Anti-Leishmania amazonensis activity of the marine sponge Dercitus (Stoeba) latex (Porifera) from São Pedro and São Paulo Archipelago, Pernambuco, Brazil.

The search for new therapeutic strategies for leishmaniasis treatment is essential due to the side effects of available drugs and the increasing incidence of resistance to them. Marine sponges use chemical compounds as a defense mechanism, and several of them present interesting pharmacological properties. The aim of this study was to evaluate the in vitro activity of the aqueous extract of the marine sponge Dercitus (Stoeba) latex against Leishmania amazonensis. MIC and toxicity against mammal cells were evaluated through broth microdilution assays. Transmission electron microscopy analysis was performed to assess possible effects on L. amazonensis ultrastructure. Arginase and proteolytic activities were measured by spectrometric methodologies. The extract of Dercitus (Stoeba) latex displayed antileishmanial activity and moderate toxicity against peritonial macrophages. Ultrastructural changes were observed after the growth of L. amazonensis promastigotes in the presence of the extract at 150 µg.ml-1 (IC50), mainly on acidocalcysomes. The extract was able to inhibit the activity of arginase and serine proteases. This study shows that Dercitus (Stoeba) latex aqueous extract may be a novel potential source of protozoa protease inhibitors and drugs that are less toxic to be used in the treatment of L. amazonensis infections.

Enzymes in the time of COVID-19: An overview about the effects in the human body, enzyme market, and perspectives for new drugs.

The rising pandemic caused by a coronavirus, resulted in a scientific quest to discover some effective treatments against its etiologic agent, the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2). This research represented a significant scientific landmark and resulted in many medical advances. However, efforts to understand the viral mechanism of action and how the human body machinery is subverted during the infection are still ongoing. Herein, we contributed to this field with this compilation of the roles of both viral and human enzymes in the context of SARS-CoV-2 infection. In this sense, this overview reports that proteases are vital for the infection to take place: from SARS-CoV-2 perspective, the main protease (Mpro ) and papain-like protease (PLpro ) are highlighted; from the human body, angiotensin-converting enzyme-2, transmembrane serine protease-2, and cathepsins (CatB/L) are pointed out. In addition, the influence of the virus on other enzymes is reported as the JAK/STAT pathway and the levels of lipase, enzymes from the cholesterol metabolism pathway, amylase, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, and glyceraldehyde 3-phosphate dehydrogenase are also be disturbed in SARS-CoV-2 infection. Finally, this paper discusses the importance of detailed enzymatic studies for future treatments against SARS-CoV-2, and how some issues related to the syndrome treatment can create opportunities in the biotechnological market of enzymes and the development of new drugs.

Bitis arietans Snake Venom and Kn-Ba, a Snake Venom Serine Protease, Induce the Production of Inflammatory Mediators in THP-1 Macrophages.

Bitis arietans is a snake of medical importance found throughout sub-Saharan Africa and in savannas and pastures of Morocco and western Arabia. The effects of its venom are characterized by local and systemic alterations, such as inflammation and cardiovascular and hemostatic disturbances, which can lead to victims' death or permanent disability. To better characterize the inflammatory process induced by this snake's venom, the participation of eicosanoids and PAF (platelet- activating factor) in this response were demonstrated in a previous study. In addition, edema and early increased vascular permeability followed by an accumulation of polymorphonuclear (PMN) cells in the peritoneal cavity were accompanied by the production of the eicosanoids LTB4, LTC4, TXB2, and PGE2, and local and systemic production of IL-6 and MCP-1. In this context, the present study focused on the identification of inflammatory mediators produced by human macrophages derived from THP-1 cells in response to Bitis arietans venom (BaV), and Kn-Ba, a serine protease purified from this venom. Here, we show that Kn-Ba, and even the less intensive BaV, induced the production of the cytokine TNF and the chemokines RANTES and IL-8. Only Kn-Ba was able to induce the production of IL-6, MCP-1, and IP-10, whereas PGE2 was produced only in response to BaV. Finally, the release of IL-1ß in culture supernatants suggests the activation of the inflammasomes by the venom of Bitis arietans and by Kn-Ba, which will be investigated in more detail in future studies.

Expression of recombinant protease MarP from Mycobacterium tuberculosis in Pichia pastoris and its effect on human monocytes.

OBJECTIVE: Mycobacterial acid-resistant protease (MarP) is a membrane-associated serine protease involved in the survival of Mycobacterium tuberculosis in macrophages; here we produced MarP in the yeast Pichia pastoris and study its involvement in macrophage immune modulation. RESULTS: Pichia pastoris vectors, harboring a full-length or a partial sequence of MarP, were constructed. GS115 clones were selected, and homologous recombination at the AOX1 locus was assessed by PCR. Protein was purified by nickel affinity chromatography, and its effect on the cytokine profile was tested in human monocytes. Only the partial MarP protein (121-397 a.a.) lacking the transmembrane domain was successfully expressed as an N-glycosylated proteolytically active protease. In vitro stimulation of THP-1 cells with MarP promoted the release of TNF-α and IL-10. CONCLUSION: Mycobacterial MarP was successfully expressed in P. pastoris, and it is capable of cytokine release in vitro.

Functional Mining of the Crotalus Spp. Venom Protease Repertoire Reveals Potential for Chronic Wound Therapeutics.

Chronic wounds are a major health problem that cause millions of dollars in expenses every year. Among all the treatments used, active wound treatments such as enzymatic treatments represent a cheaper and specific option with a fast growth category in the market. In particular, bacterial and plant proteases have been employed due to their homology to human proteases, which drive the normal wound healing process. However, the use of these proteases has demonstrated results with low reproducibility. Therefore, alternative sources of proteases such as snake venom have been proposed. Here, we performed a functional mining of proteases from rattlesnakes (Crotalus ornatus, C. molossus nigrescens, C. scutulatus, and C. atrox) due to their high protease predominance and similarity to native proteases. To characterize Crotalus spp. Proteases, we performed different protease assays to measure and confirm the presence of metalloproteases and serine proteases, such as the universal protease assay and zymography, using several substrates such as gelatin, casein, hemoglobin, L-TAME, fibrinogen, and fibrin. We found that all our venom extracts degraded casein, gelatin, L-TAME, fibrinogen, and fibrin, but not hemoglobin. Crotalus ornatus and C. m. nigrescens extracts were the most proteolytic venoms among the samples. Particularly, C. ornatus predominantly possessed low molecular weight proteases (P-I metalloproteases). Our results demonstrated the presence of metalloproteases capable of degrading gelatin (a collagen derivative) and fibrin clots, whereas serine proteases were capable of degrading fibrinogen-generating fibrin clots, mimicking thrombin activity. Moreover, we demonstrated that Crotalus spp. are a valuable source of proteases that can aid chronic wound-healing treatments.

New Insights on Moojase, a Thrombin-Like Serine Protease from Bothrops moojeni Snake Venom.

Snake venom serine proteases (SVSPs) are enzymes that are capable of interfering in various parts of the blood coagulation cascade, which makes them interesting candidates for the development of new therapeutic drugs. Herein, we isolated and characterized Moojase, a potent coagulant enzyme from Bothrops moojeni snake venom. The toxin was isolated from the crude venom using a two-step chromatographic procedure. Moojase is a glycoprotein with N-linked glycans, molecular mass of 30.3 kDa and acidic character (pI 5.80⁻6.88). Sequencing of Moojase indicated that it is an isoform of Batroxobin. Moojase was able to clot platelet-poor plasma and fibrinogen solutions in a dose-dependent manner, indicating thrombin-like properties. Moojase also rapidly induced the proteolysis of the Aα chains of human fibrinogen, followed by the degradation of the Bß chains after extended periods of incubation, and these effects were inhibited by PMSF, SDS and DTT, but not by benzamidine or EDTA. RP-HPLC analysis of its fibrinogenolysis confirmed the main generation of fibrinopeptide A. Moojase also induced the fibrinolysis of fibrin clots formed in vitro, and the aggregation of washed platelets, as well as significant amidolytic activity on substrates for thrombin, plasma kallikrein, factor Xia, and factor XIIa. Furthermore, thermofluor analyses and the esterase activity of Moojase demonstrated its very high stability at different pH buffers and temperatures. Thus, studies such as this for Moojase should increase knowledge on SVSPs, allowing their bioprospection as valuable prototypes in the development of new drugs, or as biotechnological tools.

A serine protease isolated from the bristles of the Amazonic caterpillar, Premolis semirufa, is a potent complement system activator.

BACKGROUND: The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called "Pararamose", characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa's bristles extract could interfere with the human complement system. RESULTS: The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly interfere with the activity of the Ps82, whereas the presence of PMSF, serine protease inhibitor, totally blocked the activity. CONCLUSION: These data show that a serine protease present in the Premolis semirufa's bristles extract has the ability to activate the complement system, which may contribute to the inflammatory process presented in humans after envenomation.

Rapid purification of serine proteinases from Bothrops alternatus and Bothrops moojeni venoms.

Envenomation by Bothrops species results, among other symptoms, in hemostatic disturbances. These changes can be ascribed to the presence of enzymes, primarily serine proteinases some of which are structurally similar to thrombin and specifically cleave fibrinogen releasing fibrinopeptides. A rapid, three-step, chromatographic procedure was developed to routinely purify serine proteinases from the venoms of Bothrops alternatus and Bothrops moojeni. The serine proteinase from B. alternatus displays an apparent molecular mass of ~32 kDa whereas the two closely related serine proteinases from B. moojeni display apparent molecular masses of ~32 kDa and ~35 kDa in SDS-PAGE gels. The partial sequences indicated that these enzymes share high identity with serine proteinases from the venoms of other Bothrops species. These proteins coagulate plasma and possess fibrinogenolytic activity but lack fibrinolytic activity.

Rapid purification and procoagulant and platelet aggregating activities of Rhombeobin: a thrombin-like/gyroxin-like enzyme from Lachesis muta rhombeata snake venom.

We report a rapid purification method using one-step chromatography of SVSP Rhombeobin (LMR-47) from Lachesis muta rhombeata venom and its procoagulant activities and effects on platelet aggregation. The venom was fractionated by a single chromatographic step in RP-HPLC on a C8 Discovery BIO Wide Pore, showing high degree of molecular homogeneity with molecular mass of 47035.49 Da. Rhombeobin showed amidolytic activity upon BA ρ NA, with a broad optimum pH (7-10) and was stable in solution up to 60°C. The amidolytic activity was inhibited by serine proteinase inhibitors and reducing agents, but not chelating agents. Rhombeobin showed high coagulant activity on mice plasma and bovine fibrinogen. The deduced amino acid sequence of Rhombeobin showed homology with other SVSPs, especially with LM-TL (L. m. muta) and Gyroxin (C. d. terrificus). Rhombeobin acts, in vitro, as a strong procoagulant enzyme on mice citrated plasma, shortening the APTT and PT tests in adose-dependent manner. The protein showed, "ex vivo", a strong defibrinogenating effect with 1 µg/animal. Lower doses activated the intrinsic and extrinsic coagulation pathways and impaired the platelet aggregation induced by ADP. Thus, this is the first report of a venom component that produces a venom-induced consumptive coagulopathy (VICC).

The long road of research on snake venom serine proteinases.

It has long been recognized that snake venom serine proteinases (SVSPs) affect various physiological functions including blood coagulation, fibrinolysis, blood pressure and platelet aggregation. Therefore, SVSPs have been used as refined tools to study molecular mechanisms involved in the activation of key factors that control hemostasis and as therapeutic agents in various thrombotic and hemostatic conditions. The aim of this review is to highlight the state of our knowledge on the advances made in SVSP research since the 18th century. It includes the personal accounts of some distinguished scientists that addressed specific problems and contributed to advance the field.

In vitro activity of a serine protease from Monacrosporium thaumasium fungus against first-stage larvae of Angiostrongylus vasorum.

A serine protease from the nematophagous fungus Monacrosporium thaumasium (NF34a) was purified, partially characterized and tested in vitro in control of the first larval stage of Angiostrongylus vasorum. NF34a grew in liquid culture medium, producing its crude extract that was purified by ion exchange chromatography. The fractions with high protease activity were collected in a pool, and elution of proteases was monitored by enzymatic assay and protein content. Purification steps were monitored by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Protease activity was determined under different pH and temperature conditions, and the inhibitor effects of metal ions and phenylmethylsulfonyl fluoride (PMSF) were assessed. In an experimental test, the infection process of NF34a on first-stage larvae of A. vasorum was investigated. A purified serine protease (Mt1) was identified, with an approximate molecular mass of 40 kDa and apparent homogeneity in SDS-PAGE, having optimal activity at pH 7.0 to 8.0 and temperature of 60°C. Mg(2+) and Zn(2+) partially inhibited the activity of Mt1 while PMSF inhibited it completely. Mt1 production was observed when NF34a was grown using first-stage larvae of A. vasorum as the only source of carbon and nitrogen. These results show that the enzyme may have a possible role in the infection process of the larvae. In the in vitro test of applicability against A. vasorum L(1), we observed a reduction in the number of larvae of 23.9% (p < 0.05) in the group treated with Mt1 compared with the control group. However, even this low reduction demonstrates that the Mt1 is important in the infection process.

Bhalternin: Functional and structural characterization of a new thrombin-like enzyme from Bothrops alternatus snake venom.

A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.

Isolation and characterization of a new serine protease with thrombin-like activity (TLBm) from the venom of the snake Bothrops marajoensis.

The thrombin-like serine protease TLBm from Bothrops marajoensis was isolated in one chromatographic step in reverse phase HPLC. Its molecular mass was 33239.95 Da, as based on the determined primary structure and confirmed experimentally by MALDI-TOF mass spectrometry (33332.5 Da) and it contains 12 half-cysteine residues. This TLBm exhibited high specificity for BArhoNA, Michaelis-Menten behavior with K(m) 2.3x10(-1)M and the V(max) 0.52x10(-1) nmoles rho-NA/lt/min for this substrate. TLBm also showed ability to coagulate bovine fibrinogen and was inhibited by soybean trypsin inhibitor, EDTA and S(Dm) from the serum of the species Didelphis marsupialis. The primary structure of TLBm showed the presence of His(45), Asp(103) and Ser(228) residues in the corresponding positions of the catalytic triad established in the serine proteases and Ser(228) are inhibited by phenylmethylsulfonyl fluoride (PMSF). Amino acid analysis showed a high content of Asp, Glu, Gly, Ser, Ala and Pro as well as 12 half-cysteine residues and calculated pI of 6.47; TLBm presented 285 amino acid residues. In this work, we investigated the ability of TLBm to degrade fibrinogen and we observed that it is able to cause alpha- and beta-chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with PMSF, a specific inhibitor of serine protease. Also, TLBm induced platelet aggregation in washed and platelet-rich plasma, and in both cases, PMSF inhibited its activity.