The crystal structure of quercerin:uPA reveals that catechol serves as the functional group in inhibiting serine proteases.

Thrombosis occurs in both macrovasculature and microvasculature, causing various cardio-cerebral vascular diseases. The lack of effective and safe antithrombotic drugs leads to a public health crisis. Mounting evidence suggests that protein disulfide isomerase (PDI) plays a critical role in the initial stage of thrombus formation, motivating the research of the feasibility of PDI inhibitors as novel anti-thrombotics. Rutin, one of the most potent PDI inhibitors, was reported to suppress platelet aggregation and thrombosis in animal models, but further studies and clinical translation were restricted due to its low aqueous solubility and oral bioavailability. In this work, we fabricated rutin-loaded lipid-based nano-formulation (NanoR) and characterized their physical–chemical properties, release profiles, pharmacokinetic process, and pharmacodynamic function against thrombosis in macrovessels and microvessels. NanoR provided increased solubility and dissolution of rutin to achieve earlier T_max and higher C_max than the sodium salt of rutin (NaR) after oral gavage. Ex vivo studies demonstrated that NanoR significantly inhibited thrombin generation and clot formation in the plasma of mice. Importantly, such effect was reversed by exogenous recombinant PDI, demonstrating the specificity of the NanoR. In direct current-induced arterial thrombosis model and ferric chloride-induced microvascular thrombosis model, NanoR exhibited greatly enhanced antithrombotic activity compared with NaR. NanoR also showed good safety performance according to tail bleeding assay, global coagulation tests, and histological analysis. Overall, our current results indicated that NanoR offers a promising antithrombotic treatment with potential for clinical translation.

Linlin Li, Mingxiang Huang, Jianshan Shen, Yao Wang, Rui Wang, Cai Yuan, Longguang Jiang, Mingdong Huang; Serum levels of soluble PECAM-1 in COVID-19 patients

There is increasing interest in developing peptides for pharmacological intervention with pathophysiological functions of serine proteases. From phage-displayed peptide libraries, we previously isolated peptidylic inhibitors of urokinase-type plasminogen activator, a potential target for intervention with cancer invasion. The two peptides, upain-1 (CSWRGLENHRMC) and mupain-1 (CPAYSRYLDC), are competitive inhibitors of human and murine urokinase-type plasminogen activator, respectively. Both have an Arg as the P1 residue, inserting into the S1 pocket in the active site of the enzymes, but their specificity depends to a large extent on interactions outside the enzymes' active sites, so-called exosite interactions. Here we describe upain-2 (CSWRGLENHAAC) and the synthesis of a number of upain-2 and mupain-1 variants in which the P1 Arg was substituted with novel non-natural Arg analogs and achieved considerable improvement in the affinity of the peptides to their targets. Using chimeras of human and murine urokinase-type plasminogen activator as well as X-ray crystallography, we delineated the relative contribution of the P1 residue and exosite interactions to the affinity and specificity of the inhibitors for their target enzyme. The effect of inserting a particular non-natural amino acid into the P1 position is determined by the fact that changes in interactions of the P1 residue in the S1 pocket lead to changed exosite interactions and vice versa. These findings are of general interest when the affinities and specificities of serine protease inhibitors to be used for pharmacological intervention are considered and could pave the way for potential drug candidates for the treatment of cancer.