Philip E. Thompson

PI 3-kinase p110β : a new target for antithrombotic therapy.

Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin αIIbβ3 (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110β isoform in regulating the formation and stability of integrin αIIbβ3 adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110β inhibitors have been developed which prevent formation of stable integrin αIIbβ3 adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110β as an important new target for antithrombotic therapy.

Structure--activity relationships of polymyxin antibiotics.

Tony Velkov,* Philip E. Thompson, Roger L. Nation, and Jian Li* School of Medicine, Deakin University, Pigdons Road, Geelong 3217, Victoria, Australia, Medicinal Chemistry and Drug Action and Facility for Anti-infective Drug Development and Innovation, Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Victoria, Australia

Cleavage and activation of proteinase‐activated receptor‐2 on human neutrophils by gingipain‐R from Porphyromonas gingivalis

Gingipain‐R, the major arginine‐specific proteinase from Porphyromonas gingivalis, a causative agent of adult periodontal disease, was found to cleave a model peptide representing the cleavage site of proteinase‐activated receptor‐2 (PAR‐2), a G‐protein‐coupled receptor found on the surface of neutrophils. The bacterial proteinase was also shown to induce an increase in the intracellular calcium concentration of enzyme‐treated neutrophils, most probably due to PAR‐2 activation. This response by neutrophils to gingipain‐R may be a mechanism for the development of inflammation associated with periodontal disease.

The major human and mouse granzymes are structurally and functionally divergent.

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even “orthologous” granzymes have species-specific functions, having evolved in distinct environments that pose different challenges.

The Phytosulfokine (PSK) Receptor Is Capable of Guanylate Cyclase Activity and Enabling Cyclic GMP-dependent Signaling in Plants

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μm and Vmax of 1800 nmol min−1 mg−1 of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg2+ or Mn2+. Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains.

RhoA sustains integrin αIIbβ3 adhesion contacts under high shear

The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, αIIbβ3. Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, U46619, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin αIIbβ3, by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin αIIbβ3-dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase (Y27632) had no effect on integrin αIIbβ3 activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin αIIbβ3 adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin αIIbβ3adhesion contacts under conditions of high shear stress.

Development of cognitive enhancers based on inhibition of insulin-regulated aminopeptidase

The peptides angiotensin IV and LVV-hemorphin 7 were found to enhance memory in a number of memory tasks and reverse the performance deficits in animals with experimentally induced memory loss. These peptides bound specifically to the enzyme insulin-regulated aminopeptidase (IRAP), which is proposed to be the site in the brain that mediates the memory effects of these peptides. However, the mechanism of action is still unknown but may involve inhibition of the aminopeptidase activity of IRAP, since both angiotensin IV and LVV-hemorphin 7 are competitive inhibitors of the enzyme. IRAP also has another functional domain that is thought to regulate the trafficking of the insulin-responsive glucose transporter GLUT4, thereby influencing glucose uptake into cells. Although the exact mechanism by which the peptides enhance memory is yet to be elucidated, IRAP still represents a promising target for the development of a new class of cognitive enhancing agents.

Teaching ‘Old’ Polymyxins New Tricks: New-Generation Lipopeptides Targeting Gram-Negative ‘Superbugs’

The antimicrobial lipopeptides polymyxin B and E (colistin) are being used as a ‘last-line’ therapy for infections caused by multidrug-resistant Gram-negative pathogens. Polymyxin resistance implies a total lack of antibiotics for the treatment of life-threatening infections caused by the Gram-negative ‘superbugs’. This report details the structure–activity relationships (SAR) based design, in toto synthesis, and preclinical evaluation of a series of novel polymyxin lipopeptides with better antibacterial activity against polymyxin-resistant Gram-negative bacteria.

Evidence for the activation of PAR‐2 by the sperm protease, acrosin: expression of the receptor on oocytes

Proteinase‐activated receptor‐2 (PAR‐2) is a member of a family of G‐protein‐coupled, seven‐transmembrane domain receptors that are activated by proteolytic cleavage. The receptor is expressed in a number of different tissues and potential physiological activators identified thus far include trypsin and mast cell tryptase. Acrosin, a trypsin‐like serine proteinase found in spermatozoa of all mammals, was found to cleave a model peptide fluorescent quenched substrate representing the cleavage site of PAR‐2. This substrate was cleaved with kinetics similar to those of the known PAR‐2 activators, trypsin and mast cell tryptase. Acrosin was also shown to induce significant intracellular calcium responses in Chinese hamster ovary cells stably expressing intact human PAR‐2, most probably due to activation of the receptor. Immunohistochemical studies using PAR‐2 specific antibodies indicated that the receptor is expressed by mouse oocytes, which suggests that acrosin may play additional role(s) in the fertilization process via the activation of PAR‐2 on oocytes.

Identification and development of specific inhibitors for insulin-regulated aminopeptidase as a new class of cognitive enhancers

Two structurally distinct peptides, angiotensin IV and LVV‐haemorphin 7, both competitive high‐affinity inhibitors of insulin‐regulated aminopeptidase (IRAP), were found to enhance aversion‐associated and spatial memory in normal rats and to improve performance in a number of memory tasks in rat deficits models. These findings provide compelling support for the development of specific, high‐affinity inhibitors of the enzyme as new cognitive enhancing agents. Different classes of IRAP inhibitors have been developed including peptidomimetics and small molecular weight compounds identified through in silico screening with a homology model of the catalytic domain of IRAP. The proof of principal that inhibition of IRAP activity results in facilitation of memory has been obtained by the demonstration that the small‐molecule IRAP inhibitors also exhibit memory‐enhancing properties.