Projects / Programmes
Exploitation of a virus-borne small protein to combat antibiotic resistance in Staphylococcus aureus
Code |
Science |
Field |
Subfield |
4.06.04 |
Biotechnical sciences |
Biotechnology |
Microbe biotechnology |
Code |
Science |
Field |
T490 |
Technological sciences |
Biotechnology |
Code |
Science |
Field |
3.04 |
Medical and Health Sciences |
Medical biotechnology |
antibiotics, antibiotic-resistance, virtual screening for inhibitors, antibiotic adjuvant, Staphyylococcus aureus, bacteriophage, SOS response
Researchers (22)
no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
1. |
24290 |
PhD Matej Butala |
Biochemistry and molecular biology |
Head |
2019 - 2022 |
235 |
2. |
53672 |
Sandra Cetin |
Pharmacy |
Researcher |
2021 |
2 |
3. |
23399 |
PhD Tomaž Curk |
Computer science and informatics |
Researcher |
2019 - 2022 |
251 |
4. |
15284 |
PhD Stanislav Gobec |
Pharmacy |
Researcher |
2019 - 2022 |
824 |
5. |
35424 |
PhD Tomaž Hočevar |
Computer science and informatics |
Researcher |
2019 - 2022 |
28 |
6. |
53283 |
Maja Hostnik |
Biochemistry and molecular biology |
Researcher |
2020 - 2022 |
17 |
7. |
52386 |
Eva Kočar |
Biochemistry and molecular biology |
Technical associate |
2019 - 2020 |
28 |
8. |
25435 |
PhD Janez Konc |
Computer intensive methods and applications |
Researcher |
2019 |
230 |
9. |
55572 |
PhD Alen Krajnc |
Pharmacy |
Researcher |
2022 |
31 |
10. |
00412 |
PhD Igor Križaj |
Biochemistry and molecular biology |
Researcher |
2019 - 2022 |
712 |
11. |
53699 |
Amela Kujović |
Biochemistry and molecular biology |
Technical associate |
2019 - 2020 |
22 |
12. |
18802 |
PhD Adrijana Leonardi |
Biochemistry and molecular biology |
Researcher |
2019 - 2022 |
151 |
13. |
39099 |
PhD Katja Molan |
Biochemistry and molecular biology |
Junior researcher |
2019 |
37 |
14. |
55449 |
Martina Mravinec |
Biochemistry and molecular biology |
Researcher |
2021 |
11 |
15. |
39090 |
PhD Anastasija Panevska |
Biochemistry and molecular biology |
Researcher |
2019 - 2021 |
53 |
16. |
51231 |
PhD Anja Pavlin |
Biochemistry and molecular biology |
Junior researcher |
2019 - 2022 |
26 |
17. |
06902 |
PhD Zdravko Podlesek |
Biochemistry and molecular biology |
Researcher |
2020 - 2022 |
137 |
18. |
52376 |
PhD Matic Proj |
Pharmacy |
Junior researcher |
2019 - 2022 |
63 |
19. |
04570 |
PhD Jože Pungerčar |
Biochemistry and molecular biology |
Researcher |
2019 - 2022 |
319 |
20. |
15328 |
PhD Kristina Sepčić |
Biochemistry and molecular biology |
Researcher |
2019 - 2021 |
719 |
21. |
52378 |
Nika Strašek Benedik |
Pharmacy |
Junior researcher |
2020 - 2022 |
0 |
22. |
21553 |
PhD Jernej Šribar |
Biochemistry and molecular biology |
Researcher |
2019 - 2022 |
108 |
Organisations (4)
Abstract
The development of drug‑resistant bacteria is an unavoidable outcome of widespread antibacterial chemotherapy of infectious diseases. Drug discovery programs directed against proteins considered essential for in vivo bacterial viability have yielded few new therapeutic classes of antibiotics, therefore treatment options for combating bacteria resistant to multiple drugs are narrowing. Hence, novel strategies to fight bacterial pathogens are urgently needed. Adaptation of bacteria to antibiotic therapy requires specific biochemical processes that may be subject to intervention. Preventing bacteria to acquire resistance to antibiotics will significantly prolong the lifetime of current‑day antibiotics. We will achieve this by an innovative strategy, with novel drugs that block antibiotic stress and resistance development in bacteria, prolonging the efficacy of licensed antibiotics. Clinically significant antibiotics induce mechanisms in pathogenic bacteria that activate the DNA damage response, designated the SOS response. Antibiotic‑induced SOS responses can (i) modulate the evolution and spread of drug resistance as well as virulence factors in pathogens, (ii) induce mutations and generate antibiotic resistance during therapy, (iii) induce persistence and multidrug tolerance in a subpopulation of bacterial cells that are not heritably resistant, (iv) induce biofilm formation and (v) toxin synthesis. The importance of regulating responses to stress suggests that modulation of SOS induction has the potential to address the problem of evolution of antibiotic resistance at its roots. Experimentally inactivating transcription factor LexA, the key regulator of the response, renders the bacteria unable to initiate the SOS response, these strains are sensitized to genotoxic antimicrobials and exhibit decreased mutation rates.
The natural products synthesized by microorganisms are privileged compounds for the discovery of antibiotics as they result from natural selection and are the source of highly effective antibiotics. Our recent results reported the discovery of a small, 50-residue bacteriophage protein, gp7, able to interact with and modulate functions of the global transcription factor LexA, a key SOS regulator involved in the development of antibiotic resistance. This is the first report of a natural molecule that interacts directly with LexA to inhibit its self-cleavage activity and enhance its DNA binding, thus inhibit the host SOS response. We obtained the crystal structure of the gp7 protein and according to SAXS data we generated a structural model of gp7 in complex with LexA. Based on the unpublished structures we aim to obtain few high-affinity LexA-inhibitory lead compounds or gp7 derivatives, that will after optimisation give rise to a safe “anti-evolutionary” therapeutics, to be used in combination with licensed antibiotics to fight Bacillus sp. and Staphlococcus aureus infections. First we will enhance our knowledge on biology of gp7 protein in vitro and on the genome-wide scale in Bacillus and Staphylococcus sp. This will enable us to better understand gp7-driven processes in the cell which is essential to be able to generate a safe anti-evolutionary therapeutics. Next, we will use computer-assisted drug design based on the gp7 protein characteristics to select for the chemical libraries which will be assayed for the Bacillus sp. and the S. aureus LexA self-cleavage inhibitors. This will provide us with lead SOS inhibitor structures to block mechanisms enabling resistance. We will determine the equilibrium dissociation constants for the leads-LexA interaction and evaluate the antibiotic-potentiated activity of molecules targeting LexA by assaying the inhibition of the antibiotic resistance accumulation in Bacillus sp. and in S. aureus. We believe that optimised leads/gp7-derivatives in combination with current‑day, licensed antibiotics will assist in the battle against the development of antibiotic resistance by ba
Significance for science
World Health Organisation called for an urgent and concerted action: »Combat drug resistance: no action today means no cure tomorrow.« We need to bring new classes of antibiotics to market to prevent people from dying from infections that have been treatable for decades, until resistance rendered our drugs ineffective. An academic-industry parthership has just few months ago reported the first LexA-targeting small molecules. Given the fact that GlaxoSmithKline entered such collaborative effort that lead to the identification of first-in-class inhibitors of LexA self-cleavage confirms the importance of recognising LexA as a novel, potent antibacterial drug target to address the rising tide of antibiotic resistance bacteria. However, they used an approach in which they targeted the LexA self-cleavage reaction. They observed that by hitting directly the catalytic domain of LexA makes it a difficult target for competitive inhibiton, given the high local concentration of the cleavage loop »substrate« around the LexA active site. Thus, only after refeiment of the certain chemotype targeting LexA the authors (their article has been just accepted) could reach the IC50 value down to 9 µM for the E. coli LexA. This indicates, that although great effort was allready invested, even further optimisation is needed which might result in an efficient inhibitor.
Here we propose a different and unique approach based on the natural gp7 protein characteristics and on our unpublished structural LexA-gp7 insight. We will design an effective allosteric inhibitors of LexA that we predict will target at least LexA of Bacillus sp. and S. aureus. Our approach is novel in that gp7 will be used as a scafold to design gp7 derivatives or small lead molecules that stabilize LexA in a DNA bound conformation and additionaly orient LexA cleavage site loop in the non-clavable state, thus firmly inhibiting repressors`s self-cleavage by precluding LexA to interact with RecA. It is of note that we have previously shown that gp7 inhibits LexA to interact with RecA and that DNA bound LexA cannot be inactivated. Thus, here we offer a promissing novel approach to design unique drugs that block antibiotic stress and resistance development in bacteria, prolonging the efficiency of licensed antibiotics.
In addition, we want to elucidate gp7 effect on processes in B. thuringiensis and S. aureus on the genome wide-scale. This is important to understand the gp7-effects in vivo and the results will surely be of great interest to the broad scientific commnity. In E. coli, ChIP-chip approach revealed that an additional factor is required for LexA binding to noncanonical sites in vivo (Wade et al., Genes Dev. 2005). Thus, we strongly believe that gp7-like LexA effectors exist also in other bacteria which could provide scafolds for potent drugs against antibiotic-resitance development in selected pathogens. Thus, our project proposal may open up a novel class of tomorrow`s antibacterials.
Significance for the country
World Health Organisation called for an urgent and concerted action: »Combat drug resistance: no action today means no cure tomorrow.« We need to bring new classes of antibiotics to market to prevent people from dying from infections that have been treatable for decades, until resistance rendered our drugs ineffective. An academic-industry parthership has just few months ago reported the first LexA-targeting small molecules. Given the fact that GlaxoSmithKline entered such collaborative effort that lead to the identification of first-in-class inhibitors of LexA self-cleavage confirms the importance of recognising LexA as a novel, potent antibacterial drug target to address the rising tide of antibiotic resistance bacteria. However, they used an approach in which they targeted the LexA self-cleavage reaction. They observed that by hitting directly the catalytic domain of LexA makes it a difficult target for competitive inhibiton, given the high local concentration of the cleavage loop »substrate« around the LexA active site. Thus, only after refeiment of the certain chemotype targeting LexA the authors (their article has been just accepted) could reach the IC50 value down to 9 µM for the E. coli LexA. This indicates, that although great effort was allready invested, even further optimisation is needed which might result in an efficient inhibitor.
Here we propose a different and unique approach based on the natural gp7 protein characteristics and on our unpublished structural LexA-gp7 insight. We will design an effective allosteric inhibitors of LexA that we predict will target at least LexA of Bacillus sp. and S. aureus. Our approach is novel in that gp7 will be used as a scafold to design gp7 derivatives or small lead molecules that stabilize LexA in a DNA bound conformation and additionaly orient LexA cleavage site loop in the non-clavable state, thus firmly inhibiting repressors`s self-cleavage by precluding LexA to interact with RecA. It is of note that we have previously shown that gp7 inhibits LexA to interact with RecA and that DNA bound LexA cannot be inactivated. Thus, here we offer a promissing novel approach to design unique drugs that block antibiotic stress and resistance development in bacteria, prolonging the efficiency of licensed antibiotics.
In addition, we want to elucidate gp7 effect on processes in B. thuringiensis and S. aureus on the genome wide-scale. This is important to understand the gp7-effects in vivo and the results will surely be of great interest to the broad scientific commnity. In E. coli, ChIP-chip approach revealed that an additional factor is required for LexA binding to noncanonical sites in vivo (Wade et al., Genes Dev. 2005). Thus, we strongly believe that gp7-like LexA effectors exist also in other bacteria which could provide scafolds for potent drugs against antibiotic-resitance development in selected pathogens. Thus, our project proposal may open up a novel class of tomorrow`s antibacterials.
Most important scientific results
Most important socioeconomically and culturally relevant results
Interim report