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PKC isoforms are involved in transmembrane signal transduction pathways, regulating a variety of cellular functions such as growth, differentiation, tumor promotion and apoptosis. Individual PKC isoforms differ in their expression patterns and substrate specificities, strongly suggesting that each isoform may be involved in distinct regulatory processes within the cell. Activation of PKC isoforms occurs in a number of pathological states. Availability of isoform-selective PKC inhibitors may provide important pharmacological agents to better define the physiological and pathological functions of each isoform. Comparative bioinformatics analysis of homologous PKC enzymes was used to study molecular determinants of selective inhibition. Subfamily specific positions (SSP) – variable amino acid residues with a tendency to be conserved only within a subfamily of enzymes, but different between subfamilies - are responsible for functional divergence within families of homologous enzymes. A library of human PKC catalytic domain structures was created using files from PDB for PKCα, PKCβ and PKCθ and homology modeling to predict three-dimensional representations of PKCγ, PKCδ, PKCε and PKCη. Multiple sequence alignment of 89 PKC enzymes from different species was created. Both conserved and specific positions in PKC family of enzymes were identified and further explored using molecular docking. Molecular docking and molecular dynamic simulation of PKC complexes with known inhibitors LY333531, Gö6978, GF109203x, Ro 31-8220 were used to train a set of geometry filters of productive binding of the inhibitor in different isoforms based on its orientation to known catalytically important conserved residues Val-423, Glu-421 and Thr-404 (in PKCβ). In silico library of 54 new potential selective inhibitors was generated to study the role of subfamily-specific positions in specific binding among different PKC isoforms using molecular docking. Ala-483 in PKCα and PKCβ was found to be the most promising specific position due to evident stereo chemical differences with homologous threonine in PKCγ. Molecular docking shows that introduction of methylamine group instead of indole ring in Gö6978 inhibitor disables the binding to PKCγ isoform (Fig.1). Thus, bioinformatics analysis and molecular modeling reveal subfamily-specific position responsible for discrimination of inhibitor binding to ATP-binding site of human protein kinases and outline the new computational strategy in modeling selective inhibitors of human PKC enzymes.