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Metastasis is the cause of death in the majority (∼90%) of malignant cancers. The oral potassium-sparing diuretic amiloride and its 5-substituted derivative 5-N,N-(hexamethylene)amiloride (HMA) reportedly show robust antitumor/metastasis effects in multiple in vitro and animal models. These effects are likely due, at least in part, to inhibition of the urokinase plasminogen activator (uPA), a key protease determinant of cell invasiveness and metastasis. This study reports the discovery of 6-substituted HMA analogs that show nanomolar potency against uPA, high selectivity over related trypsin-like serine proteases, and minimal inhibitory effects against epithelial sodium channels (ENaC), the diuretic and antikaliuretic target of amiloride. Reductions in lung metastases were demonstrated for two analogs in a late-stage experimental mouse metastasis model, and one analog completely inhibited formation of liver metastases in an orthotopic xenograft mouse model of pancreatic cancer. The results support further evaluation of 6-substituted HMA derivatives as uPA-targeting anticancer drugs.

SK (streptokinase) is a secreted plasminogen activator and virulence factor of GAS (group A Streptococcus). Among GAS isolates, SK gene sequences are polymorphic and are grouped into two sequence clusters (cluster type-1 and cluster type-2) with cluster type-2 being further classified into subclusters (type-2a and type-2b). In the present study, we examined the role of bacterial and host-derived cofactors in SK-mediated plasminogen activation. All SK variants, apart from type-2b, can form an activator complex with Glu-Plg (Glu-plasminogen). Specific ligand-binding-induced conformational changes in Glu-Plg mediated by fibrinogen, PAM (plasminogen-binding group A streptococcal M protein), fibrinogen fragment D or fibrin, were required for type-2b SK to form a functional activator complex with Glu-Plg. In contrast with type-1 and type-2a SK, type-2b SK activator complexes were inhibited by α2-antiplasmin unless bound to fibrin or to the GAS cell-surface via PAM in combination with fibrinogen. Taken together, these data suggest that type-2b SK plasminogen activation may be restricted to specific microenvironments within the host such as fibrin deposits or the bacterial cell surface through the action of α2-antiplasmin. We conclude that phenotypic SK variation functionally underpins a pathogenic mechanism whereby SK variants differentially focus plasminogen activation, leading to specific niche adaption within the host.

Streptococcus pyogenes (group A streptococcus, GAS) is a human specific pathogen responsible for a wide range of diseases. The majority of GAS infections give rise to uncomplicated disease, however, the migration of GAS from superficial to deep tissue sites can result in life-threatening invasive infections. GAS secrete streptokinase (SK), a potent plasminogen (Plg) activator which enables this bacterium to subvert the host Plg activation system to generate soluble and cell bound protease activity. Unlike the human Plg activators, u-PA and t-PA, which cleave the Plg activation bond (Arg561-VaI562) to generate the broad serine protease plasmin, SK lacks intrinsic protease activity. Instead, SK forms a stoichiometric complex with Plg and through non-proteolytic mechanisms, generates an active site in the bound Plg molecule to produce an activator complex with proteolytic activity. Among GAS isolates, SK gene (ska) sequences are polymorphic and can be grouped into two distinct sequence clusters (termed cluster type-1 and cluster type-2) with cluster type-2 being further divided into sub-clusters type-2a and type-2b. Allelic variants of SK produced by GAS isolates display unique Plg activation properties, however the biological significance of SK polymorphism among GAS isolates is yet to be elucidated. This project describes the first comprehensive phenotypic study of GAS SK variants by characterising the structural, functional and biochemical differences displayed by SK molecules. Five distinct ska alleles representing examples from the three known phylogenetic sequence clusters, along with SKc from group C streptococcus were cloned and expressed as recombinant proteins. Structural analysis using far-UV circular dichroism spectroscopy indicated that all SK variants displayed similar secondary structure despite significant variation in amino acid sequence. Active site generation in Glu-Plg by SK variants was examined using the fluorescent active site titrant 4- methylum

The larvae of Galleria mellonella (also known colloquially as the wax worm) is increasingly being used as an infection model to study virulence factors and pathogenesis of many prominent bacterial and fungal human pathogens. When compared with traditional mammalian model hosts, invertebrate infection models are cheaper to establish and maintain, are more amenable to high-throughput studies and are not subjected to the same ethical constraints as vertebrates. In addition to these benefits, G. mellonella larvae possess a number of other characteristics which make these organisms particularly useful for the study of human pathogens. Larvae are relatively large in size (12- 20 mm) which enables easy manipulation and facilitates the collection of tissue/ hemolymph samples for downstream analysis.

Streptococcus pyogenes is a major human pathogen responsible for numerous diseases ranging from uncomplicated skin and throat infections to severe, life threatening invasive disease such as necrotising fasciitis and streptococcal toxic shock syndrome. These severe invasive infections progress rapidly and produce high rates of morbidity and mortality despite the implementation of aggressive treatment plans. The activation of plasminogen and the acquisition of plasmin activity at the bacterial cell surface is critical for the invasive pathogenesis of this organism. To facilitate this process, S. pyogenes secrete streptokinase, a potent plasminogen activating protein. Here, we describe the role of streptokinase in invasive pathogenesis and discuss some potentially useful strategies for disruption of streptokinase mediated plasminogen activation which could be employed to treat severe invasive S. pyogenes infections.