Characterization of non-nitrocatechol pan and isoform specific catechol-O-methyltransferase inhibitors and substrates.

Reduced dopamine neurotransmission in the prefrontal cortex has been implicated as causal for the negative symptoms and cognitive deficit associated with schizophrenia; thus, a compound which selectively enhances dopamine neurotransmission in the prefrontal cortex may have therapeutic potential. Inhibition of catechol-O-methyltransferase (COMT, EC 2.1.1.6) offers a unique advantage, since this enzyme is the primary mechanism for the elimination of dopamine in cortical areas. Since membrane bound COMT (MB-COMT) is the predominant isoform in human brain, a high throughput screen (HTS) to identify novel MB-COMT specific inhibitors was completed. Subsequent optimization led to the identification of novel, non-nitrocatechol COMT inhibitors, some of which interact specifically with MB-COMT. Compounds were characterized for in vitro efficacy versus human and rat MB and soluble (S)-COMT. Select compounds were administered to male Wistar rats, and ex vivo COMT activity, compound levels in plasma and cerebrospinal fluid (CSF), and CSF dopamine metabolite levels were determined as measures of preclinical efficacy. Finally, novel non-nitrocatechol COMT inhibitors displayed less potent uncoupling of the mitochondrial membrane potential (MMP) compared to tolcapone as well as nonhepatotoxic entacapone, thus mitigating the risk of hepatotoxicity.

Competitive binding of protein kinase Calpha to membranes and Rho GTPases.

Previously, we have shown that protein kinase C (PKC) forms a direct high-affinity, isozyme-specific and membrane lipid-independent interaction with Rho GTPases [Slater, S. J., Seiz, J. L., Stagliano, B. A., and Stubbs, C. D. (2001) Biochemistry 40, 4437-4445]. Since the cellular activation of PKCalpha involves an initial translocation from cytosolic to membrane compartments, the present study investigates the interdependence between the direct protein-protein interaction of PKCalpha with the Rho GTPase, Cdc42, and the protein-lipid interactions of PKCalpha with membranes. It was hypothesized that the interaction of PKCalpha with membrane-bound Cdc42 would contribute to the overall membrane-binding affinity of the kinase by providing an additional anchor. However, it was found that the incorporation of isoprenylated Cdc42 into membranes resulted in an apparent decrease in the membrane-binding affinity of PKCalpha, whereas the association of PKCbetaI, PKCdelta, PKCepsilon, and PKCzeta was each unaffected. The presence of membrane-bound Cdc42 resulted in a rightward shift in both the PS- and Ca2+-concentration response curves for PKCalpha membrane association and for the ensuing activation, whereas the maximal levels of binding and activation attained at saturating PS and Ca2+ concentrations were in each case unaffected. Overall, these findings suggest that PKCalpha undergoes a isozyme-specific interaction with membrane-bound Cdc42 to form a PKCalpha-Cdc42 complex, which possesses a membrane-binding affinity that is reduced relative to that of the individual components due to competition between Cdc42 and PS/Ca2+ for binding to PKCalpha. Consistent with this, it was found that the interaction of PKCalpha with membrane-bound Cdc42 was accompanied by the physical dissociation of the PKCalpha-Cdc42 complex from membranes. Thus, the study provides a novel mechanism by which the membrane association and activation of PKCalpha and Cdc42 may be regulated by competing protein-protein and protein-lipid interactions.

Regulation of PKC alpha activity by C1-C2 domain interactions.

In this study, the role of interdomain interactions involving the C1 and C2 domains in the mechanism of activation of PKC was investigated. Using an in vitro assay containing only purified recombinant proteins and the phorbol ester, 4 beta-12-O-tetradecanoylphorbol-13-acetate (TPA), but lacking lipids, it was found that PKC alpha bound specifically, and with high affinity, to a alpha C1A-C1B fusion protein of the same isozyme. The alpha C1A-C1B domain also potently activated the isozyme in a phorbol ester- and diacylglycerol-dependent manner. The level of this activity was comparable with that resulting from membrane association induced under maximally activating conditions. Furthermore, it was found that alpha C1A-C1B bound to a peptide containing the C2 domain of PKC alpha. The alpha C1A-C1B domain also activated conventional PKC beta I, -beta II, and -gamma isoforms, but not novel PKC delta or -epsilon. PKC delta and -epsilon were each activated by their own C1 domains, whereas PKC alpha, -beta I, -beta II, or -gamma activities were unaffected by the C1 domain of PKC delta and only slightly activated by that of PKC epsilon. PKC zeta activity was unaffected by its own C1 domain and those of the other PKC isozymes. Based on these findings, it is proposed that the activating conformational change in PKC alpha results from the dissociation of intra-molecular interactions between the alpha C1A-C1B domain and the C2 domain. Furthermore, it is shown that PKC alpha forms dimers via inter-molecular interactions between the C1 and C2 domains of two neighboring molecules. These mechanisms may also apply for the activation of the other conventional and novel PKC isozymes.