Sandra E. Wilkinson

Therapeutic potential of protein kinase C inhibitors

The serine/threonine protein kinase, protein kinase C (PKC) is a family of closely related isoforms which are physiologically activated by diacylglycerol generated by the binding of a variety of agonists to their cellular receptors. Free fatty ccids may also play a role in activating PKC. The enzyme apparently mediates a wide range of signal transduction processes in cells and, therefore, inhibitors directed selectively against PKC may have wide-ranging therapeutic potential. This review highlights the evidence that inappropriate activation of PKC occurs in a number of disease states. Such evidence, however, is often seriously flawed because it relies on the use of phorbol esters, which are potent and direct PKC activators but may not mimic the physiological triggering of the enzyme in cells, or on the use of non-selective protein kinase inhibitors such as H7 and staurosporine. A new generation of bis-indolylmaleimides, derived from the lead provided by staurosporine, shows a high degree of selectivity for PKC over closely related protein kinases and such agents may provide more appropriate tools to investigate the role of PKC in cellular processes.

The effect of new potent selective inhibitors of protein kinase C on the neutrophil respiratory burst

New potent inhibitors of protein kinase C were found to inhibit protein kinase C isolated from rat brain and human neutrophils, with a large degree of selectivity over cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase. These novel compounds were potent inhibitors of the fluoride, diC8- and formyl-methionyl-leucyl-phenylalanine-mediated respiratory bursts in intact neutrophils. The opsonized zymosan-stimulated burst was only marginally affected by the compounds. These results differ from those obtained in studies with H7 and CI, (which are less potent and less specific protein kinase C inhibitors) and are consistent with the hypothesis that protein kinase C has a role in the transduction mechanism for the neutrophil oxidative burst stimulated with fluoride, formyl-methionyl-leucyl-phenylalanine and diC8.

A novel conformationally restricted protein kinase C inhibitor, Ro 31-8425, inhibits human neutrophil superoxide generation by soluble, particulate and post-receptor stimuli

A novel, bis‐indolylmaleimide, Ro 31‐8425, bearing a conformationally restricted side chain, inhibits protein kinase C isolated from rat brain and human neutrophils with a high degree of selectivity over cAMP‐dependent kinase and Ca2−/calmodulin‐dependent kinase. It also inhibits phorbol ester‐induced intracellular events known to be mediated by protein kinase C(p47 phosphorylation in intact platelets, CD3 and CD4 down‐regulation in T‐cells). Ro 31‐8425 inhibited superoxide generation in human neutrophils activated by both receptor stimuli (formyl‐methionyl‐leucylphenylalanine, opsonized zymosan. IgG and heat aggregated IgG) and post‐rcceptor stimuli (1,2‐dioctanoylglycerol and fluoride). The compound also blocked antigen driven, but not IL‐2 induced, T‐cell proliferation. These results support a central role for protein kinase C in the activation of the respiratory burst and antigen‐driven T‐cell proliferation.

Modulation of cellular processes by H7, a non-selective inhibitor of protein kinases.

H7 has been described as a potent inhibitor of protein kinase C (PKC) and has been widely used to investigate the regulatory role of this enzyme in intact cell systems. In this comparative study between H7 and the microbial alkaloid, staurosporine, we found that the former inhibited rat brain PKC and cAMP dependent protein kinase with IC50 values of 18 and 16 μM respectively whereas the latter was a much more potent inhibitor of both kinases with IC50 values of 9.5 nM and 42 nM respectively. H7, at concentrations up to 100 μM, failed to block cellular events induced by phorbol esters, agents which specifically stimulate PKC, yet was a potent inhibitor of IL-2 induced T cell proliferation with an IC50 value of 19 μM. In contrast, staurosporine was a potent inhibitor of both phorbol ester induced p47 phosphorylation in platelet (I50 value=540 nM) and also CD3 and CD4 down-regulation in T cells (I50 values 200 nM and 50 nM respectively). Staurosporine was also a potent inhibitor of IL-2 induced T cell proliferation I50 value=9 nM). These results provide a strong argument against the use of H7 to probe for PKC involvement in cellular processes.

Selective tyrosine kinase inhibitors

The tyrosine specific protein kinases (TK) are a subgroup of the largest known gene family, the kinases. Latest estimates suggest that there are over 2000 kinases encoded in the human genome [1]. TKs catalyse the transfer of phosphate to the phenolic hydroxyl of tyrosine residues in substrate proteins, consequently modifying the target protein properties. By working in concert with tyrosine phosphatases, which drive the reverse process, the TKs provide a switching system resulting in the transduction of signals from cell surface receptor to the nucleus. Inappropriate activation of TKs can lead to abnormal, dysregulated cellular proliferation and many of the known oncogenes are kinases. Naturally, there has been great interest in TKs as potential molecular targets for developing drugs for the treatment of cancer and results from the first clinical trials are now being published. Preclinical research is also focused on other therapeutic applications of TK inhibitors. This review concentrates on TK inhibitors which are either already in the clinic or likely to enter Phase I studies in the near future.

Oral, anti-inflammatory activity of a potent, selective, protein kinase C inhibitor

The protein kinase C family of enzymes is thought to be important in mediating signal transduction. Ro 31-8830 is a novel, potent inhibitor of protein kinase C, derived from the non-selective protein kinase inhibitor staurosporine. In this paper we demonstrate the selectivity of Ro 31-8830 for protein kinase C over other protein kinases and its ability to inhibit protein kinase-C-mediated events in platelets and lymphocytes. In addition, we describe a novel system for thein vivo evaluation of inhibitors of protein kinase C, and we demonstrate the oral anti-inflammatory activity of Ro 31-8830. This finding has implications for the treatment of inflammatory disorders in the clinic.

FKBP, thought to be identical to PKCI-2, does not inhibit protein kinase C

Abstract The FK506 and rapamycin binding protein (FKBP), recently shown to be identical to PKCI-2, and its ligands FK506 and rapamycin either acting alone or complexed to FKBP do not inhibit the kinase activity of isolated protein kinase C or protein kinase C-mediated events in cells.

The inhibitory profiles of hog pancreatic and human rheumatoid synovial cell phospholipases A2

ConclusionsThe protective effect of Harmons and Rosenthals agents, and of the Upjohn inhibitors [4] is most simply ascribed to their shielding of His-48 by reversible binding at the enzymes active site. However, protection by an allosteric mechanism cannot be excluded for any of these compounds. The lack of protection shown by other reversible inhibitors suggests that these agents do not bind to the enzyme active site. Some of these agents e.g. mepacrine, may inhibit PLA2 by interacting with its membrane substrate, so perturbing its architecture and electrostatic properties, and thus interfering with enzyme approach and/or penetration [5]. Further information on enzyme-inhibitor interactions may be derived from n.m.r. studies.A crude extract of HRS enzyme was used in this study. Nevertheless, it seems that this enzyme is similar in many respects to hog pancreatic PLA2. In particular, the similar protection profiles and pH dependencies of pBPB inactivation suggest that the pBPB modification site is the same for both enzymes. Also, the lack of preferential inhibition of either enzyme by active-site directed inhibitors suggests a degree of similarity in the architecture of their catalytic centres; however, studies with more potent inhibitors may uncover differences here.

Substrate specificity and inhibitor profile of human recombinant p56lck from a baculovirus expression vector

Abstractp56lck, a member of the src family of nonreceptor protein receptor kinases, is required for normal signal transduction through the T cell receptor. Inappropriate T cell activation and proliferation has been identified as an early event in auto-immune disease —agents which control T cell activation through modulation of p56lck kinase activity could therefore be potential therapeutic agents for a range of pathological conditions. To identify p56lck inhibitors, we have established an assay system suitable for the high throughput screening of compound libraries. The assay uses enzyme purified from baculovirus infected SF9 cells, and a novel peptidic substrate identified by L. Cantley from a degenerate combinatorial peptide library. We have used this assay system to screen a number of different compounds as potential inhibitors of p56lck. In addition, peptides based on the substrate sequence were also tested to identify a sequence that could be used in the rational design of peptide inhibitors of p56lck.