Periodic Reporting for period 2 - ProMeTeus (Membrane protein integrated technologies development for drug design)
Berichtszeitraum: 2022-01-01 bis 2023-12-31
Prometeus addressed the need to provide academic groups in Europe that target membrane proteins with relevance in health and technology global issues (antibiotic design, prostatic cancer, cancer theranostics, nanotechnology) with training in key advanced technologies provided by SME or TC large facilities.
The scientific aim of the Prometeus RISE-H2020-MSCA project was to study membrane proteins to solve health and technology global issues and the scientific activity was carried out in the context of the MSCA-RISE Actions, mainly by seconding personnel in a trans-national and trans-sectiorial fashion.
The consortium consists of 9 beneficiaries and 2 partners of which 7 are academic and 4 are SME. The results were achieved by harnessing complementary skills from Academic and SME participants. We had the contribution of Partner Organizations from the USA, committed to training personnel from the Prometeus Partners: the New York Structural Biology Center and the Advanced Scientific Research Center of City University of New York.
Our scientific goal was the structural and functional analysis of membrane protein. Based on this information we carried out an in silico search of ligands to be tested experimentally using activity assays devised in the course of the project. Finally, we the achieved the sythesis of new compounds that could be the substrates or inhibitors and potentially, leads for new drugs.
The targets enzymes have been i) Arabinofuranosyltransferases (AraTs), membrane enzymes involved in the biosynthesis of arabinan, an important component of the unique cell-envelope of Mtb, and drug targets for tuberculosis therapy; ii) Steroid alpha-dihydrogenases (SRD5A), membrane enzymes involved in steroid enzymes activation and modification, actively investigated as targets for prostatic diseases iii) odorant receptor proteins involved in perception of volatile and non-volatile odorants (chemicals and molecules) in vertebrates.
In response to the Covid-19 pandemic challenge some of the Partners have worked in collaboration to contribute to the body of knowledge on SARS-CoV-2, utilizing the synergy of different know-how present in the Consortium.
The scientific aim of the Prometeus RISE-H2020-MSCA project was to study membrane proteins to solve health and technology global issues and the scientific activity was carried out in the context of the MSCA-RISE Actions, mainly by seconding personnel in a trans-national and trans-sectiorial fashion.
The consortium consists of 9 beneficiaries and 2 partners of which 7 are academic and 4 are SME. The results were achieved by harnessing complementary skills from Academic and SME participants. We had the contribution of Partner Organizations from the USA, committed to training personnel from the Prometeus Partners: the New York Structural Biology Center and the Advanced Scientific Research Center of City University of New York.
Our scientific goal was the structural and functional analysis of membrane protein. Based on this information we carried out an in silico search of ligands to be tested experimentally using activity assays devised in the course of the project. Finally, we the achieved the sythesis of new compounds that could be the substrates or inhibitors and potentially, leads for new drugs.
The targets enzymes have been i) Arabinofuranosyltransferases (AraTs), membrane enzymes involved in the biosynthesis of arabinan, an important component of the unique cell-envelope of Mtb, and drug targets for tuberculosis therapy; ii) Steroid alpha-dihydrogenases (SRD5A), membrane enzymes involved in steroid enzymes activation and modification, actively investigated as targets for prostatic diseases iii) odorant receptor proteins involved in perception of volatile and non-volatile odorants (chemicals and molecules) in vertebrates.
In response to the Covid-19 pandemic challenge some of the Partners have worked in collaboration to contribute to the body of knowledge on SARS-CoV-2, utilizing the synergy of different know-how present in the Consortium.
Overview
The first overall scientific results of the Project have been the production and the determination of the structures of eight protein structures, mainly membrane enzymes and complexes of receptors with ligands.
We have established novel protocols for activity assays of Arabinofuranosyltransferases from mycobacteria and for human steroid alpha-dihydrogenases. The information on protein structures and the possibility of assaying their activity led to the identification of inhibitors and model substrates. Finally, possible leads for the design of tuberculosis infection have been identified.
Three European institution have gained training in cryo electron microscopy, implementing this advanced methodology in-house. High-throughput approaches in membrane protein expression and methods for their stabilization have been developed and became a part of the technology portfolio of European Partners.
List of scientific results
- The expression ofmembers of the SRD5A family has been achieved in HEK293 mammalian cells in amounts compatible with structural studies.
- The expression and the purification protocolof the human transferrin receptor 1 was established. This sample was utilized for structural studies by single particle cryo-electron microscopy (CryoEM).
- To unravel the mechanism of drug resistance in tuberculosis, that is mainly due to a highly impermeable hydrophobic mesh in its cell wall, two families of membrane enzymes were selected (Arabinosyltransferase EmbA-C, and Arabinofuranosyltransferase AftA-D), genes were cloned, proteins were expressed and purified.
-Given the threat of SARS-Cov-2 we carried out work by SAXS on proteins from the virus, we designed protein nanoparticles for vaccine development and studied the transfer on antibodies from mother to children in breast milk upon vaccination. We have also explored by protein engineering the potential of ferritin as a nanoparticle for vaccine development purposes.
- We determined, mainly by CryoEM, the structures of 8 proteins, including three AraTs, a complex betewwen ferritin and its receptor, a domain of an SRD5A enzyme and a complex of the ErbB3 receptor with lead therapeutic antibodies.
-We have developed new protocols and compounds to achieve proteins solubilization and stabilization, which are especially critical form membrane proteins.
- Assays have been established for SRD5A activity on testosterone and for bacterial cell-wall synthesizing enzymes.
- Virtual screening based on AftA, EmbB and humanSRD5A2 yielded a set of compounds that could be tested as inhibitors using the developed assays.
- Synthesis of model substrates and lead inhibitors for Arabinofuranosyltransferases.
Training and knowledge transfer results
Equally relevant in the MSCA-RISE program is training of staff and acquisition of advanced methodologies by Partners. Under this respect the project has been extremely successful, COMPPA/NYSBC has provided training to Sapienza, UnivPM and UNL/ITQB with the transfer of skills on membrane production and handling. Both COMPPA/NSBC and CUNY allowed the acquisition of expertise in CryoEM. This is of strategic relevance for the Partners Sapienza, UnivPM ITQB/UNL since now this new methodology is fully established for the benefit of other projects of both Institutes
26 scientists have carried out secondments contributing to the achievement of scientific results and gained training in methods and approaches not present in the departing Institute. Twelve of them were young scientists.
Prometeus carried out dissemination activities in the scientific community, we organized an international Symposium on CryoEM, an international workshop on memebrane protein technology, final workshop for knowledge dissemination and a final international conference where the Project results have been communicated. Other dissemination and communication activities include the hosting of students from high schools, press releases on scientific results, and information on the Prometeus Project in the context of the MSCA-H2020 at institutional open days and several other events of the different Partners.
SME partner Extremochem was acquired by a larger company and is exploiting results on synthesis of new compound for protein chemistry, partner UNL/ITQB is pursuing the pharmaceutical exploitation for new leads against mycobacterial infections.
The first overall scientific results of the Project have been the production and the determination of the structures of eight protein structures, mainly membrane enzymes and complexes of receptors with ligands.
We have established novel protocols for activity assays of Arabinofuranosyltransferases from mycobacteria and for human steroid alpha-dihydrogenases. The information on protein structures and the possibility of assaying their activity led to the identification of inhibitors and model substrates. Finally, possible leads for the design of tuberculosis infection have been identified.
Three European institution have gained training in cryo electron microscopy, implementing this advanced methodology in-house. High-throughput approaches in membrane protein expression and methods for their stabilization have been developed and became a part of the technology portfolio of European Partners.
List of scientific results
- The expression ofmembers of the SRD5A family has been achieved in HEK293 mammalian cells in amounts compatible with structural studies.
- The expression and the purification protocolof the human transferrin receptor 1 was established. This sample was utilized for structural studies by single particle cryo-electron microscopy (CryoEM).
- To unravel the mechanism of drug resistance in tuberculosis, that is mainly due to a highly impermeable hydrophobic mesh in its cell wall, two families of membrane enzymes were selected (Arabinosyltransferase EmbA-C, and Arabinofuranosyltransferase AftA-D), genes were cloned, proteins were expressed and purified.
-Given the threat of SARS-Cov-2 we carried out work by SAXS on proteins from the virus, we designed protein nanoparticles for vaccine development and studied the transfer on antibodies from mother to children in breast milk upon vaccination. We have also explored by protein engineering the potential of ferritin as a nanoparticle for vaccine development purposes.
- We determined, mainly by CryoEM, the structures of 8 proteins, including three AraTs, a complex betewwen ferritin and its receptor, a domain of an SRD5A enzyme and a complex of the ErbB3 receptor with lead therapeutic antibodies.
-We have developed new protocols and compounds to achieve proteins solubilization and stabilization, which are especially critical form membrane proteins.
- Assays have been established for SRD5A activity on testosterone and for bacterial cell-wall synthesizing enzymes.
- Virtual screening based on AftA, EmbB and humanSRD5A2 yielded a set of compounds that could be tested as inhibitors using the developed assays.
- Synthesis of model substrates and lead inhibitors for Arabinofuranosyltransferases.
Training and knowledge transfer results
Equally relevant in the MSCA-RISE program is training of staff and acquisition of advanced methodologies by Partners. Under this respect the project has been extremely successful, COMPPA/NYSBC has provided training to Sapienza, UnivPM and UNL/ITQB with the transfer of skills on membrane production and handling. Both COMPPA/NSBC and CUNY allowed the acquisition of expertise in CryoEM. This is of strategic relevance for the Partners Sapienza, UnivPM ITQB/UNL since now this new methodology is fully established for the benefit of other projects of both Institutes
26 scientists have carried out secondments contributing to the achievement of scientific results and gained training in methods and approaches not present in the departing Institute. Twelve of them were young scientists.
Prometeus carried out dissemination activities in the scientific community, we organized an international Symposium on CryoEM, an international workshop on memebrane protein technology, final workshop for knowledge dissemination and a final international conference where the Project results have been communicated. Other dissemination and communication activities include the hosting of students from high schools, press releases on scientific results, and information on the Prometeus Project in the context of the MSCA-H2020 at institutional open days and several other events of the different Partners.
SME partner Extremochem was acquired by a larger company and is exploiting results on synthesis of new compound for protein chemistry, partner UNL/ITQB is pursuing the pharmaceutical exploitation for new leads against mycobacterial infections.
We delivered a platform for developing new leads for key health issues (design of new antibiotics or new strategies for theranostics) and fostered a synergistic approach tbetween academic and non-academic institutions and SMEs.
We achieved an impact at different levels:
i) scientific knowledge on membrane proteins, with the identification of new targets and leads for prostatic diseases, oncologic pathologies and microbial infections;
ii) acquiring of the CryoEM methodology by three European Partners
iii) disseminating the Prometeus model of integrated collaboration between academia and SME;
iv) young and experienced scientists are increasing their skills, that will make them able to pursue their scientific and career objectives;
i) we are informing the general public on the rational design approach to drugs and of the synergistic approach between academia and SME, with the support of the MSCA
We achieved an impact at different levels:
i) scientific knowledge on membrane proteins, with the identification of new targets and leads for prostatic diseases, oncologic pathologies and microbial infections;
ii) acquiring of the CryoEM methodology by three European Partners
iii) disseminating the Prometeus model of integrated collaboration between academia and SME;
iv) young and experienced scientists are increasing their skills, that will make them able to pursue their scientific and career objectives;
i) we are informing the general public on the rational design approach to drugs and of the synergistic approach between academia and SME, with the support of the MSCA