Inhibition of SARS-CoV-2 Mpro with Vitamin C, L-Arginine and a Vitamin C/L-Arginine Combination.

BACKGROUND: Drug resistance is a critical problem in health care that affects therapy outcomes and requires new approaches to drug design. SARS-CoV-2 Mpro mutations are of concern as they can potentially reduce therapeutic efficacy. Viral infections are amongst the many disorders for which nutraceuticals have been employed as an adjunct therapy. The aim of this study was to examine the potential in vitro activity of L-arginine and vitamin C against SARS-CoV-2 Mpro. METHODS: The Mpro inhibition assay was developed by cloning, expression, purification, and characterization of Mpro. Selected compounds were then screened for protease inhibition. RESULTS: L-arginine was found to be active against SARS-CoV-2 Mpro, while a vitamin C/L-arginine combination had a synergistic antiviral action against Mpro. These findings confirm the results of our previous in silico repurposing study that showed L-arginine and vitamin C were potential Mpro inhibitors. Moreover, they suggest a possible molecular mechanism to explain the beneficial effect of arginine in COVID patients. CONCLUSIONS: The findings of the current study are important because they help to identify COVID-19 treatments that are efficient, inexpensive, and have a favorable safety profile. The results of this study also suggest a possible adjuvant nutritional strategy for COVID-19 that could be used in conjunction with pharmacological agents.

Virtual Screen for Repurposing of Drugs for Candidate Influenza a M2 Ion-Channel Inhibitors.

Influenza A virus (IAV) matrix protein 2 (M2), an ion channel, is crucial for virus infection, and therefore, an important anti-influenza drug target. Adamantanes, also known as M2 channel blockers, are one of the two classes of Food and Drug Administration-approved anti-influenza drugs, although their use was discontinued due to prevalent drug resistance. Fast emergence of resistance to current anti-influenza drugs have raised an urgent need for developing new anti-influenza drugs against resistant forms of circulating viruses. Here we propose a simple theoretical criterion for fast virtual screening of molecular libraries for candidate anti-influenza ion channel inhibitors both for wild type and adamantane-resistant influenza A viruses. After in silico screening of drug space using the EIIP/AQVN filter and further filtering of drugs by ligand based virtual screening and molecular docking we propose the best candidate drugs as potential dual inhibitors of wild type and adamantane-resistant influenza A viruses. Finally, guanethidine, the best ranked drug selected from ligand-based virtual screening, was experimentally tested. The experimental results show measurable anti-influenza activity of guanethidine in cell culture.

Lipoprotein lipase: A bioinformatics criterion for assessment of mutations as a risk factor for cardiovascular disease.

Lipoprotein lipase (LPL) is a key enzyme in lipid metabolism. Decrease of the LPL enzymatic activity leads to elevated triglycerides (TG) and reduced high-density lipoprotein (HDL-C levels), both risk factors for cardiovascular disease (CVD). Therefore, mutations, which decrease the LPL activity, may confer susceptibility to CVD. Here, the informational spectrum method (ISM), a virtual spectroscopy method for structure/function analysis of nucleotide and protein sequences, is applied for identification of evolutionary highly conserved information encoded by the primary structure of LPL. It was demonstrated that mutations, which alter the LPL enzymatic activity also alter this information. On the basis of this finding, an efficient and simple bioinformatics criterion for assessment of the pathogenic effect of LPL nonsynonymous single nucleotide substitution as a risk factor of CVD has been proposed.

Discovery of new therapeutic targets by the informational spectrum method.

The field of bioinformatics has become a major part of the drug discovery pipeline playing a key role in improvement and acceleration of this time and money consuming process. Here we review the application of the informational spectrum method (ISM), a virtual spectroscopy method for structure/function analysis of proteins, in identification of functional protein domains representing candidate therapeutic targets for drugs against human immunodeficiency virus (HIV)-1, anthrax, highly pathogenic influenza virus H5N1 and cardiovascular diseases.

EMILINs interact with anthrax protective antigen and inhibit toxin action in vitro.

The informational spectrum method (ISM) is a virtual spectroscopy method for the fast analysis of potential protein-protein relationships. By applying the ISM approach to the GeneBank protein database the vascular proteins EMILIN1 (Elastin Microfibril Interface Located ProteIN), EMILIN2, MMN1, and MMN2 were identified as additional anthrax PA antigen interacting molecules. This virtual molecular interaction was formally proven by solid phase assays using recombinant proteins. The interaction is independent of the presence of divalent cations and does not involve PA aspartic residue at 683, a critical residue in receptor binding. In fact, the D683A point mutation fully prevented the cell intoxication ability of PA in the presence of Lethal Factor, but it was fully ineffective on the binding of mutated PA to EMILIN1 and EMILIN2. The ISM approach also led to the identification of the potential interaction sites between PA and EMILINs. A PA mutant with a deletion at residue D425 and solid phase protein-protein interaction studies as well as deletion mutant of EMILIN2 confirmed the hypothesized interaction site. Our findings imply that the PA-cell surface receptor interaction is not likely to provide the full explanation for the vascular lesions and prominent hemorrhages that follow Bacillus anthracis infection and spreading and call into play vascular associated proteins such as EMILINs as potential inhibitory proteins.

Improving attrition rates in Ebola virus drug discovery.

INTRODUCTION: The Ebola 2014/2015 outbreak has had devastating effects on the people living in West Africa. The spread of the disease in endemic countries and the potential introduction of sporadic cases in other continents points out the global health threat of Ebola virus disease (EVD). Despite the urgent need for treating EVD, there are no approved treatments. Given the lack of treatments available, alternative therapeutic strategies have had to be used. AREAS COVERED: This article summarizes the unregistered therapeutics that were used to treat patients during the Ebola 2014/2015 outbreak, in addition to approaches used for the selection of candidate drugs. The article also proposes potential theoretical criterion for use in virtual screening of molecular libraries for candidate Ebola drugs. EXPERT OPINION: In the absence of approved therapeutics for EVD, experimental drugs have had to be used. The repurposing of approved drugs for the treatment of EVD, as an alternative therapeutic strategy, has also been suggested. Screening in vitro- and in silico-approved drugs revealed several promising candidates but further testing is required to test their efficacy. All these therapeutic approaches are, however, only short-term solutions and there is still an urgent need for the development of specific drugs for the current and future outbreaks.

Evolution of 2014/15 H3N2 Influenza Viruses Circulating in US: Consequences for Vaccine Effectiveness and Possible New Pandemic.

A key factor in the effectiveness of the seasonal influenza vaccine is its immunological compatibility with the circulating viruses during the season. Here we propose a new bioinformatics approach for analysis of influenza viruses which could be used as an efficient tool for selection of vaccine viruses, assessment of the effectiveness of seasonal influenza vaccines, and prediction of the epidemic/pandemic potential of novel influenza viruses.

Recombination property of the HIV-1 gp120 gene.

Using a chimeric primer consisting of the nucleotide sequence derived from the HIV-1 envelope gene coding for the second conserved region of gp120, and the highly conserved sequence derived from the human immunoglobulin gene coding for the VHIII domain, it has been identified in sera of AIDS patients HIV-1 field isolates carrying the complete and active Chi recombination hot spot (GCTGGTGG). The recombination between the HIV-1 gene coding for the central portion of gp120 and the bacterial gene coding for the clp protease was also demonstrated in vivo. These results point out serious concern that vectored AIDS vaccine candidates carrying the HIV-1 env gene on viral and bacterial vectors could become the source of potentially new infectious diseases rather than an effective instrument for AIDS prevention.

Safety and ethical consideration of AIDS vaccine.

It has been demonstrated that HIV-1 gp120 resembles several important properties of immunoglobulins allowing it strong influence on the human immune system, especially through induction of the deceptive imprinting and deregulation of the immune network. On the other hand there are many unanswered questions concerning properties and control of the genetically modified viruses and bacteria used as vectors in AIDS vaccines. This situation opens a serious question about the safety of vectored AIDS vaccine and the ethics of their trials in humans.

Antibodies reactive with C-terminus of the second conserved region of HIV-1gp120 as possible prognostic marker and therapeutic agent for HIV disease.

It has been reported that antibodies reactive with peptide RSANFTDNAKTIIVQLNQSVEIN (peptide NTM) derived from the C-terminus of the second conserved domain of HIV-1 envelope glycoprotein gp120 could represent an important factor in control of the HIV disease. In order to check this notion we (i) tested reactivity with peptide NTM serum samples collected from 310 consecutive HIV-1 infected patients with a CD4(+) lymphocyte count ranging from 10 to 800/microL and (ii) performed the longitudinal study that included 107 sera samples collected from 29 HIV patients. Results of these studies demonstrated correlation between presence of anti-NTM antibodies in sera of HIV patients and disease progression measured by the CD4(+) cell count. Based on these findings we proposed the anti-NTM antibodies as useful prognostic marker for HIV disease.

Simple and general criterion for "in silico" screening of candidate HIV drugs.

Highly active antiretroviral therapy (HAART) dramatically has changed the course of HIV infection. Currently, this therapy involves the use of agents from at least two distinct classes of antivirals: a protease inhibitor in combination with two nucleoside/nucleotide reverse transcriptase inhibitors (N(t)RTIs), or a non-nucleoside reverse transcriptase inhibitor (NNRTI) in combination with NRTIs. Recently, the third family of antivirals started to be used clinically, with the advent of enfuvirtide, the first fusion inhibitor. This broad spectrum of anti-HIV agents recently was extended with compounds inhibiting HIV integrase and vital entry. But these advances did not come without a cost: there were the short- and long-term drug toxicities, emergence of drug resistance, and persistence of viral reservoirs. For these reasons, there is a pressing need for novel anti-HIV drugs, particularly those that have a novel action mechanism, as these might be less likely to show cross-resistance with current therapies. The field of bioinformatics has become a major part of the drug discovery pipeline playing a key role in improvement and acceleration of the time and money consuming process of the drug development. Here we review the application of the EIIP/AQVN (Electron-Ion Interaction Potential, EIIP; Average Quasi Valence Number, AQVN) bioinformatics concept in the development of new anti-HIV drugs and propose a simple theoretical criterion for a virtual screening of molecular libraries for promising lead anti-HIV compounds and refinement of selected lead compounds in order to increase their biological activity.

New in silico and conventional in vitro approaches to advance HIV drug discovery and design.

INTRODUCTION: Recently, the new concept of the long-range intermolecular interactions in biological systems has been proposed. Combined use of molecular modeling techniques and the screening techniques based on the long-range interaction concept could significantly improve and accelerate discovery of new HIV drugs. However, any hit identified in silico needs to be characterized with respect to its biological target by enzymatic studies. Combined use of the in silico screening and the enzymatic studies allows an efficient selection of new anti-HIV drugs. AREAS COVERED: The focus of this article is on the in silico screening of molecular libraries for candidate new HIV drugs, which is based on the molecular descriptors determining the long-range interaction between the drugs and their therapeutic target. This article also reviews the techniques for enzyme kinetic studies which are required for optimization of in silico selected candidate anti-HIV drugs. EXPERT OPINION: The novel approach of combining in silico screening techniques with enzymatic studies enables the accurate measurement of the quantitative descriptors of ligand-enzyme interactions. This novel method is a powerful tool for new anti-HIV drug discovery which can also reduce the drug development costs.

Assessment of hepatitis C virus protein sequences with regard to interferon/ribavirin combination therapy response in patients with HCV genotype 1b.

Hepatitis C virus (HCV) infection is a major and rising global health problem, affecting about 170 million people worldwide. The current standard of care treatment with interferon alpha and ribavirin in patients with the genotype 1 infection, the most frequent genotype in the USA and Western Europe, leads to a successful outcome in only about 50% of individuals. Accurate prediction of hepatitis C treatment response is of great benefit to patients and clinicians. The informational spectrum method, a virtual spectroscopy method for structure/function analysis of nucleotide and protein sequences, is applied here for the identification of the conserved information of the HCV proteins that correlate with the combination therapy outcome. Among the HCV proteins that we have analyzed the informational property of the p7 of HCV genotype 1b was best related to the therapy outcome. On the basis of these results, a simple bioinformatics criterion that could be useful in assessment of the response of HCV-infected patients to the combination therapy has been proposed.

A bioinformatics approach to identify natural autoantibodies from healthy blood donors' sera reactive with the HCV NS5A-derived peptide by immunoassay.

Natural autoantibodies (NAbs) are continually produced throughout life and have an ability to recognize self and altered self, as well as foreign antigens, by recognizing cellular pattern recognition receptors. Sometimes NAb specificity demonstrates overlap between human and pathologic proteomes. This information can be useful in selecting target sequences for screening purposes. In this study we undertook a multi-step bioinformatics search to predict a virus-derived peptide that can be recognized by NAbs in sera of uninfected individuals. We selected protein hepatitis C virus (HCV) NS5A as a target sequence, motivated by the fact that the HCV proteome is characterized by extensive sequence similarities to the human proteome, and because screening for anti-HCV antibodies, including anti-NS5A, is important clinically, particularly in screening of potential blood donors. The virus-specific peptide P1, and the homologous human peptide derived from enzyme-inducible nitric oxide synthase (iNOS), P2, exhibiting not only simple homology, but also complementarities of physicochemical patterns, were synthesized and 80 HCV-negative and 50 HCV-positive blood donor sera were tested by ELISA. These peptides reacted similarly (p<0.001) with HCV-negative sera, and in several cases the measured reactivity was significantly above the cut-off value of commercial anti-HCV screening assays. In HCV-positive sera, the titers of antibodies reactive with analyzed HCV NS5A peptide were not significantly increased (p<0.001) compared to host peptide, the implications of which are unclear, but may be consistent with these antibodies being "naturally produced." Finally, we extended our bioinformatics analyses to the dataset of human self-binding sequences, and propose a general approach for the selection of specific diagnostic and screening antigens for use in immunoassays.