Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods

Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one-pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against alpha-thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg(-1). The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions, in addition to an active site

Site-selective photocatalytic functionalization of peptides and proteins at selenocysteine

The importance of modified peptides and proteins for applications in drug discovery, and for illuminating biological processes at the molecular level, is fueling a demand for efficient methods that facilitate the precise modification of these biomolecules. Herein, we describe the development of a photocatalytic method for the rapid and efficient dimerization and site-specific functionalization of peptide and protein diselenides. This methodology, dubbed the photocatalytic diselenide contraction, involves irradiation at 450 nm in the presence of an iridium photocatalyst and a phosphine and results in rapid and clean conversion of diselenides to reductively stable selenoethers. A mechanism for this photocatalytic transformation is proposed, which is supported by photoluminescence spectroscopy and density functional theory calculations. The utility of the photocatalytic diselenide contraction transformation is highlighted through the dimerization of selenopeptides, and by the generation of two families of protein conjugates via the site-selective modification of calmodulin containing the 21st amino acid selenocysteine, and the C-terminal modification of a ubiquitin diselenide.Luke J. Dowman, Sameer S. Kulkarni, Juan V. Alegre-Requena, Andrew M. Giltrap, Alexander R. Norman, Ashish Sharma, Liliana C. Gallegos, Angus S.Mackay, Adarshi P. Welegedara, Emma E. Watson, Damian van Raad, Gerhard Niederacher, Susanne Huhmann, Nicholas Proschogo, Karishma Patel, Mark Larance, Christian F. W. Becker, Joel P. Mackay, Girish Lakhwani, Thomas Huber, Robert S. Paton, Richard J. Payn

Synthesis and evaluation of peptidic thrombin inhibitors bearing acid-stable sulfotyrosine analogues

Tyrosine sulfation is an important post-translational modification of peptides and proteins which underpins and modulates many protein-protein interactions. In order to overcome the inherent instability of the native modification, we report the synthesis of two sulfonate analogues and their incorporation into two thrombin-inhibiting sulfopeptides. The effective mimicry of these sulfonate analogues for native sulfotyrosine was validated in the context of their thrombin inhibitory activity and binding mode, as determined by X-ray crystallography

Total Synthesis of Glycosylated Human Interferon-y

Interferon-γ (IFN-γ) is a glycoprotein that is responsible for orchestrating numerous critical immune induction and modulation processes and is used clinically for the treatment of a number of diseases. Herein, we describe the total chemical synthesis of homogeneously glycosylated variants of human IFN-γ using a tandem diselenide-selenoester ligation-deselenization strategy in the C- to N-terminal direction. The synthetic glycoproteins were successfully folded, and the structures and antiviral functions were assessed.Xiaoyi Wang, Anneliese S. Ashhurst, Luke J. Dowman, Emma E. Watson, Henry Y. Li, Antony J. Fairbanks, Mark Larance, Ann Kwan, and Richard J. Payn

Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates

Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics

Loss of 5α-Reductase Type 1 accelerates the development of hepatic steatosis but protects against hepatocellular carcinoma in male mice

Nonalcoholic fatty liver disease (NAFLD) has been associated with glucocorticoid excess and androgen deficiency, yet in the majority of patients with steatohepatitis, circulating cortisol and androgen levels are normal. The enzyme 5α-reductase (5αR) has a critical role in androgen and glucocorticoid action. We hypothesize that 5αR has an important role in the pathogenesis of steatohepatitis through regulation of intracrine/paracrine hormone availability. Human liver samples from patients with NAFLD and normal donor tissue were used for gene expression and immunohistochemical analysis. NAFLD samples were scored using the Kleiner classification. In addition, 5αR1-/-, 5αR2-/-, and wild-type (WT) mice were fed normal chow or American lifestyle-induced obesity syndrome (ALIOS) diet for 6 or 12 months. Liver histology was graded and staged. Hepatic and circulating free fatty acid and triglyceride levels were quantified, and gene and protein expression was measured by real-time PCR and immunohistochemistry. 5αR1 and -2 were highly expressed in human liver, and 5αR1 protein expression increased with severity of NAFLD. 5αR1-/- (but not 5αR2-/-) mice fed an ALIOS diet developed greater hepatic steatosis than WT mice, and hepatic mRNA expression of genes involved in insulin signaling was decreased. Furthermore, 60% of WT mice developed focal hepatocellular lesions consistent with hepatocellular carcinoma after 12 months of the ALIOS diet, compared with 20% of 5αR2-/- and 0% of 5αR1-/- mice (P < .05). 5αR1 deletion accelerates the development of hepatic steatosis but may protect against the development of NAFLD-related hepatocellular neoplasia and therefore has potential as a therapeutic target. Copyrigh