Potent Thrombin Inhibitor Research Articles

Aeruginosin 525 (AER525) from Cyanobacterium Aphanizomenon Sp. (KUCC C2): A New Serine Proteases Inhibitor.

Aeruginosins (AERs) are one of the most common classes of cyanobacterial peptides synthesised through a hybrid non-ribosomal peptide synthase/polyketide synthase pathway. They have been found in Microcystis, Nodularia spumigena, Oscillatoria/Plantothrix, and Nostoc. The presence of AER in Aphanizomenon isolated from the Curonian Lagoon was reported for the first time in our previous work. Here, the structure of aeruginosin 525 (AER525), isolated from Aphanizomenon sp. KUCC C2, was characterised based on high-resolution mass spectrometry. This new AER variant shows potent activity against thrombin. It also inhibits trypsin and carboxypeptidase A but has no effect on elastase and chymotrypsin. In terms of the N-terminal residue and biological activity, AER525 displaces some similarity to dysinosins, which belongs to the most potent inhibitors of thrombin among AERs. The findings underline the potential of AER525 as a new anticoagulant agent.

Therapeutic Potentials of Medicinal Leech in Chinese Medicine.

The use of medicinal leeches in clinical therapy has been employed for a long time, as it was originally recognized for exerting antithrombin effects. These effects were due to the ability of the leech to continuously suck blood while attached to human skin. According to Chinese Pharmacopoei, leeches used in traditional Chinese medicine mainly consist of Whitmania pigra Whitman, Hirudo nipponia Whitman, and Whitmania acranulata, but the latter two species are relatively scarce. The main constituents of leeches are protein and peptide macromolecules. They can be categorized into two categories based on their pharmacological effects. One group consists of active ingredients that directly target the coagulation system, such as hirudin, heparin, and histamine, which are widely known. The other group comprises protease inhibitor components like Decorsin and Hementin. Among these, hirudin secreted by the salivary glands of the leech is the most potent thrombin inhibitor and served as the sole remedy for preventing blood clotting until the discovery of heparin. Additionally, leeches play a significant role in various traditional Chinese medicine formulations. In recent decades, medicinal leeches have been applied in fields including anti-inflammatory treatment, cardiovascular disease management, antitumor treatment, and many other medical conditions. In this review, we present a comprehensive overview of the historical journey and medicinal applications of leeches in various medical conditions, emphasizing their pharmaceutical significance within traditional Chinese medicine. This review offers valuable insights for exploring additional therapeutic opportunities involving the use of leeches in various diseases and elucidating their underlying mechanisms for future research.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation

AbstractMacrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol‐containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol‐containing elements and used them for the combinatorial synthesis of a 2,688‐member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X‐ray structure analysis of macrocycle‐target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Development of supramolecular anticoagulants with on-demand reversibility.

Drugs are administered at a dosing schedule set by their therapeutic index, and termination of action is achieved by clearance and metabolism of the drug. In some cases, such as anticoagulant drugs or immunotherapeutics, it is important to be able to quickly reverse the drug's action. Here, we report a general strategy to achieve on-demand reversibility by designing a supramolecular drug (a noncovalent assembly of two cooperatively interacting drug fragments held together by transient hybridization of peptide nucleic acid (PNA)) that can be reversed with a PNA antidote that outcompetes the hybridization between the fragments. We demonstrate the approach with thrombin-inhibiting anticoagulants, creating very potent and reversible bivalent direct thrombin inhibitors (Ki = 74 pM). The supramolecular inhibitor effectively inhibited thrombus formation in mice in a needle injury thrombosis model, and this activity could be reversed by administration of the PNA antidote. This design is applicable to therapeutic targets where two binding sites can be identified.

Exploring a Hirudin variant from nonhematophagous leeches: Unraveling full-length sequence, alternative splicing, function, and potential as a novel anticoagulant polypeptide

Ethnopharmacological relevanceLeeches exhibit robust anticoagulant activity, making them useful for treating cardiovascular diseases in traditional Chinese medicine. Whitmania pigra, the primary source species of leech-derived medicinal compounds in China, has been demonstrated to possess formidable anticoagulant properties. Hirudin-like peptides, recognized as potent thrombin inhibitors, are prevalent in hematophagous leeches. Considering that W. pigra is a nonhematophagic leech, the following question arises: does a hirudin variant exist in this species? Aim of the studyIn this study we identified the hirudin-encoding gene (WP_HV1) in the W. pigra genome. The goal of this study was to assess its anticoagulant activity and analyze the related mechanisms. Materials and methodsIn this study, a hirudin-encoding gene, WP_HV1, was identified from the W. pigra genome, and its accurate coding sequence (CDS) was validated through cloning from cDNA extracted from fresh W. pigra specimens. The structure of WP_HV1 and the amino acids associated with its anticoagulant activity were determined by sequence and structural analysis and prediction of its binding energy to thrombin. E. coli was used for the expression of WP_HV1 and recombinant proteins with various structures and mutants. The anticoagulant activity of the synthesized recombinant proteins was then confirmed using thrombin time (TT). ResultsValidation of the WP_HV1 gene was accomplished, and three alternative splices were discovered. The TT of the blank sample exceeded that of the recombinant WP_HV1 sample by 1.74 times (0.05 mg/ml), indicating positive anticoagulant activity. The anticoagulant activity of WP_HV1 was found to be associated with its C-terminal tyrosine, along with the presence of 9 acidic amino acids on both the left and right sides. A significant reduction in the corresponding TT was observed for the mutated amino acids compared to those of the wild type, with decreases of 4.8, 6.6, and 3.9 s, respectively. In addition, the anticoagulant activity of WP_HV1 was enhanced and prolonged for 2.7 s when the lysine-67 residue was mutated to tryptophan. ConclusionOnly one hirudin-encoding variant was identified in W. pigra. The active amino acids associated with anticoagulation in WP_HV1 were resolved and validated, revealing a novel source for screening and developing new anticoagulant drugs.

Serpin peptidase inhibitor, clade E, member 2 in physiology and pathology: recent advancements.

Serine protease inhibitors (serpins) are the most numerous and widespread multifunctional protease inhibitor superfamily and are expressed by all eukaryotes. Serpin E2 (serpin peptidase inhibitor, clade E, member 2), a member of the serine protease inhibitor superfamily is a potent endogenous thrombin inhibitor, mainly found in the extracellular matrix and platelets, and expressed in numerous organs and secreted by many cell types. The multiple functions of serpin E2 are mainly mediated through regulating urokinase-type plasminogen activator (uPA, also known as PLAU), tissue-type plasminogen activator (tPA, also known as PLAT), and matrix metalloproteinase activity, and include hemostasis, cell adhesion, and promotion of tumor metastasis. The importance serpin E2 is clear from its involvement in numerous physiological and pathological processes. In this review, we summarize the structural characteristics of the Serpin E2 gene and protein, as well as its roles physiology and disease.

Structure-based virtual screening towards the discovery of novel thrombin inhibitors with Anti-HCC activities.

Hepatic carcinoma (HCC) is one of the most lethal malignant tumors in the world, and new treatment regimens for this disease are urgently needed. Studies have shown that thrombin stimulates tumor progression by forming fibrin and activating platelets. Dabigatran etexilate, a thrombin inhibitor, can inhibit the activity of thrombin and prevent the proliferation and metastasis of HCC in cells and nude mice. The present study was designed to find thrombin inhibitors with novel skeletons, and further confirm the correlation between thrombin inhibition and HCC prevention to identify potential anti-HCC drug leads. The potential thrombin inhibitors were firstly screened in the Topscience Database, and 20 potential active molecules were found by molecular docking. The effect of these molecules on thrombin inhibition, coagulation and tumor proliferation were evaluated, and the definite activity of ZXX-4 was identified. Further in vivo assays in nude mice showed that ZXX-4 inhibited tumor proliferation in nude mice, reduced tumor metastasis, and enhanced the clinical efficacy of first-line drug sorafenib for the treatment of HCC. ZXX-4 can be further explored as an anti-tumor lead compound with a novel skeleton, and inhibition of thrombin can serve as a potential treatment strategy for HCC.

From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm).

Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land-living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite-II and therefore (besides the free protease) it also blocks thrombomodulin-bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild-type haemadin and the oxidation resistant Met5 → nor-Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site-directed mutagenesis, spectroscopic, ligand-displacement, and Hydrogen/Deuterium Exchange-Mass Spectrometry techniques to map the αT regions relevant for the interaction with full-length haemadin and with the synthetic N- and C-terminal peptides Haem(1-10) and Haem(45-57). Haem(1-10) competitively binds to/inhibits αT active site (KI = 1.9 μM) and its potency was enhanced by 10-fold after Phe3 → β-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (KD = 1.6 μM). Hence, we synthesized a peptide in which the sequences 1-9 and 45-57 were joined together through a 3-Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like αT inhibitors with potential pharmacological applications.

Probing naphthalene diimide and 3-hydroxypropylphosphate as end-conjugating moieties for improved thrombin binding aptamers: Structural and biological effects

The limitations associated with the in vivo use of the thrombin binding aptamer (TBA or TBA15) have dramatically stimulated the search of suitable chemically modified analogues in order to discover effective and reversible inhibitors of thrombin activity. In this context, we previously proposed cyclic and pseudo-cyclic TBA analogues with improved stability that proved to be more active than the parent aptamer.Herein, we have investigated a novel library of TBA derivatives carrying naphthalene diimide (NDI) moieties at the 3′- or 5′-end. In a subset of the investigated oligonucleotides, additional 3-hydroxypropylphosphate (HPP) groups were introduced at one or both ends of the TBA sequence. Evaluation of the G-quadruplex thermal stability, serum nuclease resistance and in vitro anticoagulant activity of the new TBA analogues allowed rationalizing the effect of these appendages on the activity of the aptamer on the basis of their relative position. Notably, most of the different TBA analogues tested were more potent thrombin inhibitors than unmodified TBA. Particularly, the analogue carrying an NDI group at the 5′-end and an HPP group at the 3′-end, named N-TBA-p, exhibited enhanced G-quadruplex thermal stability (ΔTm + 14° C) and ca. 10-fold improved nuclease resistance in serum compared to the native aptamer. N-TBA-p also induced prolonged and dose-dependent clotting times, showing a ca. 11-fold higher anticoagulant activity compared to unmodified TBA, as determined by spectroscopic methods. Overall, N-TBA-p proved to be in vitro a more efficient thrombin inhibitor than all the best ones previously investigated in our group. Its interesting features, associated with its easy preparation, make it a very promising candidate for future in vivo studies.

Aptamer-thrombin loaded magnetic microspheres for bio-specific extraction and precise detection of hirudin

Hirudin, that is naturally occurring in leeches (Hirudo medicinalis) and known as the most potent natural inhibitor of thrombin, exerts double-edged effects in clinic application. It can be used as a therapeutic ingredient for cardiovascular disease, while it can be regarded as a toxic polypeptide with bleeding risk. Effective detection of hirudin in biological samples contributes greatly to reasonable therapy. In this study, we proposed a smart adsorbent based on affinity magnetic microspheres, where thrombin was immobilized for capturing hirudin in the animal serum. Aptamer was introduced as a ligand for linking the magnetic agarose microspheres and thrombin, thereby avoiding loss of biological activity of the enzyme to hirudin. Taken recombinant hirudin variant 2-Lys47 (rHV2) as a model, we established a rapid and bio-specific extraction method coupled with liquid chromatography and quadrupole-time-of-flight mass spectrometry (LC-QTOF/MS) for determination of hirudin in the serum. Owing to this strategy, a low limit of detection (LOD) of rHV2 (0.5 nM), a good linearity with correlation coefficient of 0.9975, an acceptable precision with relative standard deviation (RSD) below 3.6% (n = 6) and acceptable recoveries ranging from 85.7% to 90.2% were achieved. Moreover, the functionalized magnetic composite could be reused for at least nine cycles. Our work combined the merits of affinity separation and advanced instrument analysis for hirudin, providing a new vision to precise determination of hirudin in medical and pharmaceutical fields.

A neutralizable dimeric anti-thrombin aptamer with potent anticoagulant activity in mice

Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote. Using an anti-thrombin DNA aptamer M08s-1 as a promising anticoagulant, its homodimer and heterodimer with TBA29 linked by a conformationally flexible linker or a rigid duplex linker were designed. The dimerized M08s-1-based aptamers had about 100-fold increased binding affinity to human and mouse thrombin compared with the monomer counterparts. Administration of these bivalent aptamers into mice revealed that the anticoagulant activity of the dimers significantly surpassed that of an approved drug for HIT treatment, argatroban. Moreover, adding protamine sulfate as an antidote against the most potent bivalent aptamer completely suppressed the anticoagulant activity of the dimer. Emerging potent and neutralizable anticoagulant aptamers will be promising candidates for HIT treatment with a higher safety profile.

Integration of fluorescence and MALDI imaging for microfluidic chip-based screening of potential thrombin inhibitors from natural products

The microfluidic technology provides an ideal platform for in situ screening of enzyme inhibitors and activators from natural products. This work described a surface-modified ITO glass-PDMS hybrid microfluidic chip for evaluating thrombin interaction with its potential inhibitors by fluorescence imaging and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). The fluorescence-labeled substrate was immobilized on a conductive ITO glass slide coated with gold nanoparticles/thiol-β-cyclodextrin modified TiO2 nanowires (Au-β-CD@TiO2 NWs) via Au–S bonds. A PDMS microchannel plate was placed on top of the modified ITO slide. The premixed solutions of thrombin and candidate thrombin inhibitors were infused into the microchannels to form a microreactor environment. The enzymatic reaction was rapidly monitored by fluorescence microscopy, and MALDI MS was used to validate and quantify the enzymatic hydrolysate of thrombin to determine the enzyme kinetic process and inhibitory activities of selected flavonoids. The fluorescence and MALDI MS results showed that luteolin, cynaroside, and baicalin have good thrombin inhibitory activity and their half-maximal inhibitory concentrations (IC50) were below 30 μM. The integration of fluorescence imaging and MALDI MSI for in situ monitoring and quantifying the enzymatic reaction in a microfluidic chip is capable of rapid and accurate screening of thrombin inhibitors from natural products.

Purification and characterization of thrombin from camel plasma: interaction with camel tick salivary gland thrombin inhibitor

BackgroundThrombin is the most important enzyme in the hemostatic process by permitting rapid and localized coagulation in case of tissue damage. Camel thrombin is the natural and proper target enzyme for the previously purified camel tick salivary gland thrombin inhibitor. ResultsIn this study, the camel thrombin was purified homogenously in a single affinity chromatographic step on the heparin-agarose affinity column with a specific activity of 3242 NIH units/mg proteins. On SDS-PAGE, the purified camel thrombin contained two forms, 37 kDa α-thrombin and 28 kDa β-thrombin, and the camel prothrombin was visualized as 72 kDa. The camel thrombin Km value was found out as 60 µM of N-(p-Tosyl)-Gly-Pro-Arg-p-nitroanilide acetate and displayed its optimum activity at pH 8.3. The PMSF was the most potent inhibitor of camel thrombin. Camel tick salivary gland thrombin inhibitor has two binding sites on camel thrombin and inhibited it competitively with Ki value of 0.45 µM. ConclusionsThe purified camel thrombin was found to be more susceptible toward the camel tick salivary gland thrombin inhibitor than bovine thrombin.

Discovery of novel N-acylpyrazoles as potent and selective thrombin inhibitors

Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.

Unleashing the potential of noncanonical amino acid biosynthesis to create cells with precision tyrosine sulfation

Despite the great promise of genetic code expansion technology to modulate structures and functions of proteins, external addition of ncAAs is required in most cases and it often limits the utility of genetic code expansion technology, especially to noncanonical amino acids (ncAAs) with poor membrane internalization. Here, we report the creation of autonomous cells, both prokaryotic and eukaryotic, with the ability to biosynthesize and genetically encode sulfotyrosine (sTyr), an important protein post-translational modification with low membrane permeability. These engineered cells can produce site-specifically sulfated proteins at a higher yield than cells fed exogenously with the highest level of sTyr reported in the literature. We use these autonomous cells to prepare highly potent thrombin inhibitors with site-specific sulfation. By enhancing ncAA incorporation efficiency, this added ability of cells to biosynthesize ncAAs and genetically incorporate them into proteins greatly extends the utility of genetic code expansion methods.

Tyrosine-O-sulfation is a widespread affinity enhancer among thrombin interactors.

Tyrosine-O-sulfation is a common post-translational modification (PTM) of proteins following the cellular secretory pathway. First described in human fibrinogen, tyrosine-O-sulfation has long been associated with the modulation of protein-protein interactions in several physiological processes. A number of relevant interactions for hemostasis are largely dictated by this PTM, many of which involving the serine proteinase thrombin (FIIa), a central player in the blood-clotting cascade. Tyrosine sulfation is not limited to endogenous FIIa ligands and has also been found in hirudin, a well-known and potent thrombin inhibitor from the medicinal leech, Hirudo medicinalis. The discovery of hirudin led to successful clinical application of analogs of leech-inspired molecules, but also unveiled several other natural thrombin-directed anticoagulant molecules, many of which undergo tyrosine-O-sulfation. The presence of this PTM has been shown to enhance the anticoagulant properties of these peptides from a range of blood-feeding organisms, including ticks, mosquitos and flies. Interestingly, some of these molecules display mechanisms of action that mimic those of thrombin's bona fide substrates.

Discovery of Novel N-Acylpyrazoles as Potent and Selective Thrombin Inhibitors

Discovery of Novel N-Acylpyrazoles as Potent and Selective Thrombin Inhibitors

In vivo Anticoagulant Activity of Immediate Release Tablets of Dabigatran Etexilate Mesylate Cocrystals

Dabigatran Etexilate Mesylate (DEM), a salt of prodrug dabigatran etexilate, is a potent, oral, reversible and direct thrombin inhibitor with low oral bioavailability. The present research investigation focused on the formulation of immediate release (IR) tablets of DEM cocrystals and evaluation of In vivo anticoagulant activity. The results of the study showed that the formulated IR tablets of DEM showed improved efficacy in comparison with the plain drug by enhancing the pre-compression parameters such as bulk density, tap density, Carr's index, angle of repose and Hausner's ratio and post-compression parameters like thickness and weight variation, hardness and friability, In vitro dissolution parameters. The improved efficacy was confirmed by improvement in the pharmacodynamic parameters such as cutaneous bleeding time and clotting time indicative of enhanced bioavailability of dabigatran. Thus, it can be concluded that the IR tablets of dabigatran cocrystals can be proven to be more effective in producing the anticoagulant effect in clinical practice as compared to the plain drug resulting in more patient compliance.

Abstract 13192: Local Delivery of Thrombin Inhibiting Nanoparticles Preserves Cardiac Function and Modulates NLRP3 Inflammasome Processes in Acute Myocardial Infarction

Introduction: The role of thrombin in acute myocardial infarction (AMI) has been well described, with recent attention having turned to its effect on NLRP3 inflammasome activation and the role that plays in the pathogenesis of AMI. Perfluorocarbon nanoparticles conjugated to phenylalanine-proline-arginine-chloromethylketone (PPACK NPs) are extremely potent local inhibitors of thrombin, but their role in therapy for AMI remains unclear. We hypothesize that pre-treatment with sustained locally acting PPACK NPs preserves cardiac function by attenuating thrombin-induced inflammatory processes, including those driven by the NLRP3 inflammasome. Methods: Nine mice underwent left anterior descending (LAD) occlusion for 45 minutes. At 30 minutes before LAD occlusion, mice were injected with PPACK (n=5) or control NPs (n=4). 24 hours post-reperfusion, hearts were excised and perfused. LAD was re-occluded and Lectin-488 perfused through the excised heart. Echo was performed pre- and post-reperfusion to assess cardiac function. Hearts were sectioned for follow up histological and protein analysis. Results: PPACK NP treated mice exhibited evidence of preserved cardiac function 24 hours post-reperfusion (Figure 1, A-B), reduced nuclear translocation of p65 in infarcted myocardium (Figure 1, C-D), and reduced inflammatory markers with better vessel preservation in the area adjacent to uninjured myocardium (Figure 1, E-F). The PPACK NP group also demonstrated suppression of NLRP3 inflammasome activation (Figure 2). Conclusion: Local inhibition of thrombin with anti-inflammatory PPACK NPs preserves cardiac function when administered systemically as pre-treatment.