by ITASSER as the templates in the modeling. The five most reliable models respectively were used for docking. As illustrated despite moderate sequence identities between PKD1 and their templates, their 3D structures present similar topologies and overall shapes. Specifically, conserved structure elements of the kinase domain fold into a bi-lobed catalytic core structure, with ATP binding in a deep cleft located between these two lobes. These observations reinforced our strategy to utilize the structural conservation in the PKD1 kinase domain to identify the key residues for inhibitor-protein interactions. In this study, a small focused library of kinase inhibitors was screened against PKD1 and revealed new scaffolds for selective disruption of ATP binding to this serine/threonine kinase. Starting with 235 compounds, twenty-eight potent inhibitors were further investigated for selectivity against the closely related PKCs and CAMKs, resulting in six highly selective PKD1 inhibitors. Two new scaffold types were represented in the final lead structures, and a representative member of each type was further evaluated in secondary assays. The concentration dependence in vitro and ex vivo as well as the kinetic profile was determined. The selectivity of 4-azaindoles was evaluated by a kinetic profile of 353 diverse protein kinases in the human kinome.
