Metal-Bound Methisazone; Novel Drugs Targeting Prophylaxis and Treatment of SARS-CoV-2, a Molecular Docking Study.

SARS-CoV-2 currently lacks effective first-line drug treatment. We present promising data from in silico docking studies of new Methisazone compounds (modified with calcium, Ca; iron, Fe; magnesium, Mg; manganese, Mn; or zinc, Zn) designed to bind more strongly to key proteins involved in replication of SARS-CoV-2. In this in silico molecular docking study, we investigated the inhibiting role of Methisazone and the modified drugs against SARS-CoV-2 proteins: ribonucleic acid (RNA)-dependent RNA polymerase (RdRp), spike protein, papain-like protease (PlPr), and main protease (MPro). We found that the highest binding interactions were found with the spike protein (6VYB), with the highest overall binding being observed with Mn-bound Methisazone at -8.3 kcal/mol, followed by Zn and Ca at -8.0 kcal/mol, and Fe and Mg at -7.9 kcal/mol. We also found that the metal-modified Methisazone had higher affinity for PlPr and MPro. In addition, we identified multiple binding pockets that could be singly or multiply occupied on all proteins tested. The best binding energy was with Mn-Methisazone versus spike protein, and the largest cumulative increases in binding energies were found with PlPr. We suggest that further studies are warranted to identify whether these compounds may be effective for treatment and/or prophylaxis.

In vitro and in vivo evaluation of isatin-beta-thiosemicarbazone and marboran against vaccinia and cowpox virus infections.

It has been reported previously that some thiosemicarbazone compounds have prophylactic activity against smallpox disease and therapeutic activity against vaccinia virus (VV) infections. In these studies, isatin-beta-thiosemicarbazone (IBT) and marboran were administered once daily by intraperitoneal (ip) injection to mice using 30, 10 or 3 mg/kg for 5 days beginning 24, 48 or 72 h after inoculation with VV or cowpox virus (CV). Both compounds were highly effective (p < 0.01) at preventing mortality due to VV even when treatment was delayed up to 72 h postinfection. In CV-infected mice, neither IBT nor Marboran were effective in preventing mortality at any dosage tested when administered at 24 h postinoculation. Viral replication in liver, spleen and kidney was delayed or reduced by 100-to 10,000-fold by 10 mg/kg of marboran, but not IBT, in VV infections. Neither compound was effective against CV infection. Neither IBT nor marboran treatment of mice cutaneously infected with VV or CV reduced viral replication or clinical disease. These results suggest that this class of compound has little therapeutic potential for orthopoxvirus infections since the in vivo activity against CV, a surrogate virus for variola, is lacking.

[Antiviral activity of different drugs in vitro against viruses of bovine infectious rhinotracheitis and bovine diarrhea]. / Protivovirusnaia aktivnost' razlichnykh preparatov in vitro v otnoshenii virusov infektsionnogo rinotrakheita i virusnoi diarei krupnogo rogatogo skota.

In vitro experiments studied the antiviral activity of 11 different drugs against viruses of bovine infective rhinotracheitis (BIRT) and bovine viral diarrhea (BVD). The 50% inhibiting concentrations of the test agents were determined in the monolayers of MDBK and KCT cell cultures. Only did phosprenyl show a virucidal activity against BIRT virus. All the tested drugs significantly inhibited the reproduction of BIRT virus in the sensitive MDBK cell cultures. Thus, bromuridin, acyclovir, ribavirin and methisazonum inhibited the virus by > or = 100,000 times; liposomal ribavirin, gossypolum, anandinum, polyprenolum, phosprenyl, by 1000-10,000 times; eracond and argovit, by 100 times. In experiments on BVD virus, the cultured KCT cells displayed the antiviral activity of bromuridin, phosprenil, polyprenolum, methisazonum, acyclovir, gossypolum, argovit, and ribavirin (in two variants), which caused a statistically significant (100-10,000-fold) decrease in the productive activity of this virus. Eracond and anandid proved to be ineffective.

Progressive vaccinia.

The resumption of smallpox vaccination for health care workers and other first responders has raised concern about the occurrence of complications in people with immunodeficiency disorders, including those infected with human immunodeficiency virus. During the era of universal vaccination, roughly 1 person per million vaccinees in the general population developed progressive vaccinia, which is characterized by the relentless outward spread of infection from the vaccination site and eventual dissemination to other areas on the body. Review of 56 cases reported in the English-language medical literature from 1893 through 1997 indicates that the condition occurred only in persons with severe cell-mediated immunodeficiency. Progressive vaccinia was found to be lethal in infants who completely lacked cellular immune function, but infection resolved in many adults with acquired immunodeficiency. Almost all cases were treated with vaccinia immune globulin, but its efficacy has never been tested in a placebo-controlled trial. Further research is needed to develop effective forms of therapy.

Inhibition of human immunodeficiency virus by N-methylisatin-beta 4':4'-diethylthiosemicarbazone and N-allylisatin-beta-4':4'-diallythiosemicarbazone.

N-methylisatin-beta 4':4'-diethylthiosemicarbazone(M-IBDET) and N-allylisatin-beta-4':4'-diallylthiosemicarbazone(A-IBDAT ) inhibit the production of Human Immunodeficiency virus (HIV). Virus inhibition was related to the thiosemicarbazone derivative (TSCD) concentrations and time of treatment. Inhibition of HIV production was confirmed by various parameters of virus assay employing reverse transcriptase activity, plaque forming units (PFU) and levels of viral structural proteins. Effective antiviral TSCD concentrations ranged from 0.17 microM to 2.04 microM for M-IBDET, and from 1.45 microM to 17.4 microM for A-IBDAT. Treatment of the chronic HIV-infected cells for 48 h with 0.34 microM M-IBDET or 2.9 microM A-IBDAT caused about 50% inhibition in as virus yield ED50 as assayed by the PFU method. Almost 2 logs of virus infectivity (PFU) was suppressed after 48 h of treatment with 17.4 microM A-IBDAT. Therapeutic index values of 20 and 30 were found for M-IBDET and A-IBDAT, respectively. A significant selective inhibition of HIV structural protein synthesis was shown by both M-IBDET and A-IBDAT.

Synthesis of some acylphosphonate thiosemicarbazones and evaluation of their antiviral activity.

Reaction of dimethyl acylphosphonates (1) with thiosemicarbazide afforded the corresponding dimethyl acylphosphonate thiosemicarbazones (4). Lithium methyl acylphosphonate thiosemicarbazones (5) could be prepared either by monodealkylating compounds 4 by lithium bromide or by reacting lithium methyl acylphosphonates with thiosemicarbazide. Dihydrogen acylphosphonate thiosemicarbazones (6) could be obtained by reacting acylphosphonic acids (3) with thiosemicarbazide. The alternative approach to 6 by di-dealkylating compounds 4 using bromotrimethylsilane was limited by the solubility properties of esters 4. Compounds of types 5 and 6 did not show antiviral activity against herpes simplex type 1 and 2, or toward vaccinia virus.

Selective repression of v-abl-encoded protein by N-methylisatin-beta-4',4'-diethylthiosemicarbazone and N-allylisatin-beta-4',4'-diallylthiosemicarbazone.

N-Methylisatin-beta-4',4'-diethylthiosemicarbazone (M-IBDET) and N-allylisatin-beta-4',4'-diallylthiosemicarbazone (A-IBDAT) selectively inhibited v-abl protein (P120), an oncogene product associated with tyrosine kinase activity. Concentrations of M-IBDET ranging between 0.17 and 0.64 microM and concentrations of A-IBDAT from 1.45 to 2.9 microM reduced tyrosine kinase activity significantly, whereas 0.64 microM M-IBDET and 2.9 microM A-IBDAT blocked P120 production. Cellular growth rate, protein production, and synthesis of p45 actin and p53 nuclear oncogene were not affected at these conditions. M-IBDET and A-IBDAT selectively suppress the v-abl oncogene as well as Moloney murine leukemia virus production.

The efficacy of amantadine and other antiviral compounds against two salmonid viruses in vitro.

Various compounds, with known clinical efficacy against human viruses, were evaluated for their ability to inhibit the growth of infectious hematopoietic necrosis virus (IHNV, a rhabdovirus), and infectious pancreatic necrosis virus (IPNV, a birnavirus), in rainbow trout cell cultures. Amantadine inhibited the plaque forming ability of IHNV, at concentrations which did not affect cell growth or morphology, although it was not active against IPNV. Metisazone and bis-benzimidazole were also effective against IHNV; but they were slightly cytotoxic. Ribavirin, as expected, was active against IPNV, but was also equally effective against IHNV, although it was cytotoxic. Several other compounds were also tested but they were not inhibitory to either virus. The attraction of amantadine is the fact that relatively easy administration should be feasible.

N-methylisatin-beta-4',4'-diethylthiosemicarbazone, an inhibitor of Moloney leukemia virus protein production: characterization and in vitro translation of viral mRNA.

The mode of inhibition of N-methylisatin-beta-4',4'-diethylthiosemicarbazone (M-IBDET) on Moloney leukemia virus production was studied. Drug treatment of infected cells did not alter the amounts or sizes of the 35S and 22S subgenomic viral RNAs. The translation abilities of poly(A)+ RNA derived from M-IBDET-treated cells was also unaffected, as judged by cell-free translation analysis. Poly(A)+ RNA derived from M-IBDET-treated cells directed translation of equal amounts of viral gag precursors, gPr-80gag and Pr-65gag, as did poly(A)+ RNA prepared from untreated cells. The addition of M-IBDET to a cell-free translation system programmed with either total poly(A)+ RNA extracted from infected cells or hybrid-selected viral RNA inhibited the synthesis of viral protein precursors. An examination of the effect of M-IBDET on polysomes engaged in the translation of viral proteins revealed a fourfold accumulation of polysomal virus-specific RNA in drug-treated cells. These results suggest that the inhibition of Moloney leukemia virus by M-IBDET involves a block in the translation of viral RNA rather than interference with viral RNA transcription.

Inhibition of vaccinia virus replication in RK-13 cells by N,N'-bis(methylisatin-beta-thiosemicarbazone)-2-methylpiperazine.

N,N'-bis(methylisatin-beta-thiosemicarbazone)-2-methylpiperazine in a 100 mumol/l concentration inhibited the reproduction of vaccinia virus in RK-13 cells by about 90%. This compound (bis-IBTMP) had no influence on virus adsorption and on early stages of virus multiplication, but affected virus reproduction from 12 to 24 hr post-infection (p.i.). The incorporation of 3H-thymidine into infected cells increased during first 10 hr p.i., decreasing gradually afterwards. In the infected cells treated with bis-IBTMP the same tendency was observed up to 10 hr p.i., but later on the incorporation level remained unchanged. The uptake of 14C-amino acids in the presence of bis-IBTMP was reduced both in vaccinia virus-infected and non-infected RK-13 cells.

Effect of N-methylisatin-beta-4':4'-diethylthiosemicarbazone on intracellular Moloney leukemia virus constituents.

N-Methylisatin-beta-4':4'-diethylthiosemicarbazone (M-IBDET) inhibits intracellular production of viral constituents in a mouse cell line, 3T3/MLV, chronically infected with Moloney leukemia virus. Electron microscopic observations confirmed that inhibition of virus production by the drug was not associated with any structural changes in the cell morphology or any damage to the plasma membrane, the site of viral assembly and 'budding'. Treatment of the cells with 17 microM M-IBDET for 6 h inhibited extracellular virus production by 80% but did not affect the level of viral RNA in the cytoplasm or in the plasma membrane. Intracellular reverse transcriptase activity and levels of viral structural proteins were significantly inhibited. Thus, although the drug did not affect viral RNA, it reduced viral protein synthesis.

Immune response in vaccinia virus infected mice treated with N,N'-bis/methylisatin-beta-thiosemicarbazone/-2-methylpiperazine.

The effect of the compound N,N'-bis/methylisatin-beta-thiosemicarbazone/-2-methylpiperazine (bis-MIBTP) on immune response in BALB/c and Swiss mice have been studied in the course of vaccinia virus infection. Humoral response tested by neutralization and hemagglutination inhibiting antibodies was similar in compound-treated mice to this of untreated mice. Cell-mediated immune response, examined by spleen lymphocytes migration inhibition test, has been delayed or temporally depressed in bis-MIBTP treated mice as compared with the control group. High protective activity of the compound in vaccinia infected mice in spite of impairment of cellular immunity may indicate that antibodies have played an important role in recovery process from vaccinia infection.

Inhibition of the synthesis of Moloney leukemia virus structural proteins by N-methylisatin-beta-4',4'-diethylthiosemicarbazone.

The mechanism of inhibition of Moloney leukemia virus by N-methylisatin-beta-4',4'-diethylthiosemicarbazone was studied. Experiments that used [3H]leucine for short-pulse labeling in the presence of the drug resulted in a 71% inhibition in the synthesis of Pr-80, the polypeptide precursor of the gag viral proteins. The radioactive pulse products of the polypeptide precursors after a further 2-h chase period showed a normal cleavage of the precursors, with the formation of a reduced amount of final mature viral structural proteins. The experimental evidence indicated that at the inhibitory concentration of 17 microM N-methylisatin-beta-4',4'-diethylthiosemicarbazone, the amount of intracellular viral RNA was not affected, whereas the activities of reverse transcriptase and the other viral protein syntheses were suppressed.

Site of action of N,N'-bis(methylisatin-beta-thiosemicarbazone)-2-methylpiperazine in the vaccinia virus replication cycle.

N,N'-Bis(methylisatin-beta-thiosemicarbazone)-2-methylpiperazine inhibits late function or synthesis of a late component in the replication cycle of vaccinia virus. The kinetics of formation of the component sensitive to the inhibition with N,N'-bis(methylisatin-beta-thiosemicarbazone)-2-methylpiperazine precedes that of appearance of infectious virus by 30 min. The finding is in accord with the site of action of unsubstituted isatin-beta-thiosemicarbazone.