A Cell-based Screen in Actinomyces oris to Identify Sortase Inhibitors (original) (raw)
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In-Silico Identified New Natural Sortase A Inhibitors Disrupt S. aureus Biofilm Formation
International Journal of Molecular Sciences
Sortase A (SrtA) is a membrane-associated enzyme that anchors surface-exposed proteins to the cell wall envelope of Gram-positive bacteria such as Staphylococcus aureus. As SrtA is essential for Gram-positive bacterial pathogenesis but dispensable for microbial growth or viability, SrtA is considered a favorable target for the enhancement of novel anti-infective drugs that aim to interfere with key bacterial virulence mechanisms, such as biofilm formation, without developing drug resistance. Here, we used virtual screening to search an in-house natural compound library and identified two natural compounds, N1287 (Skyrin) and N2576 ((4,5-dichloro-1H-pyrrol-2-yl)-[2,4-dihydroxy-3-(4-methyl-pentyl)-phenyl]-methanone) that inhibited the enzymatic activity of SrtA. These compounds also significantly reduced the growth of S. aureus but possessed moderate mammalian toxicity. Furthermore, S. aureus strains treated with these compounds exhibited reduction in adherence to host fibrinogen, as ...
The Journal of Antibiotics
Staphylococcus aureus is one of the most dangerous pathogens commonly associated with high levels of morbidity and mortality. Sortase A is considered as a promising molecular target for the development of antistaphylococcal agents. Using hybrid virtual screening approach and FRET analysis, we have identified five compounds able to decrease the activity of sortase A by more than 50% at the concentration of 200 µM. The most promising compound was 2-(2-amino-3-chlorobenzoylamino)-benzoic acid which was able to inhibit S. aureus sortase A at the IC 50 value of 59.7 µM. This compound was selective toward sortase A compared to other four cysteine proteasescathepsin L, cathepsin B, rhodesain, and the SARS-CoV2 main protease. Microscale thermophoresis experiments confirmed that this compound bound sortase A with K D value of 189 µM. Antibacterial and antibiofilm assays also confirmed high specificity of the hit compound against two standard and three wild-type, S. aureus hospital infection isolates. The effect of the compound on biofilms produced by two S. aureus ATCC strains was also observed suggesting that the compound reduced biofilm formation by changing the biofilm structure and thickness.
Sortase A: An ideal target for anti-virulence drug development
Microbial Pathogenesis, 2014
Sortase A is a membrane enzyme responsible for the anchoring of surface-exposed proteins to the cell wall envelope of Gram-positive bacteria. As a well-studied member of the sortase subfamily catalysing the cell wall anchoring of important virulence factors to the surface of staphylococci, enterococci and streptococci, sortase A plays a critical role in Gram-positive bacterial pathogenesis. It is thus considered a promising target for the development of new anti-infective drugs that aim to interfere with important Gram-positive virulence mechanisms, such as adhesion to host tissues, evasion of host defences, and biofilm formation. The additional properties of sortase A as an enzyme that is not required for Grampositive bacterial growth or viability and is conveniently located on the cell membrane making it more accessible to inhibitor targeting, constitute additional reasons reinforcing the view that sortase A is an ideal target for anti-virulence drug development. Many inhibitors of sortase A have been identified to date using high-throughput or in silico screening of compound libraries (synthetic or natural), and while many have proved useful tools for probing the action model of the enzyme, several are also promising candidates for the development into potent inhibitors. This review is focused on the most promising sortase A inhibitor compounds that are currently in development as leads towards a new class of antiinfective drugs that are urgently needed to help combat the alarming increase in antimicrobial resistance.
Frontiers in Microbiology, 2020
Staphylococcus aureus is one of the most notorious pathogens and is frequently associated with nosocomial infections imposing serious risk to immune-compromised patients. This is in part due to its ability to colonize at the surface of indwelling medical devices and biofilm formation. Combating the biofilm formation with antibiotics has its own challenges like higher values of minimum inhibitory concentrations. Here, we describe a new approach to target biofilm formation by Gram positive bacteria. Sortase A is a transpeptidase enzyme which is responsible for tagging of around ∼22 cell surface proteins onto the outer surface. These proteins play a major role in the bacterial virulence. Sortase A recognizes its substrate through LPXTG motif. Here, we use this approach to install the synthetic peptide substrates on S. aureus. Sortase A substrate mimic, 6His-LPETG peptide was synthesized using solid phase peptide chemistry. Incorporation of the peptide on the cell surface was measured using ELISA. Effect of peptide incubation on Staphylococcus aureus biofilm was also studied. 71.1% biofilm inhibition was observed with 100 µM peptide while on silicon coated rubber latex catheter, 45.82% inhibition was observed. The present work demonstrates the inability of surface modified S. aureus to establish biofilm formation thereby presenting a novel method for attenuating its virulence.
Title : Identification of Small Molecule Inhibitors of the Staphylococcus aureus Sortase
2008
approved: ________________________________ ________________________ Dennis Hruby The Sortase A (SrtA) enzyme is a potential target f or a new class of anti-infective drugs because it is responsible for anchoring virulence f actors to the surface of pathogenic, Gram-positive bacteria such as Staphylococcus aureus. High throughput screening yielded low molecular weight compounds that inhibit purified SrtA from S. aureus. The efficacy of these compounds at targeting SrtA in li ve bacteria was successfully determined by developing sensitive biological assay that measure inhibition of sortasedependent surface protein expression in S. aureus. Fibrinogen-clumping and fibrinogenbinding assays were developed to determine the pres nc of clumping factors (ClfA and B), a fibronectin-binding assay was used to determi ne the presence of fibrinogen-binding proteins (FnBPA and B), and dot blots were develope d to measure protein A on the cell surface. Fifty-six compounds were identified that inhibi...
Identification of novel inhibitors of bacterial surface enzyme Staphylococcus aureus Sortase A
Bioorganic & Medicinal Chemistry Letters, 2008
In-silico virtual screening of bacterial surface enzyme Staphylococcus aureus Sortase A against commercial compound libraries using FlexX software package has led to the identification of novel inhibitors. Inhibition of enzyme catalytic activity was determined by monitoring the steady state cleavage of a model peptide substrate. Preliminary structure activity relationship studies on the lead compound resulted in the identification of compounds with improved activity. The most active compound has an IC50 value of 58 μM against the enzyme.A novel class of inhibitors of Staphylococcus aureus Sortase A is discovered by in-silico virtual screening and structure activity relationship studies.
Structural Analysis of Sortase A Inhibitors
Molecules, 2016
Bacterial sortases are cysteine transpeptidases that regulate the covalent linkage of several surface protein virulence factors in Gram-positive bacteria. Virulence factors play significant roles in adhesion, invasion of host tissues, biofilm formation and immune evasion, mediating the bacterial pathogenesis and infectivity. Therefore, sortases are emerging as important targets for the design of new anti-infective agents. We employed a computational study, based on structure derived descriptors and molecular fingerprints, in order to develop simple classification methods which could allow predicting low active or high active SrtA inhibitors. Our results indicate that a highly active SrtA inhibitor has a molecular weight ranging between 180 and 600, contains one up to four nitrogen atoms, up to three oxygen atoms and under 18 hydrogen atoms. Also the hydrogen acceptor number and the molecular flexibility, as assessed by the number of rotatable bounds, have emerged as the most relevant descriptors for SrtA affinity. The Bemis-Murcko scaffolding revealed favoured scaffolds as containing at least two ring structures bonded directly or merged in a condensed cycle. This data represent a valuable tool for identifying new potent SrtA inhibitors, potential anti-virulence agents targeted against Gram-positive bacteria, including multiresistant strains.
Pharmaceuticals
Increasingly ineffective antibiotics and rapid spread of multi- and pan-resistant bacteria represent a global health threat; hence, the need of developing new antimicrobial medicines. A first step in this direction is identifying new molecular targets, such as virulence factors. Sortase A represents a virulence factor essential for the pathogenesis of Gram-positive pathogens, some of which have a high risk for human health. We present here an exhaustive collection of sortases inhibitors grouped by relevant chemical features: vinyl sulfones, 3-aryl acrylic acids and derivatives, flavonoids, naphtoquinones, anthraquinones, indoles, pyrrolomycins, isoquinoline derivatives, aryl β-aminoethyl ketones, pyrazolethiones, pyridazinones, benzisothiazolinones, 2-phenyl-benzoxazole and 2-phenyl-benzofuran derivatives, thiadiazoles, triazolothiadiazoles, 2-(2-phenylhydrazinylidene)alkanoic acids, and 1,2,4-thiadiazolidine-3,5-dione. This review focuses on highlighting their structure–activity re...
Journal of Molecular Graphics and Modelling, 2015
Staphylococcus aureus sortase A is an attractive target of Gram-positive bacteria that plays a crucial role in anchoring of surface proteins to peptidoglycan present in bacterial cell wall. Inhibiting sortase A is an elementary and essential effort in preventing the pathogenesis. In this context, in silico virtual screening of in-house database was performed using ligand based pharmacophore model as a filter. The developed pharmacophore model AAHR 11 consists of two acceptors, one hydrophobic and one ring aromatic feature. Top ranked molecule KKR1 was docked into the active site of the target. After profound analysis, it was analyzed and optimized based on the observations from its binding pose orientation. Upgraded version of KKR1 was KKR2 and has improved docking score, binding interactions and best fit in the binding pocket. KKR1 along with KKR2 were further validated using 100 ns molecular dynamic studies. Both KKR1 and KKR2 contain Indole-thiazolidine moiety and were synthesized. The disk diffusion assay has good initial results (ZI of KKR1, KKR2 were 24, 38 mm at 10 g/mL and ZI of Ampicillin was 22 at 10 g/mL) and calculated MICs of the molecules (KKR1 5.56 ± 0.28 g/mL, KKR2 1.32 ± 0.12 g/mL, Ampicillin 8 ± 1.1 g/mL) were in good agreement with standard drug Ampicillin. KKR1 has shown IC 50 of 1.23 ± 0.14 M whereas the optimized lead molecule KKR2 show IC 50 of 0.008 ± 0.07 M. Results from in silico were validated by in vitro studies and proved that indole-thiazolidine molecules would be useful for future development as lead molecules against S. aureus sortase A.
Chemical Data Collections, 2018
Briefly describe the contentof thisdata article. If data is supplied with the data article, please explicitly state this or indicate the reference number and repository name for the data stored elsewhere. If this article is related to a published full research article, please also directly mention the article in the abstract] Novel molecules were designed against Staphylococcus aureus sortase A, an imperative and vital target involved in the bacterial virulence. Structure guided de novo designing was performed using different chemical fragments. These fragments were extended based on the requirements of the binding cavity. All the compounds in its binding orientation has formed good interaction network with amino acids HIS 120 and ARG 197. Molecules designed contain indole-chroman and indolin-chroman moieties and their docking scores were ranging from-6.92 to-5.67. Designed molecules were selected for synthesis and tested in vitro against Staphylococcus aureus sortase A using Fluorescence resonance energy transfer assay. The highest active molecule KK4 ((Z)-3-((5-nitro-1H-indol-3-yl) methylene) chroman-2,4-dione)