TMPRSS2 is an androgen-regulated cell surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastasis. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-Met receptor tyrosine kinase signaling, and initiated a pro-invasive EMT phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment.

Conjugation to macromolecular carriers is a proven strategy for improving the pharmacokinetics of drugs, with many stable polyethylene glycol conjugates having reached the market. Stable conjugates suffer several limitations: loss of drug potency due to conjugation, confining the drug to the extracellular space, and the requirement for a circulating conjugate. Current research is directed toward overcoming such limitations through releasable conjugates in which the drug is covalently linked to the carrier through a cleavable linker. Satisfactory linkers that provide predictable cleavage rates tunable over a wide time range that are useful for both circulating and noncirculating conjugates are not yet available. We describe such conjugation linkers on the basis of a nonenzymatic β-elimination reaction with preprogrammed, highly tunable cleavage rates. A set of modular linkers is described that bears a succinimidyl carbonate group for attachment to an amine-containing drug or prodrug, an azido group for conjugation to the carrier, and a tunable modulator that controls the rate of β-eliminative cleavage. The linkers provide predictable, tunable release rates of ligands from macromolecular conjugates both in vitro and in vivo, with half-lives spanning from a range of hours to >1 y at physiological pH. A circulating PEG conjugate achieved a 56-fold half-life extension of the 39-aa peptide exenatide in rats, and a noncirculating s.c. hydrogel conjugate achieved a 150-fold extension. Using slow-cleaving linkers, the latter may provide a generic format for once-a-month dosage forms of potent drugs. The releasable linkers provide additional benefits that include lowering C max and pharmacokinetic coordination of drug combinations. metronomic chemotherapy | implant M any drugs and drug candidates are suboptimal or ineffective because of a short duration of action. For example, peptides and proteins with promising therapeutic value often have serum half-lives of only minutes to hours. One solution to this problem involves conjugation to carriers such as polyethylene glycol (PEG), the Fc portion of IgG, serum albumin, and other longlived macromolecules. The large carriers retard kidney filtration and hence increase plasma half-life of the attached drug. Much success has been realized thus far with PEG as the macromolecular carrier. PEG is nontoxic and nonimmunogenic, and the plasma half-life is a function of hydrodynamic size. Conjugation of drugs to PEG with molecular mass ∼40 kDa has been successfully used with peptides, nucleic acids, and small molecules and can afford half-lives of up to ∼7 d in humans; as of 2011, 10 PEGylated peptide-based drugs were approved by the Food and Drug Administration (FDA) and ∼40 were in clinical trials (1, 2).All of the currently marketed PEG-protein conjugates are permanently PEGylated. Attachment of large PEG moieties often reduces activity of the drug, and higher concentrations of conjugate are necessary to achieve the required biological activity. A further limitation is that permanen...

The aim of this study was to identify fibrogenic mediators stimulating activation, proliferation, and/or matrix synthesis of rat pancreatic stellate cells (PSC). PSC were isolated from the pancreas of normal Wistar rats and from rats with cerulein pancreatitis. Cell activation was demonstrated by immunofluorescence microscopy of smooth muscle alpha-actin (SMA) and real-time quantitative RT-PCR of SMA, fibronectin, and transforming growth factor (TGF)-beta(1). Proliferation was measured by bromodeoxyuridine incorporation. Matrix synthesis was demonstrated on the protein and mRNA level. Within a few days in primary culture, PSC changed their phenotype from fat-storing to SMA-positive myofibroblast-like cells expressing platelet-derived growth factor (PDGF) alpha- and PDGF beta-receptors. TGF-beta(1) and tumor necrosis factor (TNF)-alpha accelerated the change in the cells' phenotype. Addition of 50 ng/ml PDGF and 5 ng/ml basic fibroblast growth factor (bFGF) to cultured PSC significantly stimulated cell proliferation (4.37 +/- 0.49- and 2.96 +/- 0.39-fold of control). Fibronectin synthesis calculated on the basis of DNA was stimulated by 5 ng/ml bFGF (3.44 +/- 1.13-fold), 5 ng/ml TGF-beta(1) (2.46 +/- 0.89-fold), 20 ng/ml PDGF (2.27 +/- 0.68-fold), and 50 ng/ml TGF-alpha (1.87 +/- 0.19-fold). As shown by RT-PCR, PSC express predominantly the splice variant EIII-A of fibronectin. Immunofluorescence microscopy and Northern blot confirmed that in particular bFGF and TGF-beta(1) stimulated the synthesis of fibronectin and collagens type I and III. In conclusion, our data demonstrate that 1) TGF-beta(1) and TNF-alpha accelerate the change in the cell phenotype, 2) PDGF represents the most effective mitogen, and 3) bFGF, TGF-beta(1), PDGF, and, to a lesser extent, TGF-alpha stimulate extracellular matrix synthesis of cultured rat PSC.

Ticks are ectoparasitic blood-feeders and important vectors for pathogens including arboviruses, rickettsiae, spirochetes and protozoa. As obligate blood-feeders, one possible strategy to retard disease transmission is disruption of the parasite's ability to digest host proteins. However, the constituent peptidases in the parasite gut and their potential interplay in the digestion of the blood meal are poorly understood. We have characterised a novel asparaginyl endopeptidase (legumain) from the hard tick Ixodes ricinus (termed IrAE), which we believe is the first such characterisation of a clan CD family C13 cysteine peptidase (protease) in arthropods. By RT-PCR of different tissues, IrAE mRNA was only expressed in the tick gut. Indirect immunofluorescence and EM localised IrAE in the digestive vesicles of gut cells and within the peritrophic matrix. IrAE was functionally expressed in Pichia pastoris and reacted with a specific peptidyl fluorogenic substrate, and acyloxymethyl ketone and aza-asparagine Michael acceptor inhibitors. IrAE activity was unstable at pH > or = 6.0 and was shown to have a strict specificity for asparagine at P1 using a positional scanning synthetic combinatorial library. The enzyme hydrolyzed protein substrates with a pH optimum of 4.5, consistent with the pH of gut cell digestive vesicles. Thus, IrAE cleaved the major protein of the blood meal, hemoglobin, to a predominant peptide of 4kDa. Also, IrAE trans-processed and activated the zymogen form of Schistosoma mansoni cathepsin B1 -- an enzyme contributing to hemoglobin digestion in the gut of that bloodfluke. The possible functions of IrAE in the gut digestive processes of I. ricinus are compared with those suggested for other hematophagous parasites.

We have developed a unique long-acting drug-delivery system for the GLP-1 agonist exenatide. The peptide was covalently attached to Tetra-PEG hydrogel microspheres by a cleavable β-eliminative linker; upon s.c. injection, the exenatide is slowly released at a rate dictated by the linker. A second β-eliminative linker with a slower cleavage rate was incorporated in polymer cross-links to trigger gel degradation after drug release. The uniform 40 μm microspheres were fabricated using a flow-focusing microfluidic device and in situ polymerization within droplets. The exenatide-laden microspheres were injected subcutaneously into the rat, and serum exenatide measured over a one-month period. Pharmacokinetic analysis showed a t1/2,β of released exenatide of about 7 days which represents over a 300-fold half-life extension in the rat and exceeds the half-life of any currently approved long-acting GLP-1 agonist. Hydrogel-exenatide conjugates gave an excellent Level A in vitro-in vivo correlation of release rates of the peptide from the gel, and indicated that exenatide release was 3-fold faster in vivo than in vitro. Pharmacokinetic simulations indicate that the hydrogel-exenatide microspheres should support weekly or biweekly subcutaneous dosing in humans. The rare ability to modify in vivo pharmacokinetics by the chemical nature of the linker indicates that an even longer acting exenatide is feasible.