Revisiting antithrombotic therapeutics; sculptin, a novel specific, competitive, reversible, scissile and tight binding inhibitor of thrombin.
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ABSTRACT:
SUBMITTER: Iqbal A
PROVIDER: S-EPMC5431157 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The development of new anticoagulants is an important goal for the improvement of thrombosis treatment. Recent studies have suggested the importance of thrombin inhibitors in the modulation of thromboembolic disorders. The aim of this study was to discover a new small-molecule thrombin inhibitor. In this study, the compound JJ1, which has a novel scaffold, was selected by structure-based docking simulation to determine its potential inhibitory activity against thrombin. JJ1 was shown to inhibit the catalytic activity of human α-thrombin with a K i of 0.019 μM by direct binding to the active site and with at least 10,000-fold selectivity relative to that reported for the inhibition of other biologically important serine proteases. JJ1 prolonged clotting times (activated partial thromboplastin time and prothrombin time) and inhibited the activity and production of thrombin. Furthermore, it inhibited thrombin-catalyzed fibrin polymerization and platelet aggregation. Similar to its in vitro antithrombotic activities, JJ1 showed enhanced antithrombotic effects in an in vivo pulmonary embolism and arterial thrombosis model. It also exhibited anticoagulant effects in mice. Collectively, these results demonstrated that JJ1 was a potent, direct, and selective thrombin inhibitor that may be useful in the management of various thrombotic disorders.
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Project description:The micromolar equilibrium constants for heme dissociation from IsdG and IsdI reported in the literature call into question whether these enzymes are actually members of the iron-regulated surface determinant system of Staphylococcus aureus, which harvests heme iron from a host during infection. In order to address this question, the heme dissociation constants for IsdG and IsdI were reevaluated using three approaches. The heme dissociation equilibrium constants were measured using a UV/Vis absorption-detected assay analyzed with an assumption-free model, and using a newly developed fluorescence-detected assay. The heme dissociation rate constants were estimated using apomyoglobin competition assays. Analyses of the UV/Vis absorption data revealed a critical flaw in the previous measurements; heme is 99.9% protein-bound at the micromolar concentrations needed for UV/Vis absorption spectroscopy, which renders accurate equilibrium constant measurement nearly impossible. However, fluorescence can be measured for more dilute samples, and analyses of these data resulted in dissociation equilibrium constants of 1.4 ± 0.6 nM and 12.9 ± 1.3 nM for IsdG and IsdI, respectively. Analyses of the kinetic data obtained from apomyoglobin competition assays estimated heme dissociation rate constants of 0.022 ± 0.002 s-1 for IsdG and 0.092 ± 0.008 s-1 for IsdI. Based upon these data, and what is known regarding the post-translational regulation of IsdG and IsdI, it is proposed that only IsdG is a member of the heme iron acquisition pathway and IsdI regulates heme homeostasis. Furthermore, the nanomolar dissociation constants mean that heme is bound tightly by IsdG and indicates that competitive inhibition of this protein will be difficult. Instead, uncompetitive inhibition based upon a detailed understanding of enzyme mechanism is a more promising antibiotic development strategy.
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Project description:Mosquito saliva carries a large number of factors with anti-hemostatic, anti-inflammatory and immuno-modulatory activities. The cE5 protein was initially identified during an Anopheles gambiae salivary gland transcriptome study and later shown to share sequence similarity with anophelin, a thrombin inhibitor from the saliva of the New World mosquito Anopheles albimanus. The cE5 gene was found to encode different mRNA isoforms coexisting in several tissues of both male and female mosquitoes, a highly unusual profile for a gene potentially encoding an anti-thrombin and involved in blood feeding. Expression of the cE5 protein and assessment of its activity and inhibitory properties showed that it is a highly specific and tight-binding thrombin inhibitor, which differs from the A. albimanus orthologue for the fast-binding kinetics. Despite the widespread occurrence of cE5 transcripts in different mosquito tissues the corresponding protein was only found in female salivary glands, where it undergoes post-translational modification. Therefore, tissue-specific restriction of the A. gambiae cE5 is not achieved by transcriptional control, as common for mosquito salivary genes involved in hematophagy, but by post-trascriptional gene regulatory mechanisms. Our observations provide a paradigm of post-transcriptional regulation as key determinant of tissue specificity for a protein from an important disease vector and point out that transcriptomic data should be interpreted with caution in the absence of concomitant proteomic support.
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Project description:IntroductionIn recent years, the traditional treatments for thrombotic diseases, heparin and warfarin, are increasingly being replaced by novel oral anticoagulants offering convenient dosing regimens, more predictable anticoagulant responses, and less frequent monitoring. However, these drugs can be contraindicated for some patients and, in particular, their bleeding liability remains high.MethodsWe have developed a new class of direct thrombin inhibitors (VE-DTIs) and have utilized kinetics, biochemical, and X-ray structural studies to characterize the mechanism of action and in vitro pharmacology of an exemplary compound from this class, Compound 1.ResultsWe demonstrate that Compound 1, an exemplary VE-DTI, acts through reversible covalent inhibition. Compound 1 inhibits thrombin by transiently acylating the active site S195 with high potency and significant selectivity over other trypsin-like serine proteases. The compound inhibits the binding of a peptide substrate with both clot-bound and free thrombin with nanomolar potency. Compound 1 is a low micromolar inhibitor of thrombin activity against endogenous substrates such as fibrinogen and a nanomolar inhibitor of the activation of protein C and thrombin-activatable fibrinolysis inhibitor. In the thrombin generation assay, Compound 1 inhibits thrombin generation with low micromolar potency but does not increase the lag time for thrombin formation. In addition, Compound 1 showed weak inhibition of clotting in PT and aPTT assays consistent with its distinctive profile in the thrombin generation assay.ConclusionCompound 1, while maintaining strong potency comparable to the current DTIs, has a distinct mechanism of action which produces a differentiating pharmacological profile. Acting through reversible covalent inhibition, these direct thrombin inhibitors could lead to new anticoagulants with better combined efficacy and bleeding profiles.
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Project description:Dose control of warfarin is a major complication in anticoagulation therapy and overdose is reversed by the vitamin K antidote. Improving the dosage management and antidotal efficacy requires mechanistic understanding. Here we find that effects of the major predictor of warfarin dosage, SNP -1639 G>A, follow a general correlation that warfarin 50% inhibitory concentration decreases with cellular level of vitamin K epoxide reductase (VKORC1), suggesting stoichiometric inhibition. Characterization of the inhibition kinetics required the use of microsomal VKORC1 with a native reductant, glutathione, that enables effective warfarin inhibition in vitro. The kinetics data can be fitted with the Morrison equation, giving a nanomolar inhibition constant and demonstrating that warfarin is a tight-binding inhibitor. However, warfarin is released from purified VKORC1-warfarin complex with increasing amount of vitamin K, indicating competitive inhibition. The competition occurs also in cells, resulting in rescued VKORC1 activity that augments the antidotal effects of vitamin K. Taken together, warfarin is a competitive inhibitor that binds VKORC1 tightly and inhibits at a stoichiometric (1:1) concentration, whereas exceeding the VKORC1 level results in warfarin overdose. Thus, warfarin dosage control should use VKORC1 level as a major indicator, and improved antidotes may be designed based on their competition with warfarin.
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Project description:Monoamine oxidase-B (MAO-B) inhibitors are widely used in the treatment of Parkinson's disease. They increase vital monoamine neurotransmitters in the brain. However, there is a need for safer natural reversible MAO inhibitors with MAO-B selectivity. Our previous studies showed that Psoralea corylifolia seeds (PCS) extract contains compounds that inhibit monoamine oxidase-B.In this study, six of PCS constituents sharing a benzopyrone structure were investigated. The compounds Biochanin-A (BIO-A), isopsoralen, 6-prenylnaringenin, neobavaisoflavone, psoralen, and psoralidin, were tested for their ability to inhibit recombinant human MAO-A and B (hMAO-A and hMAO-B) isozymes. The ability of these compounds to inhibit MAO-A and MAO-B were compared to that of PCS ethanolic extract (PCSEE) using spectrophotometric assays and confirmed by luminescence assays. The highly potent and selective MAO-B inhibitor, BIO-A, was further investigated for both isozymes reversibility and enzyme kinetics. Molecular docking studies were used to predict the bioactive conformation and molecular interactions of BIO-A with both isozymes.The data obtained indicate that benzopyrones inhibited hMAO-A and hMAO-B with different degrees as confirmed with the luminescence assay. BIO-A inhibited hMAO-B with high potency and selectivity in the present study (IC50?=?0.003 ?g/mL) and showing 38-fold more selectivity than PCSEE (hMAO-B IC50?=?3.03 ?g/mL, 17-fold selectivity) without affecting hydrogen peroxide. Furthermore, BIO-A reversibly and competitively inhibited both hMAOs with significantly lower inhibitory constant (Ki) in hMAO-B (3.8 nM) than hMAO-A (99.3 nM). Our docking studies indicated that the H-bonds and hydrophobic interactions at the human MAO-A and MAO-B active sites contributed to the reversibility and selectivity of BIO-A.The data obtained indicate that BIO-A is a potent, reversible and selective MAO-B inhibitor and may be recommended for further investigation in its possible use in the therapeutic management of Parkinson's and Alzheimer's diseases.
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Project description:Signal Transduction by Ion NanoGating (STING) is a label-free technology based on functionalized quartz nanopipettes. The nanopipette pore can be decorated with a variety of recognition elements and the molecular interaction is transduced via a simple electrochemical system. A STING sensor can be easily and reproducibly fabricated and tailored at the bench starting from inexpensive quartz capillaries. The analytical application of this new biosensing platform, however, was limited due to the difficult correlation between the measured ionic current and the analyte concentration in solution. Here we show that STING sensors functionalized with aptamers allow the quantitative detection of thrombin. The binding of thrombin generates a signal that can be directly correlated to its concentration in the bulk solution.
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