grant:KP-06-H63/1/13.12.2022, Comparative analysis of the effectiveness of new antibacterial agents based on different types of antisense oligonucleotides using different molecular mechanisms of RNA inhibition awarded to Prof. Robert Penchovsky by the Bulgarian National Science Fund (BNSF)

The project "Comparative analysis of the effectiveness of new antibacterial agents based on different types of antisense oligonucleotides using different molecular mechanisms of RNA inhibition" is a continuation of long-term research in the field of synthetic biology and the development of new antibacterial agents to be tested and optimized, through appropriate modifications or different molecular mechanisms. The planned fundamental experiments include applying the theoretically possible methods for optimizing the effectiveness of antibacterial agents through modifications in their structure and the application of various molecular mechanisms of RNA inhibition. The main focus of our project is the well-known antisense oligonucleotides to our team, with which we have been actively working for the last decade in the laboratory of Prof. Penchovski at the Faculty of Biology of Sofia University "St. Kliment Ohridski” as a continuation of his experience from his work with bacterial riboswitches at Yale University, USA, together with Ronald Breaker (discoverer of riboswitches). Antisense oligonucleotides are agents that bind specifically to the molecule they target. There are currently three generations of antisense oligonucleotides, each with its characteristics, advantages, and limitations. For our project, we will design different antisense oligonucleotides that will be representatives of different generations, as well as those that will be chimeric. We will apply them in bacterial cells to perform genetic control through their specific binding to bacterial riboswitches. Bacterial riboswitches are structural RNA domains, most commonly located in the 5'-untranslated region of messenger RNAs that specifically recognize and bind metabolites. To date, no evidence of riboswitches has been found in the human genome, making bacterial riboswitches potentially suitable targets for developing antibacterial agents. The structure of most riboswitches is known, and in international bioinformatics databases: GenBank, Rfam, including in the RSwitch database created by us the sequences of individual species and their representatives are also available. As a result, specific binding of antisense oligonucleotides and riboswitches results in conformational changes that enable messenger RNAs to regulate their expression without needing regulatory proteins. This leads to the impossibility of vital metabolites for the bacteria to be synthesized by the cells and/or transported inside it from the external environment, leading to a bacteriostatic or bactericidal effect.

The comparison of individual variants of antisense oligonucleotides will allow us to analyze them and, as a result, answer several open questions for the scientific community regarding the design of antisense oligonucleotides - their basic nucleotide skeleton, the generation to which they belong, and the possible modifications of the structure them As a separate task within the project, we aim to expand our knowledge of the types of cell-penetrating peptides and their behavior when loaded with antisense oligonucleotides by conducting experiments with selected antisense oligonucleotides and different cell-penetrating peptides. As a result of the experiments, we will observe the influence on the effectiveness of individual complexes of cell-penetrating peptides and antisense oligonucleotides. We will compare the data obtained and give a conclusion about the effectiveness and applicability of each of them. This newly obtained information will be helpful in the further studies of colleagues around the world. Through these data, we will propose an efficiency ranking and reduce inefficient attempts to import an antisense oligonucleotide into a bacterium, the time to create new therapeutic agents, and the cost of experiments and personnel. After completing our highly successful project "Design and experimental validation of chimeric antisense oligonucleotides as antibacterial agents", the results of which were published in 20 scientific publications with a total impact factor of 84 points (Q1-4 = 274 and 86 citations) and we created 16 software products available on the official website of Prof. Robert Penchovsky, we are confident that with the creation of new antisense oligonucleotides linked to different cel-penetrating peptides, based on different molecular mechanisms of RNA inhibition, to compare their effectiveness, we will obtain fundamental data through which we will give new directions and draw new horizons, accelerating the process of creating antibacterial agents against serious, life-threatening pathogenic bacteria. The research of the project funded with 102 300 EUR.

Within the framework of the project, we carried out all the planned activities and achieved the results we expected. In summary, we have 4 published scientific articles with a total impact factor of 12.9 points, 1 doctoral student finshied her 3 year-long study with the right of Ph.D. thesis defense and 2 updated software products created. All publications have been indexed in Scopus, making a total of 85 points. We participated with presentations at three scientific forums. In addition, 3 members of the project team received a total of 3 scientific awards for the period working on the project, including the project leader prof. Penchovsky.

Dr. Martina Traykovska won an award for first place for the work of a young microbiologist in a national contest by the Foundation in the name of Acad. Prof. Stephan Angeloff on March 7, 2023.

Dr. Nikolet Pavlova won an award for second place for the work of a young microbiologist in a national contest by the Foundation in the name of Acad. Prof. Stephan Angeloff on March 7, 2023.

Publications for the first-half of the project:

1. Nikolet Pavlova, Martina Traykovska, and Robert Penchovsky*, Targeting FMN, TPP, SAM-I, and glmS Riboswitches with Chimeric Antisense Oligonucleotides for Completely Rational Antibacterial Drug Development, Antibiotics, Volume 12, Issue 11, November 2023 Article number 1607, IF:4.3, (Q1=25p, Scopus).

2. Katya Popova and Robert Penchovsky*, General and Specific Cytotoxicity of Chimeric Antisense Oligonucleotides in Bacterial Cells and Human Cell Lines, Antibiotics, Antibiotics, Volume 13, Issue 2, February 2024 Article number 122, IF: 4.3, (Q1=25p, Scopus).

3. Robert Penchovsky*, Antoniya V. Georgieva, Vanya Dyakova, Martina Traykovska and Nikolet Pavlova, Antisense and Functional Nucleic Acids in Rational Drug Development, Antibiotics, Volume 13, Issue 3, March 2024, Article number 221, IF: 4.3, (Q1=25p, Scopus).

4. Nikolet Pavlova, Dimitrios Kaloudas & Robert Penchovsky*, Computational Design of Allosteric Ribozymes via Genetic Algorithms, Methods in molecular biology (Clifton, N.J.), Volume 2822, Pages 443 – 469, 2024, IF: 2.4, (Q4=10p, Scopus).

Conference participation for the first-half of the project:

1. Robert Penchovsky has been invited to give a talk on "How did we make ribozymes to say "YES", "NO", "OR", "AND" and perform complex logical operations?" at the 14^th Sofia Science Festival, May 9^th-12^th, 2024 in Sofia, Bulgaria.

2. Nikolet Pavlova gave an invited presentation on "Bioinformatics and genomic analyses of eight riboswitches and their suitability for antibacterial drug targets" at the 7^th International Conference on Pharmaceutics & Advanced Drug Delivery SystemsMarch 27-28, 2023 in London, UK.

3. Vanya Dyakova gave a talk on "Antisense oligonucleotides as therapeutics" at the Bulgarian National Conference of Pathology, May 17-18, 2024 in Borovets, Bulgaria.

Updated software:

1. Virtual PCR

2. RSwitch database

News on our research:

BGlobal, in Bulgarian, July, 2023, A microbiologist replaces antibiotics when they do not work.