Repositioning the existing drugs for neuroinflammation: a fusion of computational approach and biological validation to counter the Parkinson's disease progression.

Parkinson's disease is caused by the deficiency of striatal dopamine and the accumulation of aggregated α-synuclein in the substantia nigra pars compacta (SNpc). Neuroinflammation associated with oxidative stress is a key factor contributing to the death of dopaminergic neurons in SNpc and advancement of Parkinson's disease. Two molecular targets, i.e., nuclear factor kappa-light-chain-enhancer (NF-kB) and α-synuclein play a substantial role in neuroinflammation progression. Therefore, the compounds targeting these neuroinflammatory targets hold a great potential to combat Parkinson's disease. Thereby, in this study, molecular docking and Connectivity Map (CMap) based gene expression profiling was utilized to reposition the approved drugs as neuroprotective agents for Parkinson's disease. With in silico screening, two drugs namely theophylline and propylthiouracil were selected for anti-neuroinflammatory activity evaluation in in vivo models of chronic neuroinflammation. The neuroinflammatory effect of the identified compounds was confirmed by quantifying the expression of three important neuroinflammatory mediators, i.e. IL-6, TNF-alpha, and IL-1 beta on brain tissue using ELISA assay. The ELISA experiment demonstrated that both compounds significantly decreased the expression of neuroinflammatory mediators, highlighting the compounds' potential in neuroinflammation management. Furthermore, the drug and disease interaction network of the two identified drugs and diseases (neuroinflammation and Parkinson's disease) suggested that the two drugs might interact with various targets namely adenosine receptors, Poly [ADP-ribose] polymerase-1, myeloperoxidase (MPO) and thyroid peroxidase through multiple pathways associated with neuroinflammation and Parkinson's disease. Computational studies suggest that a particular drug may be effective in managing Parkinson's disease associated with neuroinflammation. However, further research is needed to confirm this in biological experiments.

Setomimycin as a potential molecule for COVID­19 target: in silico approach and in vitro validation.

COVID-19 pandemic caused by the SARS-CoV-2 virus has led to a worldwide crisis. In view of emerging variants time to time, there is a pressing need of effective COVID-19 therapeutics. Setomimycin, a rare tetrahydroanthracene antibiotic, remained unexplored for its therapeutic uses. Herein, we report our investigations on the potential of setomimycin as COVID-19 therapeutic. Pure setomimycin was isolated from Streptomyces sp. strain RA-WS2 from NW Himalayan region followed by establishing in silico as well as in vitro anti-SARS-CoV-2 property of the compound against SARS-CoV-2 main protease (Mpro). It was found that the compound targets Mpro enzyme with an IC50 value of 12.02 ± 0.046 µM. The molecular docking study revealed that the compound targets Glu166 residue of Mpro enzyme, hence preventing dimerization of SARS-CoV-2 Mpro monomer. Additionally, the compound also exhibited anti-inflammatory and anti-oxidant property, suggesting that setomimycin may be a viable option for application against COVID-19 infections.

Boswellic acids, as novel inhibitor targeting peptidoglycan biosynthetic enzyme UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) in Escherichia coli.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an essential cytosolic enzyme in the biosynthesis of peptidoglycan. It becomes a potential bacterial target for screening promising antibacterial compounds as it is associated with the early phases of peptidoglycan production. MurA enzyme is conserved and necessary for bacterial viability with no mammalian homolog, which is a well-proven therapeutic research target. The present study reports the natural compounds from Boswellia serrata targeting the MurA enzyme. The identified inhibitors against MurA Escherichia coli (E. coli): ß-boswellic acid (IC50 33.65 µM), Acetyl-ß-boswellic acid (IC50 30.17 µM), and Acetyl-11-keto-ß-boswellic acid (IC50 37.67 µM). Inhibitors showed a fourfold decrease in IC50 values on pre-incubation with substrate-UDP-N-acetyl-glucosamine (UDP-GlcNAc). Mode-of-inhibition studies revealed their uncompetitive nature with both the substrates. Although these boswellic acids have been explored for their pharmacological potential, this is the first study reporting these compounds' E. coli MurA inhibiting potential.

Synthesis of 3-N-/O-/S-methyl-imidazo[1,2-a] pyridine derivatives for caspase-3 mediated apoptosis induced anticancer activity.

A library of 49 analogs of imidazo[1,2-a]pyridine with 2-halo, aryl, styryl and phenylethynyl-substitution at C-2 position and N-/O-/S-methyl linkage at C-3 position, have been synthesized and evaluated for their anti-proliferative activity against breast (MCF-7, MDA-MB-231), pancreatic (MiaPaca-2), lung (A549), prostate (PC-3) and colon (HCT-116) cancer cell lines and normal cells (HEK-293). Among the screened compounds, 5b exhibited best anticancer potential in all tested cancer cells with IC50 ranging from 3.5 to 61.1 µM and no toxicity in normal cells. Further, mechanistic study of 5b revealed concentration dependent increased generation of ROS, reduced mitochondrial membrane potential (MMP), surface and nuclear morphological alterations and inhibition of colony formation in HCT-116 cells. Western blot results had shown that the cell death in HCT-116 colon cancer cells was achieved through the induction of apoptosis via upregulation of the PTEN gene and downregulation of AKT pathway. Similarly, 5b treatment induced caspase-3 cleavage which is a hallmark of apoptosis. Molecular docking and binding energy (ΔG) studies of hit 5b with respect to three important cancer targets (EGFR, mTOR and PI3Kα) revealed strong binding of inhibitor with PI3Kα (docking score -6.932 and ΔG -56.297).

Screening of compound library identifies novel inhibitors against the MurA enzyme of Escherichia coli.

Bacterial cell has always been an attractive target for anti-infective drug discovery. MurA (UDP-N-acetylglucosamine enolpyruvyl transferase) enzyme of Escherichia coli (E.coli) is crucial for peptidoglycan biosynthetic pathway, as it is involved in the early stages of bacterial cell wall biosynthesis. In the present study we aim to identify novel chemical structures targeting the MurA enzyme. For screening purpose, we used in silico approach (pharmacophore based strategy) for 52,026 library compounds (Chembridge, Chemdiv and in house synthetics) which resulted in identification of 50 compounds. These compounds were screened in vitro against MurA enzyme and release of inorganic phosphate (Pi) was estimated. Two compounds (IN00152 and IN00156) were found to inhibit MurA enzyme > 70% in primary screening and IC50 of 14.03 to 32.30 µM respectively. These two hits were further evaluated for their mode of inhibition studies and whole-cell activity where we observed 2-4 folds increase in activity in presence of Permeabilizer EDTA (Ethylenediaminetetraacetic acid). Combination studies were also performed with known antibiotics in presence of EDTA. Hits are reported for the first time against this target and our report also support the use of OM permeabilizer in combination with antibacterial compounds to address the permeability and efficacy issue. These lead hits can be further optimized for drug discovery. KEY POINTS: • Emerging Gram negative resistant strains is a matter of concern. • Need for new screening strategies to cope with drying up antibiotics pipeline. • Outer membrane permeabilizers could be useful to improve potency of molecules to reach its target.

Discovery of a New Donepezil-like Acetylcholinesterase Inhibitor for Targeting Alzheimer's Disease: Computational Studies with Biological Validation.

Alzheimer's disorder is one of the most common worldwide health problems, and its prevalence continues to increase, thereby straining the healthcare budgets of both developed and developing countries. So far, donepezil is the only Food and Drug Administration-approved dual-binding site inhibitor of acetylcholinesterase (AChE) that can amplify the cholinergic activity and also decrease Aß aggregation in Alzheimer patients. We report herein a new donepezil-like natural compound derivative (D1) as a convincing AChE inhibitor. The in silico studies suggests that D1 exhibits a dual-binding mode of action and interacts with both the catalytic anionic site and peripheral anionic site (PAS) of human AChE. The biological studies confirm the dual-binding site character of D1 and revealed that D1 not only enhances the acetylcholine levels but also reduces the accumulation of Aß plaques in Caenorhabditis elegans. In fact, 5 µM D1 was seen more potent in elevating the acetylcholine expression than 25 µM donepezil. While most of the non-cholinergic functions of donepezil, associated with the PAS of AChE, were gradually lost at higher concentrations, D1 was more functional at similar doses. Promisingly, D1 also exerted an agonistic effect on the α7 nicotinic acetylcholine receptor.

Combining ligand- and structure-based in silico methods for the identification of natural product-based inhibitors of Akt1.

The traditional method of drug discovery process has been surpassed by a rational approach where computer-aided drug designing plays a vital role in the identification of leads from large compound databases. Further, natural products have an important role in drug discovery as these have been the source of most active ingredients of medicines. Herein, in silico structure- and ligand-based approaches have been applied to screen in-house IIIM natural product repository for Akt1 (serine/threonine protein kinases) which is a well-known therapeutic target for cancer due to its overexpression and preventing the cells from undergoing apoptosis. Combined ligand-based and structure-based strategies were applied on to the existing library comprising of about 700 pure natural products, and the compounds identified from screening were biologically evaluated for Akt1 inhibition using Akt1 kinase activity assay. Fourteen promising compounds showed significant inhibition at 500 nM through in vitro screening, and from them, eight were new for Akt1 inhibition. Through the MD studies of Akt1 with the most active compound IN00145, it was inferred that Lys179, Glu191, Glu228, Ala230, Glu234 and Asp292 are the important amino acid residues which provide stability to the Akt1-IN00145 complex. Lead optimization studies were also performed around the actives to design better and selective inhibitors for Akt1. The results emphasized the successful application of virtual screening to identify new Akt1 inhibitor scaffolds that can be developed into a drug candidate in drug discovery programme.

Design, synthesis and biological evaluation of pyrazolopyrimidinone based potent and selective PDE5 inhibitors for treatment of erectile dysfunction.

Our previous discovery of series of pyrazolopyrimidinone based PDE5 inhibitors led to find potent leads but with low aqueous solubility and poor bioavailability, and low selectivity. Now, a new series of same pyrazolopyrimidinone scaffold is designed, synthesized and evaluated for its PDE5 inhibitory potential. In this study, some of the molecules are found more potent and selective PDE5 inhibitors in vitro than sildenafil. The studies revealed that compound 5 is 20 fold selective to PDE5 against PDE6. As PDE6 enzyme is involved in the phototransduction pathway in the retina and creates distortion problem, the selectivity for PDE5 specifically against PDE6 enzyme is preferred for any development candidate and in present study, compound 5 has been found to be devoid of this liability of selectivity issue. Moreover, compound 5 has shown excellent in vivo efficacy in conscious rabbit model, it's almost comparable to sildenafil. The preclinical pharmacology including pharmacokinetic and physicochemical parameter studies were also performed for compound 5, it was found to have good PK properties and other physicochemical parameters. The development of these selective PDE5 inhibitors can further lead to draw strategies for the novel preclinical and/or clinical candidates based on pyrazolopyrimidinone scaffold.

Design and synthesis of 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-ones by Huisgen 1,3-dipolar cycloaddition with PI3Kγ isoform selective activity.

A strategy for construction of medicinally important 1,4-substituted 1H-1,2,3-triazolo-quinazolin-4(3H)-ones has been devised and presented here. The compounds have been synthesized using one-pot multicomponent strategy under microwave assisted conditions. Triazolyl-quinazolinone based D-ring modified analogs are designed based on IC87114 scaffold, which is first known isoform selective inhibitor of PI3Kδ. Herein, we identified two triazolyl-quinazolinone compounds (5a and 5l) based on same scaffold with PI3Kγ specific inhibitory potential, the selectivity towards this isoform is well supported by in silico results, wherein, these compounds show better interaction and affinity and inhibitory activity for PI3Kγ rather than PI3Kδ. This repositioning of scaffold from PI3Kδ to PI3Kγ isoform can be very useful from medicinal chemistry and drug discovery perspective to unravel molecular interactions of this new scaffold in different cellular pathways.

Fusion of Structure and Ligand Based Methods for Identification of Novel CDK2 Inhibitors.

Cyclin dependent kinases play a central role in cell cycle regulation which makes them a promising target with multifarious therapeutic potential. CDK2 regulates various events of the eukaryotic cell division cycle, and the pharmacological evidence indicates that overexpression of CDK2 causes abnormal cell-cycle regulation, which is directly associated with hyperproliferation of cancer cells. Therefore, CDK2 is regarded as a potential target molecule for anticancer medication. Thus, to decline CDK2 activity by potential lead compounds has proved to be an effective treatment for cancer. The availability of a large number of X-ray crystal structures and known inhibitors of CDK2 provides a gateway to perform efficient computational studies on this target. With the aim to identify new chemical entities from commercial libraries, with increased inhibitory potency for CDK2, ligand and structure based computational drug designing approaches were applied. A druglike library of 50,000 compounds from ChemDiv and ChemBridge databases was screened against CDK2, and 110 compounds were identified using the parallel application of these models. On in vitro evaluation of 40 compounds, seven compounds were found to have more than 50% inhibition at 10 µM. MD studies of the hits revealed the stability of these inhibitors and pivotal role of Glu81 and Leu83 for binding with CDK2. The overall study resulted in the identification of four new chemical entities possessing CDK2 inhibitory activity.

Benzothiazole Derivative as a Novel Mycobacterium tuberculosis Shikimate Kinase Inhibitor: Identification and Elucidation of Its Allosteric Mode of Inhibition.

Mycobacterium tuberculosis shikimate kinase (Mtb-SK) is a key enzyme involved in the biosynthesis of aromatic amino acids through the shikimate pathway. Since it is proven to be essential for the survival of the microbe and is absent from mammals, it is a promising target for anti-TB drug discovery. In this study, a combined approach of in silico similarity search and pharmacophore building using already reported inhibitors was used to screen a procured library of 20,000 compounds of the commercially available ChemBridge database. From the in silico screening, 15 hits were identified, and these hits were evaluated in vitro for Mtb-SK enzyme inhibition. Two compounds presented significant enzyme inhibition with IC50 values of 10.69 ± 0.9 and 46.22 ± 1.2 µM. The best hit was then evaluated for the in vitro mode of inhibition where it came out to be an uncompetitive and noncompetitive inhibitor with respect to shikimate (SKM) and ATP, respectively, suggesting its binding at an allosteric site. Potential binding sites of Mtb-SK were identified which confirmed the presence of an allosteric binding pocket apart from the ATP and SKM binding sites. The docking simulations were performed at this pocket in order to find the mode of binding of the best hit in the presence of substrates and the products of the enzymatic reaction. Molecular dynamics (MD) simulations elucidated the probability of inhibitor binding at the allosteric site in the presence of ADP and shikimate-3-phosphate (S-3-P), that is, after the formation of products of the reaction. The inhibitor binding may prevent the release of the product from Mtb-SK, thereby inhibiting its activity. The binding stability and the key residue interactions of the inhibitor to this product complex were also revealed by the MD simulations. Residues ARG43, ILE45, and PHE57 were identified as crucial that were involved in interactions with the best hit. This is the first report of an allosteric binding site of Mtb-SK, which could largely address the selectivity issue associated with kinase inhibitors.

Screening of antitubercular compound library identifies novel shikimate kinase inhibitors of Mycobacterium tuberculosis.

Shikimate kinase of Mycobacterium tuberculosis is involved in the biosynthesis of aromatic amino acids through shikimate pathway. The enzyme is essential for the survival of M. tuberculosis and is absent from mammals, thus providing an excellent opportunity for identifying new chemical entities to combat tuberculosis with a novel mechanism of action. In this study, an antitubercular library of 1000 compounds was screened against M. tuberculosis shikimate kinase (MtSK). This effort led to the identification of 20 inhibitors, among which five promising leads exhibited half maximal inhibitory concentration (IC50) values below 10 µM. The most potent inhibitor ("5631296") showed an IC50 value of 5.10 µM ± 0.6. The leads were further evaluated for the activity against multidrug-resistant (MDR)-TB, Gram-positive and Gram-negative bacterial strains, mode of action, docking simulations, and combinatorial study with three frontline anti-TB drugs. Compound "5491210" displayed a nearly synergistic activity with rifampicin, isoniazid, and ethambutol while compound "5631296" was synergistic with rifampicin. In vitro cytotoxicity against HepG2 cell line was evaluated and barring one compound; all were found to be non-toxic (SI > 10). In order to rule out mitochondrial toxicity, the promising inhibitors were also evaluated for cell cytotoxicity using galactose medium where compounds "5631296" and "5122752" appeared non-toxic. Upon comprehensive analysis, compound "5631296" was found to be the most promising MtSK inhibitor that was safe, synergistic with rifampicin, and bactericidal against M. tuberculosis.

Identification and characterization of novel small molecule inhibitors of the acetyltransferase activity of Escherichia coli N-acetylglucosamine-1-phosphate-uridyltransferase/glucosamine-1-phosphate-acetyltransferase (GlmU).

This study aims at identifying novel chemical scaffolds as inhibitors specific to the acetyltransferase domain of a bifunctional enzyme, Escherichia coli GlmU, involved in the cell wall biosynthesis of Gram-negative organisms. A two-pronged approach was used to screen a 50,000 small-molecule library. Using the first approach, the library was in silico screened by docking the library against acetyltransferase domain of E. coli GlmU studies. In the second approach, complete library was screened against Escherichia coli ATCC 25922 to identify the whole cell active compounds. Active compounds from both the screens were screened in a colorimetric absorbance-based assay to identify inhibitors of acetyltransferase domain of E. coli GlmU which resulted in the identification of 1 inhibitor out of 56 hits identified by in silico screening and 4 inhibitors out of 35 whole cell active compounds on Gram-negative bacteria with the most potent inhibitor showing IC50 of 1.40 ± 0.69 µM. Mode of inhibition studies revealed these inhibitors to be competitive with AcCoA and uncompetitive with GlcN-1-P. These selected inhibitors were also tested for their antibacterial and cytotoxic activities. Compounds 5175178 and 5215319 exhibited antibacterial activity that co-related with GlmU inhibition. These compounds, therefore, represent novel chemical scaffolds targeting acetyltransferase activity of E. coli GlmU.

4-(N-Phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines as inhibitors of mammalian target of rapamycin.

A series of 4-(N-phenyl-N'-substituted benzenesulfonyl)-6-(4-hydroxyphenyl)quinolines was designed, synthesized and evaluated for their biological potential as anticancer agents by screening the molecules against panel of five human cancer cell lines viz. HL-60, MiaPaCa-2, HCT116, PC-3 and HEP-G2. The series has shown good mTOR inhibitory activity at 0.5 µM concentration. The representative compound 7h was found to be most active with the IC50 of 613 nM against mTOR. In supportive evidence, the western blotting experiment revealed that compound 7h is more potent in inhibiting p-mTOR (S2448) activity in 2-4h at 5 and 10 µM concentrations and was selective and specific towards mTORC1 versus mTORC2. Towards understanding the mechanistic aspects we studied cell cycle analysis, mitochondrial membrane potential loss in MiaPaca-2 cells for compound 7h. The docking study for this series was performed to understand the binding mode of the compounds and its consequent effect in biological activity, the initial interaction studies were found to be useful in design of molecules, where compound 7h has shown additional H-bond interaction with Lys2171 apart from Val2240 and also a small hydrophobic cleft was observed with Leu2185, Met2345 and Ile2356.

Discovery of novel pyrazolopyrimidinone analogs as potent inhibitors of phosphodiesterase type-5.

Cyclic guanosine monophosphate (cGMP) specific phosphodiesterase type-5 (PDE5), a clinically proven target to treat erectile dysfunction and diseases associated with lower cGMP levels in humans, is present in corpus cavernosum, heart, lung, platelets, prostate, urethra, bladder, liver, brain, and stomach. Sildenafil, vardenafil, tadalafil and avanafil are FDA approved drugs in market as PDE5 inhibitors for treating erectile dysfunction. In the present study a lead molecule 4-ethoxy-N-(6-hydroxyhexyl)-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)benzenesulfonamide, that is, compound-4a, an analog of pyrazolopyrimidinone scaffold has been identified as selective PDE5 inhibitor. A series of compounds was synthesized by replacing N-methylpiperazine moiety (ring-C) of sildenafil structure with different N-substitutions towards sulfonamide end. Compound-4a showed lower IC50 value (1.5 nM) against PDE5 than parent sildenafil (5.6 nM) in in vitro enzyme assay. The isoform selectivity of the compound-4a against other PDE isoforms was similar to that of the Sildenafil. In corroboration with the in vitro data, this molecule showed better efficacy in in vivo studies using the conscious rabbit model. Also compound-4a exhibited good physicochemical properties like solubility, caco-2 permeability, cLogP along with optimal PK profile having no significant CYP enzyme inhibitory liabilities. Discovery of these novel bioactive compounds may open a new alternative for developing novel preclinical candidates based on this drugable scaffold.

Discovery of new Mycobacterium tuberculosis proteasome inhibitors using a knowledge-based computational screening approach.

Mycobacterium tuberculosis bacteria cause deadly infections in patients [Corrected]. The rise of multidrug resistance associated with tuberculosis further makes the situation worse in treating the disease. M. tuberculosis proteasome is necessary for the pathogenesis of the bacterium validated as an anti-tubercular target, thus making it an attractive enzyme for designing Mtb inhibitors. In this study, a computational screening approach was applied to identify new proteasome inhibitor candidates from a library of 50,000 compounds. This chemical library was procured from the ChemBridge (20,000 compounds) and the ChemDiv (30,000 compounds) databases. After a detailed analysis of the computational screening results, 50 in silico hits were retrieved and tested in vitro finding 15 compounds with IC50 values ranging from 35.32 to 64.15 µM on lysate. A structural analysis of these hits revealed that 14 of these compounds probably have non-covalent mode of binding to the target and have not reported for anti-tubercular or anti-proteasome activity. The binding interactions of all the 14 protein-inhibitor complexes were analyzed using molecular docking studies. Further, molecular dynamics simulations of the protein in complex with the two most promising hits were carried out so as to identify the key interactions and validate the structural stability.

Identification of novel MurA inhibitors using in silico approach, their validation and elucidation of mode of inhibition.

UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is an important enzyme involved in the first cytosolic step of bacterial cell wall synthesis. In this study a combination of ligand based and structure based in silico virtual screening methods were utilised for screening of more than 50,000 drug-like compounds from CSIR-IIIM in-house compound library in order to identify potent inhibitors of MurA. The identified hits were validated in vitro under various incubation conditions using Malachite green phosphate assay, and two potent hits viz 3772-9534 and D396-0012 were identified. Among these hits, compound 3772-9534 showed significant changes in the activity values in different assay conditions. The MD simulation study of 3772-9534 suggested a novel binding site in MurA enzyme, independent of the two-substrate binding sites. Binding of inhibitors at the allosteric site induces conformational changes in the enzyme, which leads to inhibition of enzymatic activity. Overall, the study offers new insight for targeting MurA, which may promote the discovery of novel MurA allosteric site inhibitors.

Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus.

OBJECTIVES: To delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus. METHODS: Capsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed. RESULTS: Capsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log(10). CONCLUSIONS: This study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence.