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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 “Industry-Driven 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.



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".