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: 1 June 2017 – 31 May 2020
Project costs: 583 689.55 EUR
Principle Investigator: Dr. biol. A. Leončiks
Project summary:
The overall objective of the Project is discovery and selection of new antibacterial compounds that target Gram-positive bacteria transpeptidase enzyme sortase A for treating multi-resistant infections. This strategy of discovery and characterization of new antibacterial drugs corresponds to the aim of Latvian Smart Specialization Strategy (RIS3) to support development of science and technology, creation of new knowledge, and allow stimulating the following perspective sector of the national economy as the pharmaceutical industry. As the result new products as drugs and antibiotics can emerge with high added value.
In frame of the project the following activities are planned: screening of the inhibitory compounds, their structural and biophysical characterization, a following synthesis of the optimized and modified inhibitors, end-tests in eukaryotic cell lines and animal model to elucidate the specific inhibitory activity and toxicity.
The project is not associated with commercial activity and the intended type of the study is fundamental research. The project corresponds to the following fields of science: 1.6 Biological sciences, 3.4 Medicinal biotechnologies.
A first target group is the Latvian Biomedical Research and Study Centre, a scientific institution, where project will be implemented. The main fields of activity of the institute are scientific research and dissemination of the obtained results in form of knowledge and technology transfer. Another target group that will be supported in frame of the project is the institute’s scientific personnel that will enhance their scientific qualification. A public access to results will be provided during the project. It is planned to prepare and submit three scientific articles in recognized international peer-reviewed journals.
Information published 01.06.2017.
Progress of the project
1 June 2017 – 31 August 2017
During the initial phase of the project, all three Gram-positive micro-organisms Staphylococcus aureus, Listeria monocytogenes and Bacillus anthracis sortase A (SrtA) encoding genetic structures were used for gene re-cloning works to obtain enzymatic active proteins that are intended to be used for structural studies, selection and characterisation of inhibitoro substances. Initially, all recombinant enzymes are obtained in analytical quantities with a view to optimising their synthesis in a bacterial express system in order to further increase their extraction to prefabricated quantities. In parallel, work has been carried out on chromatographic purification of the proteins obtained and optimization of all related procedures.
The first purified SrtA proteins have been subjected to initial testing to optimise the methods by which the enzymatic activity of sorthase in fluorimetric selection systems with a highlighted FRET peptide substrate and potential inhibitory compounds will be analysed, as well as the development of a single standardised standardised fluorimetric data acquisition and processing protocol. As a last step, preparations have been launched to identify the crystallisation conditions of the SrtA enzymes, which will be further used for the procurement of sortase-inhibitor complexes and for the modelling of these structures.
Information published 31.08.2017.
Progress of the project
1 September 2017 – 30 November 2017
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.
Progress of the project
1 December 2017 – 28 February 2018
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.
Progress of the project
1 March 2018 – 31 May 2018
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.
Progress of the project
1 June 2018 – 31 August 2018
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.
Progress of the project
1 September 2018 – 30 November 2018
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.
Progress of the project
1 December 2018 – 28 February 2019
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.
Progress of the project
1 March 2019 – 31 May 2019
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.
Progress of the project
1 June 2019 – 31 August 2019
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.
Progress of the project
1 September 2019 – 30 November 2019
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.
Progress of the project
1 December 2019 – 29 February 2020
During the eleventh reporting period, additional modified sortase inhibitors with further chemical substitutions were synthesized in order to characterize the role of their active groups in the enzymatic inhibition capacity. These modified compounds were analyzed and characterized in various biological and structural tests with all SrtA transpeptidases used in this study.
The most perspective inhibitory compounds 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 several 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 28.02.2020.
Progress of the project
1 March 2020- 31 May 2020
During the twelfth reporting period, additionally synthesized modified sortase inhibitors were analyzed in order to characterize the role of their active groups in interaction with the enzyme active site. These tests allow assessing the inhibitory potential of selected modified compounds and characterize their structural and biological properties for different SrtA transpeptidases.
Similarly, the most perspective compounds were further tested to identify their biological and pharmacokinetic properties necessary for drug candidates. Several bacterial strains and human adenocarcinoma cell line were used in these infection assays. The other research direction involved screening of particular Gram-positive strains, including antibiotic-resistant, for their ability to inhibit biofilm formation. In frame of all these biological tests the substantial information was continuously collected and summarized in order to compare the antivirulence potential and specificity of the selected compounds in modulation of cell adhesion processes, reduction of cell cytotoxic effects and inhibition of microorganism viability.
Information published 29.05.2020.