Quinone skeleton as a new class of irreversible inhibitors against Staphylococcus aureus sortase A

Sortase A (SrtA) anchors surface proteins to the cell wall and aids biofilm formation during infection, which functions as a key virulence factor of important Gram-positive pathogens, such as Staphylococcus aureus. At present researchers need a way in which to validate whether or not SrtA is a druggable target alternative to the conventional antibiotic targets in the mechanism. In this study, we performed a high-throughput screening and identified a new class of potential inhibitors of S. aureus SrtA, which are derived from natural products and contain the quinone skeleton. Compound 283 functions as an irreversible inhibitor that covalently alkylates the active site Cys184 of SrtA. NMR analysis confirms the direct interaction of the small-molecule inhibitor towards SrtA protein. The anchoring of protein A (SpA) to the cell wall and the biofilm formation are significantly attenuated when the S. aureus Newman strain is cultured in the presence of inhibitor. Our study indicates that compound 283 could be a potential hit for the development of new anti-virulence agents against S. aureus infections by covalently targeting SrtA.     

Interaction of sortase A and lipase 2 in the inhibition of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilm formation

Recombinant sortase A (SrtA) was used to immune rabbit, and the inhibitory activity of anti-SrtA serum on Staphylococcus aureus biofilm formation was tested. Biofilm formation was inhibited by anti-SrtA rabbit serum in S. aureus ATCC25923 and two clinical isolated strains. The antiserum was separated into two fractions, and the main component with the inhibitory activity was demonstrated to be the IgG fraction. Two proteins interact with the IgG fraction were identified by using an in vitro pull-down assay and were confirmed to be lipase 2 and γ-hemolysin by mass spectrometry. Cross-interaction between SrtA and lipase 2 was further confirmed by Western blotting. Addition of anti-lipase 2 serum in the culture medium also showed inhibitory effect against biofilm formation. Together, our study suggests anti-SrtA serum inhibits S. aureus biofilm formation and lipase 2 is one of the targets of anti-SrtA serum in this inhibition process. This is the first study to demonstrate the roles of antisera against SrtA and lipase 2 in the inhibition of biofilm formation in S. aureus.     

Improving the Efficiency of the Modified Hodge Test in KPC-Producing Klebsiella pneumoniae Isolates by Incorporating an EDTA Disk

Streptococcus mutans (S. mutans) is the main etiological agent of dental caries, and adheres to the tooth surface through the sortase A (SrtA)-mediated cell wall-anchored protein Pac. Inhibition of SrtA activity results in a marked reduction in the adhesion potential of S. mutans, and the frequency of dental caries. Morin is a natural plant extract that was previously reported to inhibit Staphylococcus aureus SrtA activity. Here, we demonstrate that morin has an inhibitory effect against S. mutans UA159 SrtA, with an IC₅₀ of 27.2 ± 2.6 μM. Western blotting demonstrated that 30 μM morin induced the partial release of the Pac protein into the supernatant. The biofilm mass of S. mutans was reduced in the presence of 30 μM morin, which was not caused by a decrease in S. mutans viability. These results indicate that morin might be important as a new agent to prevent caries.     

Synthesis,biological evaluations and computational studies of N-(3-(-2-(7-Chloroquinolin-2-yl)vinyl) benzylidene)anilines as fungal biofilm inhibitors

In the present investigation, new chloroquinoline derivatives bearing vinyl benzylidene aniline substituents at 2nd position were synthesized and screed for biofilm inhibitory, antifungal and antibacterial activity. The result of biofilm inhibition of C. albicans suggested that compounds 5j (IC₅₀ value?=?51.2?μM) and 5a (IC₅₀ value?=?66.2?μM) possess promising antibiofilm inhibition when compared with the standard antifungal drug fluconazole (IC₅₀?=?40.0?μM). Two compounds 5a (MIC?=?94.2?μg/mL) and 5f (MIC?=?98.8?μg/mL) also exhibited good antifungal activity comparable to standard drug fluconazole (MIC?=?50.0?μg/mL). The antibacterial screening against four strains of bacteria viz. E. coli, P. aeruginosa, B. subtilis, and S. aureus suggested their potential antibacterial activity and especially all the compounds except 5g were found more active than the standard drug ciprofloxacin against B. subtilis. To further gain insights into the possible mechanism of these compounds in biofilm inhibition through the agglutinin like protein (Als), molecular docking and molecular dynamics simulation studies were carried out. Molecular modeling studies suggested the clear role in inhibition of this protein and the resulting biofilm inhibitory activity.     

Interrogation of Bacillus anthracis SrtA active site loop forming open/close lid conformations through extensive MD simulations for understanding binding selectivity of SrtA inhibitors

Bacillus anthracis is a gram positive, deadly spore forming bacteria causing anthrax and these bacteria having the complex mechanism in the cell wall envelope, which can adopt the changes in environmental conditions. In this, the membrane bound cell wall proteins are said to progressive drug target for the inhibition of Bacillus anthracis. Among the cell wall proteins, the SrtA is one of the important mechanistic protein, which mediate the ligation with LPXTG motif by forming the amide bonds. The SrtA plays the vital role in cell signalling, cell wall formation, and biofilm formations. Inhibition of SrtA leads to rupture of the cell wall and biofilm formation, and that leads to inhibition of Bacillus anthracis and thus, SrtA is core important enzyme to study the inhibition mechanism. In this study, we have examined 28 compounds, which have the inhibitory activity against the Bacillus anthracis SrtA for developing the 3D-QSAR and also, compounds binding selectivity with both open and closed SrtA conformations, obtained from 100 ns of MD simulations. The binding site loop deviate in forming the open and closed gate mechanism is investigated to understand the inhibitory profile of reported compounds, and results show the closed state active site conformations are required for ligand binding specificity. Overall, the present study may offer an opportunity for better understanding of the mechanism of action and can be aided to further designing of a novel and highly potent SrtA inhibitors.     

Discovery and development of substituted thiadiazoles as inhibitors of Staphylococcus aureus Sortase A

High-throughput screening of small-molecule libraries has led to the identification of thiadiazoles as a new class of inhibitors against Staphylococcus aureus sortase A (SrtA). N-(5-((4-nitrobenzyl)thio)-1,3,4-thiadiazol-2-yl)nicotinamide (IC₅₀ = 3.8 µM) was identified as a potent inhibitor of SrtA after synthetic modification of hit compounds. Additional ligands developed in this study displayed affinities in the low micromolar range without affecting bacterial growth in vitro. The study also suggest a new mode of action through covalent binding to the active site cysteine.     

Design,synthesis and biological evaluation of novel hybrids of N-aryl pyrrothine-base α-pyrone as bacterial RNA polymerase inhibitors

Antibiotic resistance in bacteria has been an emerging public health problem, thus discovery of novel and effective antibiotics is urgent. A series of novel hybrids of N-aryl pyrrothine-base α-pyrone hybrids was designed, synthesized and evaluated as bacterial RNA polymerase (RNAP) inhibitors. Among them, compound 13c exhibited potent antibacterial activity against antibiotic-resistant S. aureus with the minimum inhibitory concentration (MIC) in the range of 1–4 μg/mL. Moreover, compound 13c exhibited strong inhibitory activity against E.coli RNAP with IC₅₀ value of 16.06 μM, and cytotoxicity in HepG2 cells with IC₅₀ value of 7.04 μM. The molecular docking study further suggested that compound 13c binds to the switch region of bacterial RNAP. In summary, compound 13c is a novel bacterial RNAP inhibitor, and a promising lead compound for further optimization.     

Syntheses,in vitro urease inhibitory activities of urea and thiourea derivatives of tryptamine,their molecular docking and cytotoxic studies

Urease is an enzyme of amidohydrolase family and is responsible for the different pathological conditions in the human body including peptic ulcers, catheter encrustation, kidney stone formation, hepatic coma, encephalopathy, and many others. Therefore, the search for potent urease inhibitors has attracted major scientific attention in recent years. Urea and thiourea derivatives of tryptamine (1–25) were synthesized via reaction of tryptamine with different substituted phenyl isocyanates/isothiocyanates. The synthetic compounds were evaluated for their urease enzyme inhibitory activity and they exhibited good inhibitory potential against urease enzyme in the range of (IC₅₀ = 11.4 ± 0.4–24.2 ± 1.5 μM) as compared to the standard thiourea (IC₅₀ = 21.2 ± 1.3 μM). Out of twenty-five compounds, fourteen were found to be more active than the standard. Limited structure-activity relationship suggested that the compounds with CH₃, and OCH₃ substituents at aryl part were the most potent derivatives. Compound 14 (IC₅₀ = 11.4 ± 0.4 μM) with a methyl substituent at ortho position was found to be the most active member of the series. Whereas, among halogen substituted derivatives, para substituted chloro compound 16 (IC₅₀ = 13.7 ± 0.9 μM) showed good urease inhibitory activity. These synthetic derivatives were found to be non-cytotoxic in cellular assay. Kinetic studies revealed that the compounds 11, 12, 14, 17, 21, 22, and 24 showed a non-competitive type of inhibition. In silico study identified the possible bindings interactions of potential inhibitors with the active site of enzyme. These newly identified inhibitors of urease enzyme can serve as leads for further research and development.     

Inhibition of sortase-mediated Staphylococcus aureus adhesion to fibronectin via fibronectin-binding protein by sortase inhibitors

The sortase enzymes are a family of Gram-positive transpeptidases responsible for anchoring surface protein virulence factors to the peptidoglycan cell wall layer. In Staphylococcus aureus, deletion of the sortase isoforms results in marked reduction in virulence and infection potential, making it an important antivirulence target. Recombinant sortase A (SrtA) and sortase B (SrtB) were incubated with peptide substrate containing either the LPETG or NPQTN motifs. (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile, β-sitosterol-3-O-glucopyranoside, berberine chloride, and psammaplin A1 showed potent inhibitory activity against SrtA and SrtB. These compounds also exhibited potent inhibitory activity against S. aureus cell adhesion to fibronectin. The fibronectin-binding activity data highlight the potential of these compounds for the treatment of S. aureus infections via inhibition of sortase activity.     

Discovery and structure–activity relationship analysis of Staphylococcus aureus sortase A inhibitors

Methicillin resistant Staphylococcus aureus (MRSA) is a major health problem that has created a pressing need for new antibiotics. Compounds that inhibit the S. aureus SrtA sortase may function as potent anti-infective agents as this enzyme attaches virulence factors to the cell wall. Using high-throughput screening, we have identified several compounds that inhibit the enzymatic activity of the SrtA. A structure–activity relationship (SAR) analysis led to the identification of several pyridazinone and pyrazolethione analogs that inhibit SrtA with IC₅₀ values in the sub-micromolar range. Many of these molecules also inhibit the sortase enzyme from Bacillus anthracis suggesting that they may be generalized sortase inhibitors.     

Synthesis and in vitro urease inhibitory activity of benzohydrazide derivatives,in silico and kinetic studies

Benzohydrazide derivatives 1–43 were synthesized via “one-pot” reaction and structural characterization of these synthetic derivatives was carried out by different spectroscopic techniques such as ¹H NMR and EI-MS. The synthetic molecules were evaluated for their in vitro urease inhibitory activity. All synthetic derivatives showed good inhibitory activities in the range of (IC₅₀ = 0.87 ± 0.31–19.0 ± 0.25 µM) as compared to the standard thiourea (IC₅₀ = 21.25 ± 0.15 µM), except seven compounds 17, 18, 23, 24, 29, 30, and 41 which were found to be inactive. The most active compound of the series was compound 36 (IC₅₀ = 0.87 ± 0.31 μM) having two chloro groups at meta positions of ring A and methoxy group at para position of ring B. The structure–activity relationship (SAR) of the active compounds was established on the basis of different substituents and their positions in the molecules. Kinetic studies of the active compounds revealed that compounds can inhibit enzyme via competitive and noncompetitive modes. In silico study was also performed to understand the binding interactions of the molecules (ligand) with the active site of enzyme.     

Inhibition of biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC₅₀ = 20 μM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.     

Design,synthesis, in-silico studies and biological screening of quinazolinone analogues as potential antibacterial agents against MRSA

Type or The emergence of resistance to antibiotic has developed a complicated situation in the treatment of bacterial infections. Considering the antimicrobial resistance phenomenon as one of the greatest challenge of medicinal chemists for search of better anti-bacterial agents, which have potential narrow spectrum activity with low development of resistance potential and low toxicity to host. Cross-linking of peptidoglycan is a key step catalyze by Penicillin binding protein (PBP) to maintain integrity of cell wall in bacterial cell. However, these Penicillin binding protein (PBP) has developed resistance in methicillin-resistant Staphylococcus aureus (MRSA) due to acquisition of additional PBP2a. Various Quinazolinone analogues are reported in literature as potential anti-bacterial agents against MRSA. In present study new quinazolinone analogues has been designed, guided by molecular docking, In-silico and MM-GBSA study. Newly designed molecules have been synthesized by medicinal chemistry route and their characterization was done by using IR, NMR, & HR-MS techniques. Biological evaluation of synthesized compounds has been done on wild type Gram-negative (Escherichia coli), Gram-positive (Staphylococcus aureus) and resistant MRSA bacterial strains using Streptomycin, Kanamycin and Linezolid as standard drugs respectively. The in vitro evaluation results have shown that compound 5f is active with MIC value 15.625 μg/mL against S. aureus and with MIC value 31.25 μg/mL against MRSA.     

Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamides as alkaline phosphatase inhibitors: Synthesis,computational studies,enzyme inhibitory kinetics and DNA binding studies

Substituted phenyl[(5-benzyl-1,3,4-oxadiazol-2-yl)sulfanyl]acetates/acetamides 9a-j were synthesized as alkaline phosphatase inhibitors. Phenyl acetic acid 1 through a series of reactions was converted into 5-benzyl-1,3,4-oxadiazole-2-thione 4. The intermediate oxadiazole 4 was then reacted with chloroacetyl derivatives of phenols 6a-f and anilines derivatives 8a-d to afford the title oxadiazole derivatives 9a-j. All of the title compounds 9a-j were evaluated for their inhibitory activity against human alkaline phosphatise (ALP). It was found that compounds 9a-j exhibited good to excellent alkaline phosphatase inhibitory activity especially 9h displayed potent activity with IC₅₀ value 0.420 ± 0.012 µM while IC₅₀ value of standard (KH₂PO₄) was 2.80 µM. The enzyme inhibitory kinetics of most potent inhibitor 9h was determined by Line-weaever Burk plots showing non-competitive mode of binding with enzyme. Molecular docking studies were performed against alkaline phosphatase enzyme (1EW2) to check the binding affinity of the synthesized compounds 9a-j against target protein. The compound 9h exhibited excellent binding affinity having binding energy value (−7.90 kcal/mol) compared to other derivatives. The brine shrimp viability assay results proved that derivative 9h was non-toxic at concentration used for enzyme assay. The lead compound 9h showed LD₅₀ 106.71 µM while the standard potassium dichromate showed LD₅₀ 0.891 µM. The DNA binding interactions of the synthesized compound 9h was also determined experimentally by spectrophotometric and electrochemical methods. The compound 9h was found to bind with grooves of DNA as depicted by both UV–Vis spectroscopy and cyclic voltammetry with binding constant values 7.83 × 10³ and 7.95 × 10³ M⁻¹ respectively revealing significant strength of 9h-DNA complex. As dry lab and wet lab results concise each other it was concluded that synthesized compounds, especially compound 9h may serve as lead compound to design most potent inhibitors of human ALP.     

Synthesis,anticancer assessment on human breast,liver and colon carcinoma cell lines and molecular modeling study using novel pyrazolo[4,3-c]pyridine derivatives

The key intermediate 3-aminopyrazolo[4,3-c]pyridine-4,6-dione (2) is considered as a precursor for some novel pyrazolo[4,3-c]pyridines 4a-c, arylhydrazopyrazolo[4,3-c]pyridines 8a-e, pyrazolo[4,5,1-ij][1,6]naphthyridines 11a-e and pyrido[4′,3′:3,4]pyrazolo[1,5-a]-pyrimidines 15a-d through Knovenegal condensation, coupling reaction and Michael addition. Some of the newly synthesized pyrazolo[4,3-c]pyridine derivatives were investigated for anticancer activity. The results of the cytotoxic activity revealed that compound 6b was the most active compound against the breast and liver carcinoma cell lines which gives IC₅₀ values of 1.937 and 3.695 µg/mL, respectively compared to reference drug (doxorubicin) with IC₅₀ values of 2.527 and 4.749 µg/ml, respectively. Moreover, compound 6c was potent compound against the colon carcinoma cell line which gives the value of IC₅₀ = 2.914 µg/ml compared to doxorubicin with IC₅₀ value of 3.641 µg/ml. Some selected of the novel synthesized compounds were docked inside the active site of ERK2 enzyme and were found display a suitable binding with the active site amino acids according to their bond lengths, angles and conformational energy.     

Lipoic acid modified antimicrobial peptide with enhanced antimicrobial properties

RIWVIWRR-NH₂ (Bac8c) is a natural antimicrobial peptide (AMP) exhibiting great antibacterial activity against Gram-negative and Gram-positive bacteria. In this work, lipoic acid was used as a fatty acid hydrophobic ligand to modify Bac8c (LA-Bac8c) to further improve its antimicrobial properties. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) assays showed that LA-Bac8c exhibited lower MIC (MBC) values against Staphylococcus aureus (S. aureus) and methicillin-resistant Staphylococcus aureus (MRSA) than Bac8c. Similar results were reflected in the antibiofilm activity towards S. aureus and MRSA, and LA-Bac8c showed better activity to the biofilm which has been formed or is being formed. In addition to this, the obvious interaction between bacteria/biofilm and LA-Bac8c was observed by microscopy. LA-Bac8c displayed strong membrane depolarization and outer membrane permeabilizing ability, and the cell membrane treated with LA-Bac8c was destroyed to the leakage of bacteria cellular components. All these data indicated LA-Bac8c could be used as a useful antimicrobial peptide with wide application prospect.     

New antibacterial and 5-lipoxygenase activities of synthetic benzyl phenyl ketones: Biological and docking studies

We investigated twelve benzyl phenyl ketone derivatives which are synthetic precursors of isoflavonoids that are shown be good 5-hLOX inhibitors, especially those that have the catechol group, but these precursors never have been assayed as 5-hLOX inhibitors being a novelty as inhibitors of the enzyme, due to sharing important structural characteristics. Screening assays, half maximal inhibitory concentration (IC₅₀) and kinetic assays of all the studied molecules (5 µg/ml in media assay) showed that 1-(2,4-dihydroxy-3-methylphenyl)-2-(3-chlorophenyl)-ethanone (K205; IC₅₀ = 3.5 µM; K_i = 4.8 µM) and 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-nitrophenyl)-ethanone (K206; IC₅₀ = 2.3 µM; K_i = 0.7 µM) were potent, selective, competitive and nonredox inhibitors of 5-hLOX. Antioxidant behavior was also assayed by DPPH, FRAP, and assessing ROS production, and those with antibacterial and antiproliferative properties relating to 1-(2,4-dihydroxy-3-methylphenyl)-2-(2-chlorophenyl)-ethanone (K208) established it as the most interesting and relevant compound studied, as it showed nearly 100% inhibition of bacterial growth of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). Finally, docking studies were done that helped to characterize how the inhibitor structures correlated to decreased 5-hLOX activity.     

Novel tacrine-coumarin hybrids linked to 1,2,3-triazole as anti-Alzheimer’s compounds: In vitro and in vivo biological evaluation and docking study

A new series of tacrine-coumarin hybrids linked to 1,2,3-triazole were designed, synthesized, and tested as potent dual binding site cholinesterase inhibitors (ChEIs) for the treatment of Alzheimer’s disease (AD). Among them, compound 8e was the most potent anti-AChE derivative (IC₅₀ = 27 nM) and compound 8m displayed the best anti-BChE activity (IC₅₀ = 6 nM) much more active than tacrine and donepezil as the reference drugs. Compound 8e was also evaluated for its BACE1 inhibitory activity and neuroprotectivity against PC12 cells exposed to Aβ_25-35 which indicated low activity. Finally, in vivo studies by Morris water maze task showed that compound 8e significantly reversed scopolamine-induced memory deficit in rats.     

Therapeutic effect of (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) against Staphylococcus aureus infection in a murine model

Sortase enzymes belong to a family of transpeptidases found in Gram-positive bacteria. Sortase is responsible for the reaction that anchors surface protein virulence factors to the peptidoglycan cell wall of the bacteria. The compound (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) has previously been reported as a novel sortase inhibitor in vitro, but the in vivo effects of DMMA have not been studied. Here, we evaluated the in vivo effects of DMMA against infection by wild-type and sortase A- and/or sortase B-deficient Staphylococcus aureus in Balb/c mice. With DMMA treatment, survival rates increased and kidney and joint infection rates decreased (p < 0.01) in a dose-dependent manner. The rate of kidney infection was significantly reduced in the mice treated with sortase A knock-out S. aureus (p < 0.01). These results indicate that by acting as a potent inhibitor of sortase A and moderate inhibitor of sortase B, DMMA can decrease kidney and joint infection rates and reduce mortality in mice infected with S. aureus. These findings suggest that DMMA is a promising therapeutic compound against Gram-positive bacteria.     

New carboxamides bearing benzenesulphonamides: Synthesis,molecular docking and pharmacological properties

Ten new derivatives of benzenesulphonamide bearing carboxamide functionality were synthesized and investigated for their in vitro antimicrobial, antioxidant and in vivo anti-inflammatory activities. Compound 9d inhibited carrageenan induced rat-paw oedema at 93.81, 88.79 and 86.09% at 1 h, 2 h and 3 h administration respectively. In the antimicrobial activity, compound 9a (6.54, 6.69 and 6.64 mg/mL) was most potent against S. aureus, B. subtilis and C. albicans respectively, compound 9e (6.45 and 6.46 mg/mL) was most active against P. aeruginosa and A. niger respectively while compound 9i (6.24 mg/mL) was most active against E. coli. Only compound 9a (IC₅₀ 0.3052 mg/mL) had comparable activity with Vitamin C (IC₅₀ 0.2090 mg/mL) in the antioxidant assay.