A Crystallographic Snapshot of SARS-CoV-2 Main Protease Maturation Process.

SARS-CoV-2 is the causative agent of COVID-19. The dimeric form of the viral Mpro is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. The lack of structural information for intermediary forms of Mpro is a setback for the understanding its self-maturation process. Herein, we used X-ray crystallography combined with biochemical data to characterize multiple forms of SARS-CoV-2 Mpro. For the immature form, we show that extra N-terminal residues caused conformational changes in the positioning of domain-three over the active site, hampering the dimerization and diminishing its activity. We propose that this form preludes the cis and trans-cleavage of N-terminal residues. Using fragment screening, we probe new cavities in this form which can be used to guide therapeutic development. Furthermore, we characterized a serine site-directed mutant of the Mpro bound to its endogenous N and C-terminal residues during dimeric association stage of the maturation process. We suggest this form is a transitional state during the C-terminal trans-cleavage. This data sheds light in the structural modifications of the SARS-CoV-2 main protease during its self-maturation process.

Technetium-99m-labeled lapachol as an imaging probe for breast tumor identification. / Lapachol marcado con tecnecio 99m como sonda de imágenes para la identificación de tumores de mama.

OBJECTIVE: Breast cancer is a health problem worldwide with high incidence and mortality rates. It is well known that the development of more sensitive and specific diagnostic methods is of great importance since an early diagnosis is essential to successfully treat tumors. Lapachol is a natural compound, belonging to the naphthoquinone group that has been widely used in traditional medicine to treat various illnesses, including cancer. The aim of this study was to evaluate technetium-99m (99mTc) labeled lapachol as an imaging probe for breast cancer identification. METHODS: To achieve this purpose, lapachol was labeled with 99mTc, radiochemical purity and in vitro stability were determined. Blood clearance, in healthy mice, and biodistribution, in 4T1 tumor-bearing mice, were also evaluated. RESULTS: Lapachol was successfully labeled with 99mTc, with high values of radiochemical yield (95.9±3.4%). In vitro stability showed that the radiolabeled complex remained stable for up to 24h, with values above 90% for both saline and plasma (95.6±3.6% and 96.4±1.7%, respectively). The radiolabeled complex decays in a biphasic manner, with a half-life of distribution and elimination equal to 3.3 and 50.0min, respectively. Biodistribution and scintigraphic images showed high uptake in organs of excretion (kidneys, liver, and intestine). It could be also noted that tumor uptake was higher than the muscle at all time points. Tumor-to-muscle ratio reaches ∼4.5 at 24h after administration. CONCLUSION: These findings suggest that 99mTc-lapachol can be a potential diagnostic agent for breast tumors.

Neutralization of pharmacological and toxic activities of Bothrops jararacussu snake venom and isolated myotoxins by Serjania erecta methanolic extract and its fractions

Most of the snakebites recorded in Brazil are caused by the Bothrops genus. Given that the local tissue damage caused by this genus cannot be treated by antivenom therapy, numerous studies are focusing on supplementary alternatives, such as the use of medicinal plants. Serjania erecta has already demonstrated anti-inflammatory, antiseptic and healing properties. In the current study, the aerial parts of S. erecta were extracted with methanol, then submitted to chromatographic fractionation on a Sephadex LH20 column and eluted with methanol, which resulted in four main fractions. The crude extract and fractions neutralized the toxic activities of Bothrops jararacussu snake venom and isolated myotoxins (BthTX-I and II). Results showed that phospholipase A2, fibrinogenolytic, myotoxic and hemorrhagic activities were inhibited by the extract. Moreover, the myotoxic and edematous activities induced by BthTX-I, and phospholipase A2 activity induced by BthTX-II, were inhibited by the extract of S. erecta and its fraction. The clotting time on bovine plasma was significantly prolonged by the inhibitory action of fractions SF3 and SF4. This extract is a promising source of natural inhibitors, such as flavonoids and tannins, which act by forming complexes with metal ions and proteins, inhibiting the action of serineproteases, metalloproteases and phospholipases A2.

Procoagulant properties of human MV3 melanoma cells.

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

Procoagulant properties of human MV3 melanoma cells

A correlation between cancer and prothrombotic states has long been described. More recently, a number of studies have focused on the procoagulant mechanisms exhibited by tumor cells. In the present study, we dissected the molecular mechanisms responsible for the procoagulant activity of MV3, a highly aggressive human melanoma cell line. It was observed that tumor cells strongly accelerate plasma coagulation as a result of: i) expression of the blood clotting initiator protein, a tissue factor, as shown by flow cytometry and functional assays (factor Xa formation in the presence of cells and factor VIIa), and ii) direct activation of prothrombin to thrombin by cells, as evidenced by hydrolysis of the synthetic substrate, S-2238, and the natural substrate, fibrinogen. This ability was highly potentiated by the addition of exogenous factor Va, which functions as a co-factor for the enzyme factor Xa. In contrast, prothrombin activation was not observed when cells were previously incubated with DEGR-factor Xa, an inactive derivative of the enzyme. Moreover, a monoclonal antibody against bovine factor Xa reduced the prothrombin-converting activity of tumor cells. In conclusion, the data strongly suggest that MV3 cells recruit factor Xa from the culture medium, triggering an uncommon procoagulant mechanism.

On the molecular mechanisms for the highly procoagulant pattern of C6 glioma cells.

BACKGROUND: That there is a correlation between cancer and procoagulant states is well-known. C6 glioma cell line was originally induced in random-bred Wistar-Furth rats and is morphologically similar to glioblastoma multiforme, the most common aggressive glioma resistant to therapeutic interventions. OBJECTIVES: In this study we analyzed the molecular mechanisms responsible for the highly procoagulant properties of C6 glioma cells. METHODS: The presence of tissue factor (TF) and phosphatidylserine (PS) in C6 cells was investigated by flow-cytometric and functional analyses. The assembly of extrinsic tenase, intrinsic tenase and prothrombinase complexes on these cells was studied using enzymatic assays employing plasma or purified proteins. RESULTS: TF was identified by flow-cytometric and functional [factor (F) Xa formation in the presence of cells and FVIIa] assays. Alternatively, conversion of FX into FXa was also observed in the presence of C6 cells, FIXa and FVIIIa. This effect was both cell- and FVIIIa-dependent, being consistent with formation of the intrinsic tenase complex. C6 cells were also able to activate prothrombin in the presence of FXa and FVa, thus supporting formation of the prothrombinase complex. This ability was similar to positive controls performed with PS-containing vesicles. Accordingly, exposure of PS on C6 cells was demonstrated by flow cytometry employing specific anti-PS antibodies. In addition, annexin V, which blocks PS binding sites, inhibited FX and prothrombin conversion by their respective C6-assembled activating complexes. CONCLUSION: C6 glioma cells support all procoagulant reactions leading to robust thrombin formation. This ability results from concomitant TF exposure and from the presence of the anionic lipid PS at the outer leaflet of cell membrane. Therefore, this animal cell line may be used to explore new aspects concerning the role of blood coagulation proteins in tumor biology, especially those affecting the central nervous system.

Mutant H-ras overexpression inhibits drug and U.V. induced apoptosis.

Various studies have shown that oncogene and oncosuppressor gene activity can enhance or suppress programmed cell death (apoptosis) in various cell systems. Recent data indicates that overexpression of activated H-ras could influence that onset of apoptosis. We investigated the role of activated H-ras in the apoptotic cell death of rat fibroblast lines. We found that forced overexpression of H-ras induced resistance to U.V. and drug induced apoptosis. We examined possible mechanisms for the action of H-ras in resistance to apoptosis. It was found that both ras transfected and ras untransfected lines displayed similar endonuclease activities. In addition, it was found that the irradiated ras transfected line showed inhibited production of peroxides compared to the irradiated ras untransfected line. Drug induced apoptosis did not appear to involve peroxide production. In addition the antioxidant compound PDTC, was found to inhibit U.V. induced apoptosis but not drug induced apoptosis. In addition, we found the ras transfected line to possess elevated levels of catalase compared to the parent untransfected line. Thus we suggest that an anti-oxidant mechanism, possibly mediated by forced overexpression of activated H-ras could protect cells from apoptosis.

The repression of apoptosis by activated abl oncogenes in chronic myelogenous leukaemia.

The formation of the unique fusion gene, bcr-abl, and the resultant increase in abl tyrosine kinase activity, is seen as the major driving force in the initiation of chronic myelogenous leukaemia (CML). The deregulation of abl tyrosine kinase activity, brought about by the binding of a portion of the Scr molecule to the SH2 regulatory domain of abl, appears to play a role in promoting resistance to drug-induced apoptosis. Thus the large increase In mature myeloid cells seen in CML could be the direct result of the suppression of apoptosis by the bcr-abl fusion protein. The role and contribution of apoptosis in the progression of CML and the possible role of antisense oligonucleotides to the bcr-abl gene as therapeutic agents is discussed.

Inhibition of apoptosis by antioxidants in the human HL-60 leukemia cell line.

Cell death via apoptosis is an important event involved in a number of immunological processes. Recently, apoptosis has been associated with oxidative stress in a number of cell systems. Here we assessed the inhibitory capacity of different antioxidants on UV- and drug-induced apoptosis in the human leukemic cell line, HL-60. We found that the oxygen radical scavenger, BHA, the radioprotector cysteamine and the metal chelators, pyrrolidinedithiocarbamate (PDTC), diethyldithiocarbamate (DEDTC), and dimethyldithiocarbamate (DMDTC), were able to significantly inhibit nuclear fragmentation and reduce the formation of apoptotic bodies in UV-irradiated human leukemic cells. Both BHA and PDTC were found to reduce DNA fragmentation as assessed by in situ DNA nick-end labelling and quantification thereof using fluorescence flow cytometry. In addition to inhibiting UV-induced apoptosis, PDTC was also capable of reducing the amount of apoptosis induced by a range of cytotoxic drugs, such as actinomycin-D, camptothecin, etoposide, and melphalan, whereas BHA and cysteamine were not as effective in these cases after more than four hours in culture when compared to PDTC. To further elucidate the working mechanism of PDTC, we have looked at the effect of PDTC on DNA fragmentation in isolated nuclei, under conditions that promote activation of endogenous endonuclease involved in apoptosis. In contrast to ZnCl2, a potent inhibitor of endonuclease activity, PDTC was unable to inhibit DNA-ladder formation in this assay. Taken together, these results indicate that oxygen radicals may have a central role to play in the induction of apoptosis and that dithiocarbamates can serve as potent inhibitors of apoptosis induced by a wide variety of stimuli.

Zinc inhibits UV radiation-induced apoptosis but fails to prevent subsequent cell death.

Zinc ions inhibit the morphological and DNA fragmentation features of apoptosis in a number of systems. HL-60 cells pretreated with zinc and exposed to UV irradiation maintained their normal morphology for up to 8 h, whereas non-zinc-treated cells underwent extensive apoptosis. Zinc pretreatment also inhibited both single and double-stranded DNA fragmentation, which is characteristic of apoptosis. The most effective zinc concentration that blocked apoptosis over short incubation periods (up to 8 h) was also the most toxic over extended time periods (> or = 12 h). The mechanism of cell death at these longer time periods was akin to necrosis, but occurred in the absence of any DNA fragmentation. The effects of the nuclease inhibitor aurintricarboxylic acid (ATA) was also examined on UV-induced apoptosis in HL-60 cells. ATA had no toxic effects over the concentration range tested, but also failed to prevent DNA fragmentation in whole cells. Further analysis showed that it effectively inhibited DNA fragmentation in isolated nuclei.

Increased resistance to apoptosis associated with HL-60 myeloid differentiation status.

Morphologically HL-60 leukaemia cells largely resemble promyelocytes and can be induced to terminally differentiate in vitro. Upon reaching terminal maturation these cells rapidly undergo apoptosis. Using three chemotherapeutic agents with known apoptosis inducing capability, the susceptibility of RA - and PMA - differentiated cultures was monitored by morphological means and flow cytometry. We observed that as cells with morphological characteristics of mature granulocytes/monocytes became more prominent in the populations, there was an increased resistance to apoptosis. The inhibition of the typical internucleosomal DNA fragmentation was confirmed by agarose gel electrophoresis. However, activation of a CA+/Mg+ independent endonuclease in isolated nuclei was not affected. Flow immunocytometry revealed reduced levels of c-myc and bcl-2 oncoproteins in RA and PMA treated cells. These observations suggest that HL-60 derived granulocytes/monocytes become increasingly resistant to the induction of apoptosis and that this resistance is independent of c-myc and bcl-2 expression. Together these results demonstrate that the phenotypic changes associated with RA and PMA induced differentiation, inhibit a critical step in the progression of apoptosis.

Apoptosis or necrosis: intracellular levels of glutathione influence mode of cell death.

The effect of lowering intracellular glutathione (GSH) concentrations on the toxicity of alkylating agents, and RNA synthesis inhibitor and topoisomerase 1 and 2 inhibitors to a number of human leukaemic cell lines were evaluated. By using the GSH synthesis inhibitor DL-buthionine-(S,R)-sulfoximine (BSO), GSH levels were artificially reduced. Cells with low GSH concentrations were exposed to a number of cytotoxic agents and the resultant mode of cell death was analysed using morphological and biochemical criteria. It was found that untreated cells exposed to the above drugs underwent apoptosis to varying extents. However, the toxicity of alkylating agents was dramatically increased to all cell lines on lowering GSH levels, with the mode of cell death switching from apoptosis to necrosis. The reduction of GSH levels had no effect on the toxicity of actinomycin-D, camptothecin or etoposide, nor did it affect the mode of cell death induced by these agents. These observations suggest that modulation of GSH levels effect the toxicity of alkylating agents and that GSH influences the mode of cell death induced by alkylating agents.

Activation of a calcium magnesium independent endonuclease in human leukemic cell apoptosis.

Cytotoxic drugs induce apoptosis in human tumour cell lines and this is characterised by fragmentation of the cell's DNA into nucleosome size units or multiples thereof. In the present study we demonstrated that nuclei isolated from three human haematopoietic cell lines, HL-60, U937 and K562, contain an endonuclease that is independent of Ca++, Mg++ and Na+ ions for its activity. This contrasts with what has previously been shown for a number of rodent cell types in which apoptosis has been studied. The lack of ion sensitivity is also found in the nuclei of peripheral blood granulocytes, indicating the data are not peculiar to cell lines. In addition, this particular endonuclease activity does not appear to be sensitive to the endonuclease inhibitor aurintricarboxylic acid. The previously demonstrated lack of calcium flux in HL-60 cells undergoing apoptosis, and the current demonstration of a lack of an endonuclease dependent on this ion for its activity, suggest that the mechanism of apoptosis in human cells may be different from that in rodent cells.

[Cimetidine in the treatment of gastric ulcer]. / Cimetidine no tratamento da úlcera gástrica.

Twenty five patients with gastric ulcer were prospectively studied. They were admitted during acute phase and given cimetidine, 1000 mg/day for 6 weeks. They were maintained on cimetidine (up to 800 mg/day) for another 12 week period. The patients were studied with laboratory tests, endoscopy and biopsy during 4 steps of follow-up. A 76% healing rate was noted during the study. The incidence of side effects was low and drug tolerance and efficacy was shown to be good.