Potent and Selective Disruption of Protein Kinase D Functionality by a Benzoxoloazepinolone

Abstract

Protein kinase D (PKD) is a novel family of serine/threonine kinases targeted by the second messenger diacylglycerol. It has been implicated in many important cellular processes and pathological conditions. However, further analysis of PKD in these processes is severely hampered by the lack of a PKD-specific inhibitor that can be readily applied to cells and in animal models. We now report the discovery of the first potent and selective cell-active small molecule inhibitor for PKD, benzoxoloazepinolone (CID755673). This inhibitor was identified from the National Institutes of Health small molecule repository library of 196,173 compounds using a human PKD1 (PKC)-based fluorescence polarization high throughput screening assay. CID755673 suppressed half of the PKD1 enzyme activity at 182 nM and exhibited selective PKD1 inhibition when compared with AKT, polo-like kinase 1 (PLK1), CDK activating kinase (CAK), CAMKII␣, and three different PKC isoforms. Moreover, it was not competitive with ATP for enzyme inhibition. In cellbased assays, CID755673 blocked phorbol ester-induced endogenous PKD1 activation in LNCaP cells in a concentration-dependent manner. Functionally, CID755673 inhibited the known biological actions of PKD1 including phorbol ester-induced class IIa histone deacetylase 5 nuclear exclusion, vesicular stomatitis virus glycoprotein transport from the Golgi to the plasma membrane, and the ilimaquinone-induced Golgi fragmentation. Moreover, CID755673 inhibited prostate cancer cell proliferation, cell migration, and invasion. In summary, our findings indicate that CID755673 is a potent and selective PKD1 inhibitor with valuable pharmacological and cell biological potential. Protein kinase D (PKD)3 belongs to a subfamily of the Ca 2ϩ / calmodulin-dependent kinases (CAMKs) (1). PKD is a novel target of the second messenger diacylglycerol and phorbol esters, the natural products from plants and potent tumor promoters in mouse skin (2). Three isoforms of PKD (PKD1, -2, and -3) have been identified, which share high sequence homology (3-6). The regulatory domain of PKD contains a C1 domain that binds diacylglycerol/phorbol esters with high affinity and a PH domain that mediates protein-protein interactions. The entire regulatory domain appears to exert a negative effect on catalytic activity, possibly serving as an autoinhibitory domain for PKD (7). The activity of PKD is controlled through a protein kinase C (PKC)-dependent mechanism (8). PKC is the primary target of diacylglycerol/phorbol esters and it activates PKD by directly binding and phosphorylating PKD on two serine residues in the activation loop. In most cellular systems examined, PKD is an effector of selective PKC isoforms, acting in a canonical PKC/PKD pathway that leads to a unique set of biological responses including cell proliferation, survival, protein transport, and immune responses (2, 9).PKD regulates many fundamental cellular functions and has been implicated in the pathogenesis of several diseases. PKD is a key regulator of protein transpo...

Thyroid hormone induced angiogenesis through the integrin αvβ3/protein kinase D/histone deacetylase 5 signaling pathway

“…The wound was imaged at different intervals as indicated using an inverted phase-contrast microscope. The wound gap was measured on the images and the distance of migration was calculated as wound gap at 0 hKwound gap at x h, where x is the time point of measurement, and the average woundhealing time was calculated based on at least nine determinations of the wound (Sharlow et al 2008).…”

Profiling the NIH Small Molecule Repository for Compounds That Generate H2O2by Redox Cycling in Reducing Environments

“…15,20,21,23,24 Detailed assay descriptions, protocols, and data analysis procedures are uploaded together with the data to the PubChem database and are thus in the public domain. 15,[17][18][19][20][21][22][23][24] The presence of RCCs in any library that is to be screened in buffers containing DTT or TCEP against protein targets that are susceptible to oxidation has serious negative consequences for the probe and/or lead generation process: primary HTS campaigns may exhibit high hit rates from Sigma-Aldrich (St. Louis, MO); Hank's balanced salt solution (HBSS, catalog # SH30268.02) was from Hyclone (Logan, UT); sodium hydroxide (catalog # VW6720-1) was from VWR (West Chester, PA); and dimethyl sulfoxide (DMSO, 99.9% HPLC grade under argon, CAS # 67-68-5) was from Alfa Aesar (Ward Hill, MA). All other materials were reagent grade from Sigma-Aldrich.…”

“…15,[17][18][19][20][21] The NIH SMR distributes copies of this library to the centers of the network for screening against selected peer-reviewed assays that have been submitted by investigators throughout the broader scientifi c community. 15,[20][21][22][23] The screening centers develop, optimize, and adapt the assays for HTS, screen the NIH SMR library to identify actives, perform counter screens and secondary assay to confi rm hits, and conduct limited analog synthesis efforts to confi rm chemical structures, generate analogs with improved physiochemical properties, such as water solubility, and to explore the structure activity relationships of the probe compounds identifi ed. 15,20,21,23,24 Detailed assay descriptions, protocols, and data analysis procedures are uploaded together with the data to the PubChem database and are thus in the public domain.…”

“…15,[20][21][22][23] The screening centers develop, optimize, and adapt the assays for HTS, screen the NIH SMR library to identify actives, perform counter screens and secondary assay to confi rm hits, and conduct limited analog synthesis efforts to confi rm chemical structures, generate analogs with improved physiochemical properties, such as water solubility, and to explore the structure activity relationships of the probe compounds identifi ed. 15,20,21,23,24 Detailed assay descriptions, protocols, and data analysis procedures are uploaded together with the data to the PubChem database and are thus in the public domain. 15,[17][18][19][20][21][22][23][24] The presence of RCCs in any library that is to be screened in buffers containing DTT or TCEP against protein targets that are susceptible to oxidation has serious negative consequences for the probe and/or lead generation process: primary HTS campaigns may exhibit high hit rates from Sigma-Aldrich (St. Louis, MO); Hank's balanced salt solution (HBSS, catalog # SH30268.02) was from Hyclone (Logan, UT); sodium hydroxide (catalog # VW6720-1) was from VWR (West Chester, PA); and dimethyl sulfoxide (DMSO, 99.9% HPLC grade under argon, CAS # 67-68-5) was from Alfa Aesar (Ward Hill, MA).…”

Inducible Inhibition of Gβγ Reveals Localization-dependent Functions at the Plasma Membrane and Golgi

“…Here, we investigated a role for ␤␥ in Golgi fragmentation induced by ilimaquinone or nocodazole. Ilimaquinone treatment of cells has been demonstrated to cause Golgi fragmentation in a PKD-dependent manner, although the direct targets of ilimaquinone remain elusive (32). An early report also suggested that ilimaquinone-induced Golgi fragmentation might also require ␤␥, and thus we sought to characterize this using the inducible ␤␥ inhibitor (23).…”