ANTI-STAPHYLOCOCCAL BIOFILM ACTIVITY OF NOVEL SORTASE A (SRTA) INHIBITORS

Pathogenic staphylococci have an extraordinary ability to form biofilms. This characteristic is likely the most important virulence factor of staphylococci in the development of the chronic form of infectious diseases and in biomaterial associated infections (BAI). Staphylococcal biofilms are particularly dangerous because they are more resistant to host immune defence system and have a significantly increased tolerance to conventional antibiotics. There is undoubtedly an urgent need for novel treatments, strategies and anti-staphylococcal biofilm agents. The Sortase A (SrtA) transpeptidase is responsible for covalent anchoring to the cell wall of various surface proteins (FnbpA, FnbpB, ClfA, ClfB, Protein A, etc.) that have a direct role in the pathogenesis and in the first stage of biofilm formation and because of this it can be considered a good target candidate to design agents that could interfere with virulence mechanism including biofilm formation. With the aim to discover new SrtA inhibitors, a library of 50000 low-molecular weight compounds was screened in a high throughput assay by using the standard Dabcyl-QALPETGEE-Edans fluorescence resonance energy transfer (FRET)- peptide substrate for measurement of enzyme activity. A group of the selected 38 most potent compounds and 3 known reference inhibitors were further evaluated in an in vitro biofilm formation assay at a screening concentration of 10\ub5g/ml using three reference staphylococcal strains S.aureus 29213, 6538 and S.epidermidis RP62A. An interesting correlation between inhibition of SrtA and biofilm formation inhibition was observed in many cases especially at a concentration equal or more than IC50 determined as SrtA inhibitors

Enzymatic Activity of Circular Sortase A Under Denaturing Conditions: an Advanced Tool for Protein Ligation

Staphylococcus aureus sortase A is a transpeptidase that is extensively used in various protein research applications. Sortase A is highly selective and does not require any cofactors for the catalysis of protein ligation and, importantly, can be produced in high yields. However, the primary disadvantage of this transpeptidase is its inability to access the recognition site within the highly structured regions of folded substrates. To overcome this problem, we developed an Escherichia coli expression system that produces milligram quantities of circularly closed sortase A; efficient enzyme cyclization was achieved by Synechocystis sp. PCC6803 intein-mediated post-translational splicing. The structural integrity of circular sortase A and its biochemical characteristics were compared to those of the linear enzyme analog and were found to be similar under native conditions. Additionally, the modified sortase was active at concentrations of urea up to 3 M and was capable of efficient catalytic protein–protein coupling, as shown by the ligation of purified glutathione-S-transferase and green fluorescence protein. In contrast to the circular enzyme, linear sortase A was unable to mediate the ligation of substrate proteins under the same conditions. Therefore, the proposed circular sortase A has improved enzymatic properties and has applications in advanced protein engineering and design

Discovery and Structure-Activity Relationship Studies of Irreversible Benzisothiazolinone-Based Inhibitors against Staphylococcus Aureus Sortase A Transpeptidase

Gram-positive bacteria, in general, and staphylococci, in particular, are the widespread cause of nosocomial and community-acquired infections. The rapid evolvement of strains resistant to antibiotics currently in use is a serious challenge. Novel antimicrobial compounds have to be developed to fight these resistant bacteria, and sortase A, a bacterial cell wall enzyme, is a promising target for novel therapies. As a transpeptidase that covalently attaches various virulence factors to the cell surface, this enzyme plays a crucial role in the ability of bacteria to invade the host’s tissues and to escape the immune response. In this study we have screened a small molecule library against recombinant Staphylococcus aureus sortase A using an in vitro FRET-based assay. The selected hits were validated by NMR methods in order to exclude false positives and to analyze the reversibility of inhibition. Further structural and functional analysis of the best hit allowed the identification of a novel class of benzisothiazolinone-based compounds as potent and promising sortase inhibitors

Novel Sortase A (SrtA) inhibitors interfere with the formation of staphylococcal biofilms

Staphylococcus aureus, due to its wide arsenal of virulence factors, is a very versatile pathogen responsible for a wide variety of infectious diseases. The virulence factors include the cell-wall associated proteins that have a direct role in the first stage of pathogenesis. The Sortase A (SrtA) transpeptidase is responsible for covalent anchoring to the cell wall of various surface proteins and it is considered a good target to design new antivirulence agents. In this study, we report the identification of an inhibitor of SrtA afforded from the random screening of a small molecular library of around 150 synthetic compounds, through a high throughput assay by using the standard Dabcyl-QALPETGEE-Edans fluorescence resonance energy transfer (FRET)-peptide substrate for measurement of enzyme activity. Such hit compound showed an IC50 value of 80µM. A group of derivatives of the hit compound has been obtained and evaluated for their activity as SrtA inhibitors. The efficacy of the most interesting SrtA inhibitors needs to be evaluated by in vivo models of infection, but we used a simple methodology in vitro, such as inhibition of biofilm formation, for a preliminary assessment of antivirulence properties of some of these novel inhibitors of SrtA. An interesting correlation between inhibition of SrtA and interference with the formation of staphylococcal biofilms has been observed in many cases especially at a concentration equal or more than IC50 determined as SrtA inhibitors

Protein Macrocyclization for Tertiary Structure Stabilization

Proteins possess unique molecular recognition capabilities and enzymatic activities, features that are usually tied to a particular tertiary structure. To make use of proteins for biotechnological and biomedical purposes, it is often required to enforce their tertiary structure in order to ensure sufficient stability under the conditions inherent to the application of interest. The introduction of intramolecular crosslinks has proven efficient in stabilizing native protein folds. Herein, we give an overview of methods that allow the macrocyclization of expressed proteins, discussing involved reaction mechanisms and structural implications