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Development of new antimicrobial agents targeting Gram-positive bacterial sortase A

 

 

Project Title: „Development of new antimicrobial agents targeting Gram-positive bacterial sortase A” 

Funding: European Regional Development Fund (ERDF), Measure 1.1.1.1 “Support for applied research”

Project No.: 1.1.1.1/16/A/107

Period: 1st June 2017 – 31 st May 2020

Project costs: 583 689.55 EUR 

Principle Investigator: Dr. biol. A.Leončiks 

 

The aim of the project is to develop new antibacterial agents that target the enzyme sortase A found in most Gram-positive bacteria. Such agents with a new mode of action are urgently needed due to a notable increase of antibiotic-resistance among community- and hospital-acquired bacterial infections. Sortase A is a very attractive target for the development of new antibacterials because it plays a key role in bacterial virulence and infection by attaching important surface proteins to the cell wall, though not being essential for bacterial viability and thus reducing the possibility of developing resistance.

The aim of the project corresponds to the Latvian Smart Specialization Strategy in the field of development of human capital in science and technology and in creation of new knowledge in the following fields: biomedicine, medicinal technology, biopharmacy and biotechnology. The main and most basic part of the project that includes selection of the inhibitory compounds, structure and ligand interaction studies, and tests in animals will be carried out in the Biomedical Research and Study Centre where is the necessary infrastructure including equipment and competent scientific personnel. The additional studies of NMR structural analysis of inhibitors and all necessary chemical synthesis will be organized as outsourcing in a way as it was realized before in similar projects. It is possible to achieve the main goal of the project – discovery, selection and characterization of new antimicrobials against Gram+ pathogens Staphylococcus aureus, Listeria monocytogenes and Bacillus anthracis in a planned time frame of 36 months. By completing the project three scientific articles will be prepared and submitted.

Information published: 01.06.2017.


1st stage 2017 June to August (3 months)

Gram+ microorganism Staphylococcus aureus, Listeria monocytogenes and Bacillus anthracis sortase A encoding genetic constructs were used for the production of enzymatically active proteins for further structural research purposes, selection and characterization of new inhibitory compounds. At the beginning the recombinant proteins are synthesized in analytical scales in order to optimize the bacterial expression level and gradually increase the enzyme production to preparative amounts. Simultaneously, all recombinant proteins are chromatographically fractionated and purified to a highest degree of homogeneity. 

The first purified SrtA proteins are used to initiate and optimize the analysis of specific sortase enzymatic activity in fluorometric cleavage assays with the labeled FRET peptide substrates and selected inhibitory compounds, and to standardize the protocols used for fluorometric data collection and processing. In addition, optimization tests of crystallization conditions for several available sortase A recombinant enzymes are initiated that are planned to be used for the study of the sortase-inhibitor complexes and structural modelling.  

Information published: 31.08.2017.

 

2 stage 2017 September to November (3 months)

All previously planned sortase A expression constructs are completed and the specific proteins have been synthesized in E.coli expression system. As a result, Gram+ Staphylococcus aureus, Listeria monocytogenes and Bacillus anthracis sortases A have been acquired in preparative amounts and further purified to the level necessary for FRET analysis and structural characterization. In addition, L.monocytogenes SrtA(ΔN58) as the first candidate for NMR structural studies has been expressed as isotopically labelled protein. The untagged purified sortases are used in crystallization screens to determine the optimal conditions for protein crystal growth.

As next, these constructs are used to optimize enzymatic activity in FRET assay necessary for chemical library screenings. Currently several FRET substrates are tested with different fluorophores and quenchers. In these tests S.aureus SrtA(ΔN59) demonstrated the highest enzymatic activity while B.anthracis SrtA(ΔN56) and L.monocytogenes SrtA(ΔN58) activity were selective in regards to substrate specificity and moderate in regards to total fluorescence signal output after substrate cleavage.

Information published: 30.11.2017.

 

3 stage December to February (3 months)

In total, five novel genetic constructs that encode recombinant sortase A of B.anthracis and L.monocytogenes were tested in order to improve the efficiency of the FRET enzymatic activity assay. All isotopically labelled sortases were further investigated by NMR spectroscopy to elucidate their structures and characterize the mechanism of enzyme-inhibitor interaction. In parallel, we investigated in silico the putative interaction and optimal binding parameters of SrtA inhibitors by using molecular modeling software. We have finished the preliminary tests where several SrtA substrates with diverse amino acid composition and FRET pairs were used to evaluate the assay’s sensitivity and specificity, and have chosen the most suitable universal fluorescently-labelled oligopeptides for small chemical library screening against three different bacterial sortases A.

Information published: 28.02.2018.

 

4 stage March to May (3 months)

The initially planned three distinct Gram+ sortase A bacterial expressions and enzymatic tests with the aim to create the optimal sortase constructs were finished during the fourth reporting period. These optimized constructs are now to be used for screening of the library of small chemical compounds to select the most promising sortase inhibitors. All high-throughput screening assays at this stage are performed using the first of two planned FRET peptides with Abz fluorescent group to select the strongest hits for further confirmatory tests. The enzyme structure and interaction reactions with the selected sortase A inhibitory compounds and fragment library were analyzed and possible reaction types have been identified. In total, five different covalent and non-covalent interaction mechanisms were observed and classified in the docking studies when the selected ligands showed potent binding activity to recombinant sortase A protein, their molecular mode of action, impact and the strength of the complex formation were investigated and the interacting residues were mapped.

Information published: 31.05.2018.


5th stage June to August (3 months)

During the fifth reporting period, the analysis of the small compound library compounds was completed and preliminary data on potential sortase inhibitors were collected. In total, 8000 new chemical compounds from the focused library were screened in the HTS assay with Abz-LPETG-K(DNP) FRET peptide and three Gram+ recombinant sortases A from B.anthracis, L.monocytogenes and S.aureusmicroorganisms. The screening of the library is continuing with the next FRET peptide Dabcyl-LPETG-Edans and after completing this assay the best selected inhibitory substances will be used in the further experiments.

The structural study of sortase A is focused on the interaction of several synthesized inhibitory compounds with the active center of sortase A enzyme in order to design novel non-covalent compounds with high affinity and selectivity. In addition, all three available recombinant sortase A enzyme-inhibitor structure complexes are modelled using the 2D NMR spectroscopy.

Information published: 31.08.2018.

 

6 stage September to November (3 months)

During the sixth reporting period, the FRET analysis of the small compound library using two fluorescently labeled substrates and three recombinant Gram+ bacterial sortase A encoding enzymes has been completed. In total, several hundred compounds were identified as potential sortase A inhibitors. Further biological analysis will be performed to determine in detail their toxicity, virulence and infection control abilities.

The structural studies of several covalent and non-covalent SrtA inhibitors have revealed molecular mechanisms of interaction between selected inhibitory molecules and particular amino acids in the proteolytic domain. Additionally, 2D and 3D NMR structural information has been obtained for sortase A complexes with three different inhibitors that also will be used in further chemical modification studies in order to selectively enhance specific activity and other relevant functional properties of the best selected compounds.

 Information published: 30.11.2018.

 

7 stage December to February (3 months)

During the seventh reporting period, after completing the screening of drug-like small compound library, a first group of 600 new compounds is selected with relative inhibitory activity (%I) 70% against at least one of the sortase A (SrtA) transpeptidases used in our studies. These selected compounds are actively researched in further studies to elucidate their molecular and cellular properties needed in the search for lead molecules. In this regard we have initiated studies of cell cytotoxicity, minimal inhibitory activity, cell culture infection and other approaches for evaluation of compound properties.

Another research direction is related to chemical modification of several most perspective SrtA inhibitors by using previously acquired knowledge about the enzyme-inhibitor structure and interaction. At present, using all available data of the eight best covalent inhibitors and seven non-covalent inhibitors, for further modifications have been selected four perspective compounds whose derivatives will be analyzed in various structural and biological assays.

Information published 28.02.2019.

 

8 stage March to May (3 months)

During the eighth reporting period, several most perspective sortase inhibitors were further chemically modified by substitution of active groups and intramolecular linkers, in order to synthesize analogs of these compounds for further biological and structural studies. In total, four innovative inhibitory compounds were selected for this optimization study where after chemical modifications up to ten new derivative molecules were synthesized to every of these inhibitors.

By further analysis of the large group of sortase inhibitors that exhibited the relative inhibitory activity (%I) of 70% where several SrtA enzymes from different Gram+ microorganisms and two FRET sortase substrates were used, a smaller group of 30 hit compounds were selected. This group of sortase inhibitors is characterized by very high relative inhibitory activity (%I) of 90%. Further, all these selected hit compounds and the modified derivatives were further studied to investigate their potential efficacy and safety as antivirulence agents. In this regard, various biological test systems, including antimicrobial and cytotoxic assays, pathogen adhesion, cell invasion and biofilm formation inhibition analysis are employed to evaluate the compound potency and specificity.

Information published 31.05.2019.

 

9 stage June to August (3 months)

During the ninth reporting period, several single compounds with additional modifications, as the substitution of active groups, were synthesized. The thus obtained derivatives were further compared to each other and used to study their molecular interaction properties with sortase a homologs from several microorganisms.

Another research direction is concerned with the study of the best selected inhibitors and their newly-synthesized analogous compounds biological testing in order to compare the pharmacokinetic properties and therapeutic potential. In this regard, various tests were performed to evaluate the compounds cytotoxicity, pathogen cell adhesion and antimicrobial infection-prevention abilities. In addition, three more SrtA enzymes from different Gram-positive bacteria are used to supplement the obtained structural data and optimize selection of drug candidates with the highest activity and specificity.

Information published 30.08.2019.

 

10 stage September to November (3 months)

During the tenth reporting period, more compounds with additional modifications and substitution in their active groups were synthesized. These modified compounds were further analyzed and characterized in order to compare biological and structural data with all sortase A transpeptidases used in this study.

The selected inhibitors and their analogs were further tested to identify their biological and pharmacokinetic properties necessary for drug candidates. These tests include S.aureus and S.epidermidis human adenocarcinoma cell infection assays. The other research direction involved the screening of S.aureus, L.monocytogenes and S.epidermidis Gram-positive strains, including antibiotic-resistant, for their ability to inhibit biofilm formation processes. In the frame of these biological tests, additional research was conducted where the inhibitory activity and specificity was compared in the processes of cell adhesion modulation, reduction of the cytotoxic effects and inhibition of microorganism viability.

Information published 29.11.2019. 




Mājas lapas izstrādi finansēja ERAF 2.1.1.2. aktivitātes projekts Nr. 2010/0196/2DP/2.1.1.2.0/10/APIA/VIAA/004 "Latvijas biomedicīnas pētījumu integrācija Eiropas zinātnes telpā".