RESUMO
The coagulation cascade is the process of the conversion of soluble fibrinogen to insoluble fibrin that terminates in production of a clot. Factor Xa (FXa) is a serine protease involved in the blood coagulation cascade. Moreover, FXa plays a vital role in the enzymatic sequence which ends with the thrombus production. Thrombosis is a common causal pathology for three widespread cardiovascular syndromes: acute coronary syndrome (ACS), venous thromboembolism (VTE), and strokes. In this research a series of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives as a potential factor Xa (FXa) inhibitor were designed, synthesized, and evaluated for their FXa inhibitor activity, cytotoxicity activity and coagulation parameters. Rational design for the desired novel molecules was performed through protein-ligand complexes selection and ligand clustering. The microwave-assisted synthetic strategy of selected compounds was carried out by using Ullmann-Goldberg, N-propargylation, Mannich addition, Friedel-Crafts, and 1,3-dipolar cycloaddition type reactions under microwave irradiation. The microwave methodology proved to be an efficient way to obtain all novel compounds in high yields (73-93%). Furthermore, a thermochemical analysis, optimization and reactivity indexes such as electronic chemical potential (µ), chemical hardness (η), and electrophilicity (ω) were performed to understand the relationship between the structure and the energetic behavior of all the series. Then, in vitro analysis showed that compounds 27, 29-31, and 34 exhibited inhibitory activity against FXa and the corresponding half maximal inhibitory concentration (IC50) values were calculated. Next, a cell viability assay in HEK293 and HepG2 cell lines, and coagulation parameters (anti FXa, Prothrombin time (PT), activated Partial Thromboplastin Time (aPTT)) of the most active novel molecules were performed to determine the corresponding cytotoxicity and possible action on clotting pathways. The obtained results suggest that compounds 27 and 29 inhibited FXa targeting through coagulation factors in the intrinsic and extrinsic pathways. However, compound 34 may target coagulation FXa mainly by the extrinsic and common pathway. Interestingly, the most active compounds in relation to the inhibition activity against FXa and coagulation parameters did not show toxicity at the performed coagulation assay concentrations. Finally, docking studies confirmed the preferential binding mode of N-propargyltetrahydroquinoline and 1,2,3-triazole derivatives inside the active site of FXa.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Inibidores do Fator Xa/síntese química , Inibidores do Fator Xa/farmacologia , Fator Xa/química , Quinolinas/química , Triazóis/química , Compostos de Anilina/síntese química , Compostos de Anilina/química , Azidas/síntese química , Azidas/química , Testes de Coagulação Sanguínea , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Fator Xa/metabolismo , Inibidores do Fator Xa/química , Humanos , Concentração Inibidora 50 , Ligantes , Micro-Ondas , Simulação de Acoplamento Molecular , Quinolinas/síntese química , Triazóis/síntese químicaRESUMO
Unfractionated heparin (UFH) and their low-molecular-weight derivatives are sourced almost exclusively from porcine mucosa (HPI); however, a worldwide introduction of UFH from bovine mucosa (HBI) has been recommended to reinforce the currently unsteady supply chain of heparin products. Although HBI has different chemical composition and about half of the anticoagulant potency of HPI (â¼100 and â¼180 international unit [IU]/mg, respectively), they have been employed as interchangeable UFHs in some countries since the 1990s. However, their use as a single drug provoked several bleeding incidents in Brazil, which precipitated the publication of the first monographs exclusive for HBI and HPI by the Brazilian Pharmacopoeia. Nevertheless, we succeed in producing with high-resolution anion-exchange chromatography a novel HBI derivative with anticoagulant potency (200 IU/mg), disaccharide composition (enriched in N,6-disulfated α-glucosamine) and safety profile (bleeding and heparin-induced thrombocytopaenia potentials and protamine neutralization) similar to those seen in the gold standard HPI. Therefore, we show that it is possible to equalize the composition and pharmacological characteristics of these distinct UFHs by employing an easily implementable improvement in the HBI manufacturing.
Assuntos
Anticoagulantes/química , Heparina/química , Mucosa Intestinal/metabolismo , Tromboembolia/tratamento farmacológico , Tromboembolia/prevenção & controle , Animais , Ânions , Anticoagulantes/uso terapêutico , Bovinos , Cromatografia por Troca Iônica , Composição de Medicamentos/métodos , Fator Xa/química , Heparina/uso terapêutico , Heparina de Baixo Peso Molecular/química , Humanos , Tempo de Tromboplastina Parcial , Ligação Proteica , Protrombina/química , Suínos , Equivalência TerapêuticaRESUMO
Antithrombin (AT) is a serpin that inhibits mainly thrombin and fXa after being activated by binding to glycosaminoglycans as heparin and heparan sulfate. Upon binding, the native AT conformation, relatively inactive as a protease inhibitor, is converted to an activated form. Recently, a new compound, named TMI, was discovered in our group with nanomolar affinity to antithrombin, and shown to be able to induce a partial activation of antithrombin. As TMI represents an original scaffold for structural optimizations aiming the development of new antithrombotic drugs, the present work demonstrated, through a series of molecular dynamics simulations, that TMI is able to modulate AT reactive center loop flexibility similarly to what is observed to heparin, as well as exposing AT P1 residue, Arg393. These results represent the first atomic level indication of AT conformational activation by TMI, and may offer a predictive basis for future studies aiming TMI structural optimization.
Assuntos
Antitrombinas/química , Ativadores de Enzimas/química , Fibrinolíticos/química , Heparina/química , Heparitina Sulfato/química , Fosfatos de Inositol/química , Regulação Alostérica , Antitrombinas/metabolismo , Sítios de Ligação , Desenho de Fármacos , Ativadores de Enzimas/metabolismo , Fator Xa/química , Fibrinolíticos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Humanos , Fosfatos de Inositol/metabolismo , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Termodinâmica , Trombina/químicaRESUMO
Factor Xa (FXa), a vitamin K-dependent serine protease plays a pivotal role in the coagulation cascade, one of the most interesting targets for the development of new anticoagulants. In the present work, we performed a virtual screening campaign based on ligand-based shape and electrostatic similarity search and protein-ligand docking to discover novel FXa-targeted scaffolds for further development of inhibitors. From an initial set of 260,000 compounds from the NCI Open database, 30 potential FXa inhibitors were identified and selected for in vitro biological evaluation. Compound 5 (NSC635393, 4-(3-methyl-4H-1,4-benzothiazin-2-yl)-2,4-dioxo-N-phenylbutanamide) displayed an IC50 value of 2.02 nM against human FXa. The identified compound may serve as starting point for the development of novel FXa inhibitors.
Assuntos
Inibidores do Fator Xa/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Bases de Dados Factuais , Inibidores Enzimáticos/farmacologia , Fator Xa/química , Fator Xa/metabolismo , Simulação de Acoplamento Molecular , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Thrombin is a multifunctional enzyme with a key role in the coagulation cascade. Its functional modulation can culminate into normal blood coagulation or thrombosis. Thus, the identification of novel potent inhibitors of thrombin are of immense importance. Sculptin is the first specific thrombin inhibitor identified in the transcriptomics analysis of tick's salivary glands. It consists of 168 residues having four similar repeats and evolutionary diverged from hirudin. Sculptin is a competitive, specific and reversible inhibitor of thrombin with a Ki of 18.3 ± 1.9 pM (k on 4.04 ± 0.03 × 107 M-1 s-1 and k off 0.65 ± 0.04 × 10-3 s-1). It is slowly consumed by thrombin eventually losing its activity. Contrary, sculptin is hydrolyzed by factor Xa and each polypeptide fragment is able to inhibit thrombin independently. A single domain of sculptin alone retains ~45% of inhibitory activity, which could bind thrombin in a bivalent fashion. The formation of a small turn/helical-like structure by active site binding residues of sculptin might have made it a more potent thrombin inhibitor. In addition, sculptin prolongs global coagulation parameters. In conclusion, sculptin and its independent domain(s) have strong potential to become novel antithrombotic therapeutics.
Assuntos
Fibrinolíticos/química , Hirudinas/química , Fragmentos de Peptídeos/química , Peptídeos/química , Trombose/prevenção & controle , Animais , Ligação Competitiva , Coagulação Sanguínea/fisiologia , Domínio Catalítico , Cristalografia por Raios X , Fator Xa/química , Fator Xa/metabolismo , Fibrinolíticos/metabolismo , Expressão Gênica , Hirudinas/genética , Hirudinas/metabolismo , Humanos , Hidrólise , Ixodidae/química , Cinética , Modelos Moleculares , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Filogenia , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Trombose/sangue , Trombose/patologiaRESUMO
A series of novel tetrahydropyrazolopyridone derivatives containing 1,3,4-triazole, triazolylmethyl, and partially saturated heterocyclic moieties as P2 binding element was designed, synthesized, and evaluated in vitro for anticoagulant activity in human and rabbit plasma. All compounds showed moderate to significant potency, and compounds 15b, 15c, 20b, 20c, and 22b were further examined for their inhibitory activity against human FXa in vitro. While compounds 15c and 22b were tested for rat venous thrombosis in vivo. The most promising compound 15c, with an IC50 (FXa) value of 0.14µM and 98% inhibition rate, warranted further investigation as an FXa inhibitor.
Assuntos
Anticoagulantes/síntese química , Desenho de Fármacos , Inibidores do Fator Xa/síntese química , Fator Xa/química , Pirazóis/química , Piridinas/química , Animais , Anticoagulantes/farmacologia , Anticoagulantes/uso terapêutico , Sítios de Ligação , Coagulação Sanguínea/efeitos dos fármacos , Domínio Catalítico , Fator Xa/metabolismo , Inibidores do Fator Xa/farmacologia , Inibidores do Fator Xa/uso terapêutico , Humanos , Simulação de Acoplamento Molecular , Ligação Proteica , Pirazóis/farmacologia , Pirazóis/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Coelhos , Ratos , Relação Estrutura-Atividade , Trombose Venosa/tratamento farmacológicoRESUMO
There is a clinical need to develop safe therapeutic strategies to mitigate bleeding. Previously, we found that a novel zymogen-like factor Xa variant (FXa-I16L) was effective in correcting the coagulation defect in hemophilic mice. Here we expand the mutational framework to tune the FX(a) zymogen-like state. Alteration of FXa zymogenicity yields variants (V17M, I16L, I16M, V17T, V17S, and I16T) with a wide range (≤1000-fold) of reduced function toward physiologic substrates and inhibitors. The extent of zymogen-like character, including resistance to antithrombin III, correlates well with plasma half-life (<2 minutes to >4 hours). Importantly, biologic function, including that of the most zymogen-like variant (FXa-I16T), was greatly enhanced when bound to FVa membranes. This resulted in improvement of clotting times and thrombin generation in hemophilic plasma. The FXa variants were remarkably effective in mouse injury models. In these systems, the data show that the more active the protease, the more difficult it is to overcome the protective mechanism of circulating inhibitors to achieve a therapeutic benefit. Depending on the treatment situation, the more zymogen-like variants (V17S and I16T) were most useful when given before injury whereas variants exhibiting greater activity but shorter half-lives (I16L and I16M) were most effective when administered after injury. This new class of FXa variants provides a useful and flexible platform for selectively bioengineering biologic function and half-life to target different clinical bleeding scenarios.
Assuntos
Precursores Enzimáticos , Fator Xa , Hemostáticos/isolamento & purificação , Animais , Coagulação Sanguínea/efeitos dos fármacos , Domínio Catalítico , Análise Mutacional de DNA , Precursores Enzimáticos/química , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Fator Xa/química , Fator Xa/genética , Fator Xa/metabolismo , Meia-Vida , Hemofilia A/sangue , Hemofilia A/tratamento farmacológico , Hemostáticos/síntese química , Hemostáticos/química , Hemostáticos/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Mutantes/uso terapêutico , Protrombina/metabolismo , Tromboplastina/químicaRESUMO
fIIa and fXa are two of the main targets of antithrombin, a serine proteases inhibitor that plays a major role in the regulation of blood clotting. The formation of ternary complexes between such molecules and glycosaminoglycans, as heparin, is the main path for inhibiting those enzymes, which may occur through two distinct mechanisms of action. While these serine proteases present distinct susceptibilities to these paths, in which fIIa demands an interaction with heparin, neither the molecular basis of this differential inhibition nor the role of fIIa glycosylation on this process is fully understood. Thus, the present work evaluated through molecular dynamics simulations the effects of glycosylation on fIIa and the consequences of heparin binding to both proteases function and dynamics. Based on the obtained data, fIIa N-linked glycan promoted an increase in the active site pocket size by stabilizing regions that encircle it, while heparin binding was observed to reverse such an effect. Additionally, heparin orientation observed on the surface of fIIa, but not fXa, allows a linear long-chain heparin binding to antithrombin in ternary complexes. Finally, the enzymes catalytic triad organization was disrupted due to a strong glycosaminoglycan binding to the proteases exosite 2. Such data support an atomic-level explanation for the higher inhibition constant of the antithrombin-heparin complex over fIIa than fXa, as well as for the different susceptibilities of those enzymes for antithrombin mechanisms of action.
Assuntos
Coagulação Sanguínea , Fator Xa/química , Heparina/química , Simulação de Dinâmica Molecular , Trombina/química , Domínio Catalítico , Fator Xa/metabolismo , Inibidores do Fator Xa , Heparina/metabolismo , Humanos , Trombina/antagonistas & inibidores , Trombina/metabolismoRESUMO
The structural characterization and the anticoagulant potential of a novel heparin/heparan sulfate-like compound from the heads of Litopenaeus vannamei shrimp are described. While it is distinct from either heparin or heparan sulfate, enzymatic depolymerization and nuclear magnetic resonance spectroscopy analyses revealed that this molecule does share some structural features with heparin, such as the high degree of N- and 6-O-sulfation and minor N-acetylation, and with heparan sulfate, in the glucuronic acid content. Its ability to stabilize human antithrombin explains its significant anticoagulant activity in aPTT and Factor-Xa inhibition assays. Interestingly, in contrast to mammalian heparin, the shrimp compound displayed negligible hemorrhagic effect. Together, these findings have particular interest since they reveal a novel molecule with significant anti-Xa activity coupled with low bleeding effects which make the shrimp heparin/HS-like compound a potential alternative for mammalian heparin.
Assuntos
Anticoagulantes/química , Hemorragia/prevenção & controle , Heparina/química , Heparitina Sulfato/química , Penaeidae/química , Acetilação , Animais , Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Antitrombinas/antagonistas & inibidores , Antitrombinas/química , Antitrombinas/isolamento & purificação , Bovinos , Cromatografia por Troca Iônica , Fator Xa/química , Inibidores do Fator Xa , Ácido Glucurônico/química , Cabeça , Heparina/isolamento & purificação , Heparina/farmacologia , Heparitina Sulfato/isolamento & purificação , Heparitina Sulfato/farmacologia , Humanos , Intestinos/química , Pâncreas/química , Tempo de Tromboplastina Parcial , Ratos , Suínos , Cauda/irrigação sanguínea , Cauda/efeitos dos fármacosRESUMO
Bioactive compounds of great interest are found in the saliva of hematophagous organisms. While exploring a cDNA library derived from the salivary glands of the tick Amblyomma cajennense, a transcript that codes for a protein with unique structure (containing an N-terminal Kunitz-type domain and a C-terminus with no homology to any annotated sequences) was found. The recombinant mature form of this protein ( approximately 13.5kDa) was produced in Escherichia coli BL21 (DE3), and it was able to inhibit Factor Xa (FXa) and extend global blood clotting times in vitro and ex vivo. Static and dynamic predictions of its tertiary structure indicate regions that may be related to its FXa inhibitor function.