Natural and engineered kallikrein inhibitors: an emerging pharmacopoeia

The kallikreins and kallikrein-related peptidases are serine proteases that control a plethora of developmental and homeostatic phenomena, ranging from semen liquefaction to skin desquamation and blood pressure. The diversity of roles played by kallikreins has stimulated considerable interest in these enzymes from the perspective of diagnostics and drug design. Kallikreins already have well-established credentials as targets for therapeutic intervention and there is increasing appreciation of their potential both as biomarkers and as targets for inhibitor design. Here, we explore the current status of naturally occurring kallikrein protease-inhibitor complexes and illustrate how this knowledge can interface with strategies for rational re-engineering of bioscaffolds and design of small-molecule inhibitors.

Expression of PSA-RP2, an alternatively spliced variant from the PSA gene, is increased in prostate cancer tissues but the protein is not secreted from prostate cancer cells

PSA-RP2 is a variant transcript expressed from the PSA gene that is conserved in gorillas, chimpanzees and humans suggesting a particular relevance for this transcript in these primates. We demonstrated by qRT-PCR that PSA-RP2 is upregulated in prostate cancer compared with benign prostatic hyperplasia tissues. The PSA-RP2 protein was not detected in seminal fluid and was cytoplasmically localised but not secreted from LNCaP or transfected PC3 prostate cells, despite secretion from transfected Cos-7 and HEK293 kidney cell lines. PSA-RP2-transfected PC3 cells showed slightly decreased proliferation and increased migration towards PC3-conditioned medium that could suggest a functional role in prostate cancer.

Myocyte enhancer factor 2C : an osteoblast transcription factor identified by DMSO enhanced mineralization

Rapid mineralization of cultured osteoblasts could be a useful characteristic in stem-cell mediated therapies for fracture and other orthopaedic problems. Dimethyl sulfoxide (DMSO) is a small amphipathic solvent molecule capable of simulating cell differentiation. We report that, in primary human osteoblasts, DMSO dose-dependently enhanced the expression of osteoblast differentiation markers alkaline phosphatase (ALP) activity and extracellular matrix mineralization. Furthermore, similar DMSO mediated mineralization enhancement was observed in primary osteoblast-like cells differentiated from mouse mesenchymal cells derived from fat, a promising source of starter cells for cell-based therapy. Using a convenient mouse pre-osteoblast model cell line MC3T3-E1 we further investigated this phenomenon showing that numerous osteoblast-expressed genes were elevated in response to DMSO treatment and correlated with enhanced mineralization. Myocyte enhancer factor 2c (Mef2c) was identified as the transcription factor most induced by DMSO, among numerous DMSO-induced genes, suggesting a role for Mef2c in osteoblast gene regulation. Immunohistochemistry confirmed expression of Mef2c in osteoblast-like cells in mouse mandible, cortical and trabecular bone. shRNAi-mediated Mef2c gene silencing resulted in defective osteoblast differentiation, decreased ALP activity and matrix mineralization and knockdown of osteoblast specific gene expression, including osteocalcin and bone sialoprotein. Flow on knockdown of bone specific transcription factors, Runx2 and osterix by shRNAi knockdown of Mef2c suggests that Mef2c lies upstream of these two important factors in the cascade of gene expression in osteoblasts.

Generation of profluorescent isoindoline nitroxides using click chemistry

Novel profluorescent nitroxides bearing a triazole linker between the coumarin fluorophore and an isoindoline nitroxide were prepared in good yields using the coppercatalyzed azide�alkyne 1,3-dipolar cycloaddition reaction (CuAAC). Nitroxides containing 7-hydroxy and 7-diethylamino substitution on their coumarin rings displayed significant fluorescence suppression, and upon reaction with methyl radicals, normal fluorescence emission was returned. The fluorescence emission for the 7-hydroxycoumarin nitroxide and its diamagnetic analogue was found to be strongly influenced by pH with maximal fluorescence emission achieved in basic solution. Solvent polarity was also shown to affect fluorescence emission. The significant difference in fluorescence output between the nitroxides and their corresponding diamagnetic analogues makes these compounds ideal tools for monitoring processes involving free-radical species.