Final Report Summary - NOVRIB (Novel Insights into Multi-drug Resistance to Antibiotics and the Primordial Ribosome)
NOVRIB aimed at revealing the scope and mechanisms of pathogen’s resistance to antibiotics by structural studies (part 1) and the origin of life (part 2).
Part 1: We attempted at providing structural information as well as molecular tools for dealing with a substantial challenge in contemporary medicine, namely the rapid spread of resistance to antibiotics, which is creating a global clinical threat. We focused on bacterial ribosome since they are targeted by over 40% of the antibiotics that are currently in medical use. In order to shed light on antibiotics resistance, we progressed along the following lines:
(a) We determined high resolution structures of ribosome and their large subunits from the multi-drug resistant genuine pathogen Staphylococcus aureus (SA) in its apo form and in complex with several antibiotics, among them several new potential drugs. These include the SA large ribosomal subunit (SA50S) and the full SA ribosome complex (SA70S) with very prominent ribosomal antibiotics, such as: oxazolidinones, lincosamides , pleuromutilins, and ketolides. Consequently we revealed the binding modes, selectivity towards bacteria and the inhibition modes of these drugs. Among those antibiotics, the pleuromutilins consist of a tricyclic mutilin core, and a carboxyl group, which is essential for its antimicrobial activity. They bind to the large ribosomal subunit at the PTC with their cores at the A-site and their extensions pointing towards the P-site, thus inhibiting the formation of the peptide bonds. We found the specific interactions that were formed by new semi synthetic potent drugs developed by Nabriva Therapeutics, Vienna, Austria, with whom we are collaborating, in the binding of BC-3205 and lefamulin (formerly called BC-3781) are the key for their prominent inhibition properties.
(b) To illuminate parameters that lead to species-specific response to antibiotics, we determined the high resolution structure the ribosome of the multi drug resistant bacterium Staphylococcus aureus, in its apo form and in complex with various antibiotics. Careful analyses of these crystal structures shed light on several specific internal as well as peripheral unique structural motifs that may be potential candidates for improving known antibiotics and for the design of pathogen selective novel antibiotics, thus preserving the microbiome during treatment against infectious diseases.
(c) As resistance to erythromycin, a clinically useful antibiotic that binds to the wall of the ribosomal exit tunnel through which nascent proteins migrate from the peptidyl transferase center to the exterior of the ribosome, are associated with ribosomal proteins uL4 and uL22 that reach the walls of the exit tunnel, we determined the structures of the large ribosomal subunit from an erythromycin resistant mutant that harbors a deletion in ribosomal protein uL22 in its apo form and in complex with erythromycin. We found that resistance to erythromycin is acquired by alterations of rRNA nucleotides that directly interact with the drug or by mutations in the uL22 β-hairpin loop, which may be correlated also with the regulatory properties of the exit tunnel.
Part 2: We continued our attempts at defining the internal ribosome region suggested by us to be a vestige of a pre-biotic apparatus, which we call the proto-ribosome, by designing autonomous molecular entities with catalytic capabilities. Hence, constructs that bind RNA molecules that could serve as substrates of the proto-ribosome have been designed and an apparent correlation between their chemical properties and their expected contribution to the enhancement of the pace of peptide bond formation has been identified.
Part 1: We attempted at providing structural information as well as molecular tools for dealing with a substantial challenge in contemporary medicine, namely the rapid spread of resistance to antibiotics, which is creating a global clinical threat. We focused on bacterial ribosome since they are targeted by over 40% of the antibiotics that are currently in medical use. In order to shed light on antibiotics resistance, we progressed along the following lines:
(a) We determined high resolution structures of ribosome and their large subunits from the multi-drug resistant genuine pathogen Staphylococcus aureus (SA) in its apo form and in complex with several antibiotics, among them several new potential drugs. These include the SA large ribosomal subunit (SA50S) and the full SA ribosome complex (SA70S) with very prominent ribosomal antibiotics, such as: oxazolidinones, lincosamides , pleuromutilins, and ketolides. Consequently we revealed the binding modes, selectivity towards bacteria and the inhibition modes of these drugs. Among those antibiotics, the pleuromutilins consist of a tricyclic mutilin core, and a carboxyl group, which is essential for its antimicrobial activity. They bind to the large ribosomal subunit at the PTC with their cores at the A-site and their extensions pointing towards the P-site, thus inhibiting the formation of the peptide bonds. We found the specific interactions that were formed by new semi synthetic potent drugs developed by Nabriva Therapeutics, Vienna, Austria, with whom we are collaborating, in the binding of BC-3205 and lefamulin (formerly called BC-3781) are the key for their prominent inhibition properties.
(b) To illuminate parameters that lead to species-specific response to antibiotics, we determined the high resolution structure the ribosome of the multi drug resistant bacterium Staphylococcus aureus, in its apo form and in complex with various antibiotics. Careful analyses of these crystal structures shed light on several specific internal as well as peripheral unique structural motifs that may be potential candidates for improving known antibiotics and for the design of pathogen selective novel antibiotics, thus preserving the microbiome during treatment against infectious diseases.
(c) As resistance to erythromycin, a clinically useful antibiotic that binds to the wall of the ribosomal exit tunnel through which nascent proteins migrate from the peptidyl transferase center to the exterior of the ribosome, are associated with ribosomal proteins uL4 and uL22 that reach the walls of the exit tunnel, we determined the structures of the large ribosomal subunit from an erythromycin resistant mutant that harbors a deletion in ribosomal protein uL22 in its apo form and in complex with erythromycin. We found that resistance to erythromycin is acquired by alterations of rRNA nucleotides that directly interact with the drug or by mutations in the uL22 β-hairpin loop, which may be correlated also with the regulatory properties of the exit tunnel.
Part 2: We continued our attempts at defining the internal ribosome region suggested by us to be a vestige of a pre-biotic apparatus, which we call the proto-ribosome, by designing autonomous molecular entities with catalytic capabilities. Hence, constructs that bind RNA molecules that could serve as substrates of the proto-ribosome have been designed and an apparent correlation between their chemical properties and their expected contribution to the enhancement of the pace of peptide bond formation has been identified.