Quantitative structure-activity relationships, molecular docking and molecular dynamics simulations reveal drug repurposing candidates as potent SARS-CoV-2 main protease inhibitors.

The current outbreak of COVID-19 is leading an unprecedented scientific effort focusing on targeting SARS-CoV-2 proteins critical for its viral replication. Herein, we performed high-throughput virtual screening of more than eleven thousand FDA-approved drugs using backpropagation-based artificial neural networks (q2LOO = 0.60, r2 = 0.80 and r2pred = 0.91), partial-least-square (PLS) regression (q2LOO = 0.83, r2 = 0.62 and r2pred = 0.70) and sequential minimal optimization (SMO) regression (q2LOO = 0.70, r2 = 0.80 and r2pred = 0.89). We simulated the stability of Acarbose-derived hexasaccharide, Naratriptan, Peramivir, Dihydrostreptomycin, Enviomycin, Rolitetracycline, Viomycin, Angiotensin II, Angiotensin 1-7, Angiotensinamide, Fenoterol, Zanamivir, Laninamivir and Laninamivir octanoate with 3CLpro by 100 ns and calculated binding free energy using molecular mechanics combined with Poisson-Boltzmann surface area (MM-PBSA). Our QSAR models and molecular dynamics data suggest that seven repurposed-drug candidates such as Acarbose-derived Hexasaccharide, Angiotensinamide, Dihydrostreptomycin, Enviomycin, Fenoterol, Naratriptan and Viomycin are potential SARS-CoV-2 main protease inhibitors. In addition, our QSAR models and molecular dynamics simulations revealed that His41, Asn142, Cys145, Glu166 and Gln189 are potential pharmacophoric centers for 3CLpro inhibitors. Glu166 is a potential pharmacophore for drug design and inhibitors that interact with this residue may be critical to avoid dimerization of 3CLpro. Our results will contribute to future investigations of novel chemical scaffolds and the discovery of novel hits in high-throughput screening as potential anti-SARS-CoV-2 properties.Communicated by Ramaswamy H. Sarma.

Molecular basis for the selectivity of antituberculosis compounds capreomycin and viomycin.

Capreomycin and the structurally similar compound viomycin are cyclic peptide antibiotics which are particularly active against Mycobacterium tuberculosis, including multidrug resistant strains. Both antibiotics bind across the ribosomal interface involving 23S rRNA helix 69 (H69) and 16S rRNA helix 44 (h44). The binding site of tuberactinomycins in h44 partially overlaps with that of aminoglycosides, and they share with these drugs the side effect of irreversible hearing loss. Here we studied the drug target interaction on ribosomes modified by site-directed mutagenesis. We identified rRNA residues in h44 as the main determinants of phylogenetic selectivity, predict compensatory evolution to impact future resistance development, and propose mechanisms involved in tuberactinomycin ototoxicity, which may enable the development of improved, less-toxic derivatives.

When do adaptive plasticity and genetic evolution prevent extinction of a density-regulated population?

We study the dynamics of evolutionary recovery after an abrupt environmental shift in a density-regulated population with evolving plasticity. Maladaptation to the new environment initially causes the population to decline, until adaptive phenotypic plasticity and genetic evolution restore positive population growth rate. We assume that selection on a quantitative trait is density-independent and that the initial cost of plasticity is much lower than the benefit of the initial plastic response. The initial partially adaptive plasticity reduces the effective magnitude of the environmental shift, whereas evolution of plasticity increases the rate of adaptation. Both effects greatly facilitate population persistence. In contrast, density dependence of population growth always hinders persistence. With theta-logistic population regulation, a lower value of theta produces a faster initial population decline and a higher extinction risk.

Cocaine pre-exposure produces a sensitized and context-specific c-fos mRNA response to footshock stress in the central nucleus of the AMYGDALA.

In recent years, there has been growing interest in the putative relationship between stress and vulnerability to relapse in former drug addicts. In animal studies aimed at exploring this relationship, it has been shown that brief exposure to intermittent footshock stress produces reliable reinstatement of drug seeking after prolonged drug-free periods. Whereas footshock reinstates drug seeking, it does not reinstate behaviors maintained by non-drug reinforcers, suggesting that prior drug experience may produce a form of sensitization within neuronal systems that mediate stress-induced reinstatement. The primary objective of the present experiments was to determine whether pre-exposure to cocaine produces a long-lasting, sensitized neuronal response to footshock stress within two brain regions known to mediate footshock-induced reinstatement; the central nucleus of the amygdala (CeA) and bed nucleus of the stria terminalis (BNST). In experiment 1, animals were injected for 7 days with cocaine (days 1 and 7 in test chambers; days 2-6 in homecages) or saline. After 21 drug-free days, they were exposed to footshock or no footshock. In experiment 2, rats were injected daily for 7 days with cocaine in one of two contexts and saline in the alternate context. After 21 drug-free days, they were given footshock either in the same context that they were given cocaine in or the alternate context. In CeA, footshock produced enhanced expression of c-fos mRNA in cocaine, but not saline, pre-exposed animals. Furthermore, this effect was gated by the environmental context in which cocaine was given; footshock only enhanced c-fos mRNA expression when it was given in a context that had previously been paired with cocaine. Although footshock induced c-fos mRNA expression in the BNST, its effects in this region were not dependent on drug history. The major findings are that a history of cocaine exposure produces sensitization to an acute stressor within CeA, and this effect is gated by environmental context.

Deciphering tuberactinomycin biosynthesis: isolation, sequencing, and annotation of the viomycin biosynthetic gene cluster.

The tuberactinomycin antibiotics are essential components in the drug arsenal against Mycobacterium tuberculosis infections and are specifically used for the treatment of multidrug-resistant tuberculosis. These antibiotics are also being investigated for their targeting of the catalytic RNAs involved in viral replication and for the treatment of bacterial infections caused by methicillin-resistant Staphylococcus aureus strains and vancomycin-resistant enterococci. We report on the isolation, sequencing, and annotation of the biosynthetic gene cluster for one member of this antibiotic family, viomycin, from Streptomyces sp. strain ATCC 11861. This is the first gene cluster for a member of the tuberactinomycin family of antibiotics sequenced, and the information gained can be extrapolated to all members of this family. The gene cluster covers 36.3 kb of DNA and encodes 20 open reading frames that we propose are involved in the biosynthesis, regulation, export, and activation of viomycin, in addition to self-resistance to the antibiotic. These results enable us to predict the metabolic logic of tuberactinomycin production and begin steps toward the combinatorial biosynthesis of these antibiotics to complement existing chemical modification techniques to produce novel tuberactinomycin derivatives.

Rapid identification and characterization of hammerhead-ribozyme inhibitors using fluorescence-based technology.

The ability to rapidly identify small molecules that interact with RNA would have significant clinical and research applications. Low-molecular-weight molecules that bind to RNA have the potential to be used as drugs. Therefore, technologies facilitating the rapid and reliable identification of such activities become increasingly important. We have applied a fluorescence-based assay to screen for modulators of hammerhead ribozyme (HHR) catalysis from a small library of antibiotic compounds. Several unknown potent inhibitors of the hammerhead cleavage reaction were identified and further characterized. Tuberactinomycin A, for which positive cooperativity of inhibition in vitro was found, also reduced ribozyme cleavage in vivo. The assay is applicable to the screening of mixtures of compounds, as inhibitory activities were detected within a collection of 2,000 extracts from different actinomycete strains. This approach allows the rapid, reliable, and convenient identification and characterization of ribozyme modulators leading to insights difficult to obtain by classical methodology.

In vitro selection of a viomycin-binding RNA pseudoknot.

BACKGROUND: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection. RESULTS: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb2+-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot. CONCLUSIONS: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.

Antibiotic-induced oligomerisation of group I intron RNA.

Antibiotics act as inhibitors of various biological processes. Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs. The linear intron, which is excised during the self-splicing process, is still an active molecular capable of performing an intramolecular transesterification resulting in a circular molecule. However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers. The antibiotic stimulates the intron to react in trans instead of in cis. The phage T4-derived td intron uses the same sites for oligomerisation as for circularisation. Gel- retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic. It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA-RNA interactions and thus enabling their reaction. The tuberactinomycins are further able to induce the interaction of heterologous introns. The ligation of the T4 phage-derived td intron with the Tetrahymena rRNA intron is very efficient, resulting in molecules composed of two introns derived from different species. The td intron attacks the Tetrahymena intron at various sites, which are located within double-stranded regions. These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA-RNA interactions in a pre-protein era.

Enhancement of Neurospora VS ribozyme cleavage by tuberactinomycin antibiotics.

Several examples of inhibition of the function of a ribozyme or RNA-protein complex have shown that certain antibiotics can interact specifically with RNA. There are, however, few examples of antibiotics that have a positive, rather than a negative, effect on the function of an RNA. We have found that micromolar concentrations of viomycin, a basic, cyclic peptide antibiotic of the tuberactinomycin group, enhance the cleavage of a ribozyme derived from Neurospora VS RNA. Viomycin decreases by an order of magnitude the concentration of magnesium required for cleavage. It also stimulates an otherwise insignificant transcleavage reaction by enhancing interactions between RNA molecules. The ability of viomycin to enhance some RNA-mediated reactions but inhibit others, including translation and Group I intron splicing, demonstrates the potential for natural selection by small molecules during evolution in the 'RNA world' and may have broader implications with respect to ribozyme expression and activity in contemporary cells.

The Fate of Intractable Tuberculosis Cases Under National Tuberculosis Programme / 결핵

BACKGROUND: The natural history of bacillary tuberculosis was studied in India and results showed that at the end of the 5-year period, 49% of the patients were dead, 33% were cured and 18% remained sputum-positive. The aim of this survey is to observe the natural course of the patients with intractable tuberculosis disease who were incurable with all drug regimens of the national tuberculosis programme(NTP). METHOD: Of the patients who have been found as intractable cases in Kang-Weon Province by the supervisory medical officer during the period from January 1,1987 to December 31,1992, 179 were eligible for this study. Sputum examination was done for those who were survived until October in 1993 at the Kang-Weon provincial laboratory of KNTA. 49 out of 179 patients were transferred to the private sectors and retreated with the combination of prothionamide, cycloserine, ofloxacin, enviomycin, etc. They seemed to have been bacteriologically cured, and so they were excluded from the study. Finally 130 patients were analyzed by modified life table method to calculate the fatality rate and the survival rate during the period of 7 years. RESULTS: 1) 80.8% of intractable cases were male and 19.2%,female. 2) More than 94% of intractable cases showed moderately or far advanced Tb findings on their X-rays at the time of registration at health centres. 3) The cumulative case-fatality rate was 19.74% at the end of 1-year period and has risen to 34.55% by the end of 4-year period(increasing by 4.9% a year on an average). The case-fatality rate has shown no appreciable rise since then until the end of 7-year period. 4) The case-survival rate was 80.26% at the end of 1-year period and has decreased to 65.45% by the end of 4-year period. And then there was no appreciable change in the survival rate until the end of 7-year observation. CONCLUSION: The case-survival rate of intractable cases was higher than that of untreated pulmonary tuberculosis patients and they may have risk of spreading multidrug resistant organisms. It is time we made an effort to improve case-management qualitatively.

Peptide antibiotics of the tuberactinomycin family as inhibitors of group I intron RNA splicing.

The tuberactinomycins are a group of cyclic peptide antibiotics, which are potent inhibitors of prokaryotic protein synthesis. We report the inhibitory effect of viomycin, di-beta-lysyl-capreomycin IIA and tuberactinomycin A on group I intron self-splicing. They compete with the guanosine cofactor for the G-binding site located in the conserved core of the intron. They are 100-fold more active than all other competitive inhibitors described so far (dGTP, arginine or streptomycin), inhibiting splicing at concentrations between 10 and 50 microM. Mutation of the G-binding site leads to partial resistance, and the inhibitory effect of these drugs is dependent on Mg2+ concentration. This suggests that the tuberactinomycins have more than one contact site with the intron RNA: via the G-binding site and via additional contacts with the RNA backbone. Positioning the tuberactinomycins in the three-dimensional model of the td intron core suggests that the charged lysyl side-chain (R1) is in contact with the backbone of the P1 helix. Structure/function analyses with various tuberactinomycin analogues with different activities confirm the involvement of this sidechain in inhibition of group I self-splicing. The demonstration of a new class of splicing inhibitors, the peptide antibiotics, illustrates how antibiotics may interact with catalytic RNA.

[Relationships between ototoxicities and chemical structures of ototoxic drugs].

The relationships between degrees of ototoxicity and the chemical structures of ototoxic drugs were investigated using isolated bullfrog semicircular canals. Thirteen derivatives of tuberactinomycin (Tum), a peptide antibiotic with pharmacological characteristics similar to those of kanamycin, were prepared by replacing the R1 branch with various amino acids. The degrees of ototoxicity were measured in these derivatives as well as in different kinds of aminoglycoside (AGs) antibiotics (TOB, GM, NTL, ISP, AMK). In order to measure the degree of ototoxicity, the ampullary nerve action potential of isolated bullfrog posterior semicircular canal, in response to mechanical endolymphatic flow, was recorded in Frog Ringer's solution and in the presence of different amounts of the various drugs. The degree of ototoxicity was determined by the amount of decrease in the maximal spike account. The degrees of ototoxicity of AGs were calculated to be in the order TOB > GM > ISP > AMK > NTL. This result was in agreement with those of previous morphological investigations. The derivatives of Tum with an R1 branch containing an acidic or a basic amino acid showed greater ototoxicities than those containing aliphatic side chains. The degree of ototoxicity of each drug tended to differ with each concentration, in both AG and Tum derivatives. Thus, the result indicate that chemical structure may be closely related to the degree of ototoxicity.

Susceptibility of south Indian strains of Mycobacterium tuberculosis to tuberactinomycin.

A total of 114 strains of Mycobacterium tuberculosis isolated from sputum samples of 114 patients of pulmonary tuberculosis in south India, were coded and tested for their in vitro susceptibility to tuberactinomycin (Tum) incorporated in Lowenstein-Jensen (LJ) medium. Of these strains, 95 (83.3%) and 15 (13.2%) were susceptible to Tum at 25 and 50 mg/l respectively. Only 4 (3.5%) strains were inhibited at 100 mg/l or more. Of the 37 drug sensitive strains, 2 (5.4%) were not susceptible to Tum at 25 mg/l compared to 17 (22.1%) of 77 strains-resistant to one or more of antituberculosis drugs (P less than 0.02). The drug susceptibility pattern of the strains revealed that there was no significant association of resistance between Tum and streptomycin or rifampicin or ethambutol or ethionamide or isoniazid. However, 15 (53.6%) of 28 kanamycin (K) resistant strains were not susceptible to Tum at 25 mg/l. This cross resistance between Tum and K was further studied in 24 and 15 K sensitive and resistant strains respectively, by correlating their proportion resistance at 16 mg/l and it was found to have a significant positive correlation (r = 0.55; P less than 0.01).

Bacteriostatic and bactericidal activity of antituberculosis drugs against Mycobacterium tuberculosis, Mycobacterium avium-Mycobacterium intracellulare complex and Mycobacterium kansasii in different growth phases.

Bacteriostatic and bactericidal activities of rifampicin, isoniazid, streptomycin, enviomycin and ethambutol against Mycobacterium tuberculosis, Mycobacterium avium--M. intracellulare complex and Mycobacterium kansasii were studied in different growth phases. Bacteriostatic activities of the drugs were similar in different growth phases, except isoniazid. M. tuberculosis was much less susceptible to isoniazid in the lag phase than in the log and the stationary phases. In contrast, bactericidal activity was influenced by the growth phase. M. tuberculosis was killed by isoniazid, streptomycin and rifampicin. The bactericidal activity of isoniazid was strongest. The bactericidal activity of isoniazid and streptomycin was most marked in the log phase. M. avium complex and M. kansasii resisted the bactericidal activity, but some strains of M. avium complex were killed by streptomycin and enviomycin, and the activities of these two drugs were most marked in the lag phase.

[Chemotherapeutic regimens that were considered effective to cure pulmonary infection caused by Mycobacterium avium-Mycobacterium intracellulare complex].

During the period of 24 years from 1965 to 1988, we treated a total of 181 patients who had pulmonary infection caused by Mycobacterium avium--Mycobacterium intracellulare complex (MAI complex). Of these 181, 34 (19%) were cured showing sputum conversion and disappearance of cavity or marked reduction of cavity in the size to 1/2 or less or change of the cavity to thin-walled one. In these patients, negative culture continued at least for one year by monthly sputum examination. The most frequently used regimen in these patients was RFP + INH + SM, and the secondly RFP + INH + EVM, and thereafter multiple drug regimens including RFP + INH. The most frequently used drugs were RFP, INH, EVM, SM and EB. Based on the above results, we recommend the regimen RFP + INH + EVM + EB or RFP + INH + SM + EB, to which, if possible, were added a combination of MC + SX + KT. (As to abbreviations, refer to Table 3).

Investigation of vestibular damage by antituberculous drugs.

Vestibular function testing was performed regularly on patients who were administered streptomycin, kanamycin, or enviomycin, and vestibular damage was detected at an early stage, and quantitatively. We investigated the point in time at which the therapy should be discontinued. Subjects consisted of 204 cases of tuberculosis treated with streptomycin, kanamycin, enviomycin. They were admitted to the hospital between December 1984 and August 1989. Twenty-eight cases of vestibular dysfunction due to streptomycin, kanamycin, and enviomycin could easily be detected at an early stage by performing Meyer zum Gottesberge's head-shaking test for the evaluation of jumbling, together with Romberg's test and the stepping test. All cases who had vestibular dysfunction completely recovered because of early detection. In addition, 7 cases recovered afterwards from temporary vestibular damage shown only in Meyer zum Gottesberge's head-shaking test (abnormality of vestibulo-ocular reflex was only detected and vestibulo-spinal reflex remained intact), despite continuation of streptomycin injection. When the results of the head-shaking test are less than 50% and when a sway and/or rotation in the stepping test occurs, the injections should be discontinued.

[Bactericidal activities of rifampicin, ethambutol, enviomycin and streptomycin on Mycobacterium avium-Mycobacterium intracellulare complex strains].

Bacteriostatic and bactericidal activities of rifampicin, ethambutol, enviomycin and streptomycin on Mycobacterium avium-Mycobacterium intracellulare complex (MAI complex) strains were determined. The susceptibility testings were made in Ogawa egg medium, and minimal inhibitory concentrations (MICs) were determined as the lowest concentration of drugs, in which the growth of 20 to 100 colony-forming units was completely inhibited. The MICs correspond to the MICs that can inhibit the growth of 95 to 99% of bacterial population. Bactericidal activity was determined in a modified Dubos liquid medium (1.3 g of Dubos TB Broth Base were dissolved in 180 ml of distilled water and this solution was supplemented by 20 ml of bovine serum). A one ml-sample of bacterial suspensions (10 mg wet weight per ml) was added to 9 ml of the Dubos liquid medium, and the medium was incubated at 37 degrees C for 0, 1, 3 and 7 days under shaking condition (56 strokes per minute; 8 cm amplitude). The bactericidal activity was measured as the number of colony-forming units contained in a 0.02 ml-sample of the medium. The bactericidal activities of rifampicin and ethambutol were weak or absent even in strains 13008 and 13016, which were very susceptible to all four drugs. However, the bactericidal activities of streptomycin and enviomycin could be observed in these strains.(ABSTRACT TRUNCATED AT 250 WORDS)

[Chemotherapeutic regimens for nontuberculous mycobacterial infection based on in-vitro susceptibility test results].

Treatment of nontuberculous mycobacterial infection should be carried out by the chemotherapeutic regimens most suitable for each species. We performed in-vitro susceptibility tests for various species and determined the probability in which the mycobacteria of each species are inhibited by blood concentrations attainable by the dosages usually used. From such determinations, the following regimens have been recommended: 1) M. avium-M. intracellulare complex, Rifampicin + Enviomycin + Ethambutol; 2) M. kansasii, Ofloxacin + Enviomycin + Rifampicin; 3) M. szulgai, Enviomycin - Ethambutol + Isoniazid; 4) M. fortuitum, Ofloxacin. The effectiveness of the treatment of the infection caused by M. avium complex is less than 15% even using the above regimen, while it is high in the treatment of infections caused by M. kansasii and M. szulgai. There are no effective regimens for the treatment of infection caused by M. chelonae or M. simiae. The above recommendations are useful in practice, because, at present, reliable susceptibility testing of nontuberculous mycobacteria is not yet done commonly.