Periodic Reporting for period 4 - ReSOLUTE (Research empowerment on solute carriers (ReSOLUTE))
Okres sprawozdawczy: 2021-07-01 do 2022-06-30
Metabolism is at the center of the attention of researchers worldwide as the uptake and release of chemical matter over cellular membranes is a critical interface governing exchange with the environment. Transporters located on cellular membranes play a key role taking up and distributing nutrients and chemical matter from the environment. The largest group of cellular transporters encoded in the human genome is represented by solute carrier transporters (SLCs). Though more than half of all SLC genes are associated with human disease and despite the fact that some SLCs are prominent drug targets, the specificity and function of many individual SLCs remains unknown.
RESOLUTE’s (https://re-solute.eu) overarching goal is to trigger an escalation in the intensity of research on SLCs worldwide and to establish SLCs as a tractable target class for medical research and drug development.
One of the main goals of RESOLUTE is to produce high-quality reagents and datasets to empower the scientific community and to boost research on SLCs. The RESOLUTE reagents and tools open access collection is available in our web portal (https://re-solute.eu/resources/reagents). This collection contains i) plasmids with 894 codon-optimized sequences of human SLCs inserted in a vector compatible with the gateway system, ii) cell lines with genetically deleted or overexpressed individual SLCs, iii) data and protocols for transport assays to assess the function of individual SLCs, and iv) a dataset of SLC commercial antibodies tested on our cell lines.
Using RESOLUTE cell lines, we generated new data sets from employing omics technologies to de-orphanize SLCs in a systematic way. In the RESOLUTE web portal, researchers find the data generated by RESOLUTE (https://re-solute.eu/resources/datasets) together with the RESOLUTE knowledgebase (https://re-solute.eu/knowledgebase) which integrates data from public repositories, REsolution and RESOLUTE, hence being the reference hub for research on SLCs. Additionally, researchers can explore our transcriptomics and imaging datasets in visualization dashboards (https://re-solute.eu/resources/dashboards).
The second objective of RESOLUTE is to develop robust functional assays for SLCs. In the last 4 years, the RESOLUTE consortium has developed 25 medium or high-throughput transport assays for SLCs, some of which were implemented in our industry and SME partners and are likely used in future drug discovery endeavors. Consortium members published a review article providing a well-structured overview on cellular assay technologies for SLCs (https://doi.org/10.3389/fphar.2021.722889). We consider this an important step towards providing the scientific community with novel approaches to functionally investigate SLCs.
The RESOLUTE consortium puts emphasis on gaining visibility worldwide as well as to create awareness around SLC research. In addition to regular news updates through the Twitter account and web portal, 11 videos describing the RESOLUTE project and its activities were produced. Furthermore, the SLC family card game with information on the current SLC families was created and distributed >400 decks to date.
In summary, the outputs from the RESOLUTE consortium is expanding the knowledge and facilitating research on SLCs.
RESOLUTE’s (https://re-solute.eu) overarching goal is to trigger an escalation in the intensity of research on SLCs worldwide and to establish SLCs as a tractable target class for medical research and drug development.
One of the main goals of RESOLUTE is to produce high-quality reagents and datasets to empower the scientific community and to boost research on SLCs. The RESOLUTE reagents and tools open access collection is available in our web portal (https://re-solute.eu/resources/reagents). This collection contains i) plasmids with 894 codon-optimized sequences of human SLCs inserted in a vector compatible with the gateway system, ii) cell lines with genetically deleted or overexpressed individual SLCs, iii) data and protocols for transport assays to assess the function of individual SLCs, and iv) a dataset of SLC commercial antibodies tested on our cell lines.
Using RESOLUTE cell lines, we generated new data sets from employing omics technologies to de-orphanize SLCs in a systematic way. In the RESOLUTE web portal, researchers find the data generated by RESOLUTE (https://re-solute.eu/resources/datasets) together with the RESOLUTE knowledgebase (https://re-solute.eu/knowledgebase) which integrates data from public repositories, REsolution and RESOLUTE, hence being the reference hub for research on SLCs. Additionally, researchers can explore our transcriptomics and imaging datasets in visualization dashboards (https://re-solute.eu/resources/dashboards).
The second objective of RESOLUTE is to develop robust functional assays for SLCs. In the last 4 years, the RESOLUTE consortium has developed 25 medium or high-throughput transport assays for SLCs, some of which were implemented in our industry and SME partners and are likely used in future drug discovery endeavors. Consortium members published a review article providing a well-structured overview on cellular assay technologies for SLCs (https://doi.org/10.3389/fphar.2021.722889). We consider this an important step towards providing the scientific community with novel approaches to functionally investigate SLCs.
The RESOLUTE consortium puts emphasis on gaining visibility worldwide as well as to create awareness around SLC research. In addition to regular news updates through the Twitter account and web portal, 11 videos describing the RESOLUTE project and its activities were produced. Furthermore, the SLC family card game with information on the current SLC families was created and distributed >400 decks to date.
In summary, the outputs from the RESOLUTE consortium is expanding the knowledge and facilitating research on SLCs.
RESOLUTE has two main objectives:
Objective 1: Create a comprehensive set of long-lasting, intensely characterized tools enabling solute carrier transporter (SLC) research and packages of reliable knowledge for the majority of SLCs.
To this end we have generated the following tools in the last four years:
- 894 codon-optimized human SLC sequences inserted in a plasmid compatible with the gateway cloning system and released to the public repository Addgene.
- 1197 lentiviral CRISPR/Cas9 vectors to generate knock-out (k.o.) cell lines.
- 1090 inducible SLC expression vectors to generate two types of overexpression cell lines.
- 446 BacMam vectors for SLC expression and purification studies.
- 40 BioID vectors to generate cells for proximity proteomics studies.
- 3 plasmid libraries (1 x cDNA and 2 x CRISPR).
- 1077 cell lines overexpressing tagged SLCs (475 Ct WT-OE, 122 Nt WT-OE and 480 KO-OE).
- K.o. cell lines clones for 315 SLCs.
- Released datasets for 7 parental cell lines, high-content imaging for 431 SLCs (publicly available from Aug 22), RNA-Seq for 446 SLCs (publicly available from Aug 22); as well as reports for the systematic characterization of commercial antibodies for 6 SLCs, 7 assays released in public repositories.
- Internal large-scale datasets: metabolomics for 292 SLCs, proteomics for 321 SLCs, ionomics for 43 SLCs.
- 26 SLCs proteins purified and reconstituted in proteoliposomes / detergents for in vitro assays or binder generation.
- Positively validated high-affinity binders for 6 SLCs.
- 23 standard operation procedures developed by RESOLUTE (i.e proteomics, ionomics, targeted and untargeted metabolomics, immunofluorescence, protein expression, RNA-Seq and cell line data sheets).
- RESOLUTE web portal with updated information on the goals of each WP, communication activities, publications and information on the project progress. The RESOLUTE knowledgebase now contains information on genetic variants for each SLC. The RESOLUTE public database contains plasmids and cell lines available for the scientific community, large datasets released by the consortium and released functional assays for SLCs.
- Twitter account with more than 1,000 followers to distribute news about RESOLUTE, REsolution and scientific articles featuring SLCs.
- 48 posts in the “news” section of the RESOLUTE web portal during this reporting period (Approx. 5,000 unique visits to the web portal).
- 11 original videos produced by RESOLUTE.
Objective 2: Develop robust functional assays for at least 50 of the 72 prioritized SLCs:
To accomplish this goal, RESOLUTE has performed and developed 25 different MTS/HTS and 11 validated proof-of-principle transport assays for 25 priority SLCs.
Objective 1: Create a comprehensive set of long-lasting, intensely characterized tools enabling solute carrier transporter (SLC) research and packages of reliable knowledge for the majority of SLCs.
To this end we have generated the following tools in the last four years:
- 894 codon-optimized human SLC sequences inserted in a plasmid compatible with the gateway cloning system and released to the public repository Addgene.
- 1197 lentiviral CRISPR/Cas9 vectors to generate knock-out (k.o.) cell lines.
- 1090 inducible SLC expression vectors to generate two types of overexpression cell lines.
- 446 BacMam vectors for SLC expression and purification studies.
- 40 BioID vectors to generate cells for proximity proteomics studies.
- 3 plasmid libraries (1 x cDNA and 2 x CRISPR).
- 1077 cell lines overexpressing tagged SLCs (475 Ct WT-OE, 122 Nt WT-OE and 480 KO-OE).
- K.o. cell lines clones for 315 SLCs.
- Released datasets for 7 parental cell lines, high-content imaging for 431 SLCs (publicly available from Aug 22), RNA-Seq for 446 SLCs (publicly available from Aug 22); as well as reports for the systematic characterization of commercial antibodies for 6 SLCs, 7 assays released in public repositories.
- Internal large-scale datasets: metabolomics for 292 SLCs, proteomics for 321 SLCs, ionomics for 43 SLCs.
- 26 SLCs proteins purified and reconstituted in proteoliposomes / detergents for in vitro assays or binder generation.
- Positively validated high-affinity binders for 6 SLCs.
- 23 standard operation procedures developed by RESOLUTE (i.e proteomics, ionomics, targeted and untargeted metabolomics, immunofluorescence, protein expression, RNA-Seq and cell line data sheets).
- RESOLUTE web portal with updated information on the goals of each WP, communication activities, publications and information on the project progress. The RESOLUTE knowledgebase now contains information on genetic variants for each SLC. The RESOLUTE public database contains plasmids and cell lines available for the scientific community, large datasets released by the consortium and released functional assays for SLCs.
- Twitter account with more than 1,000 followers to distribute news about RESOLUTE, REsolution and scientific articles featuring SLCs.
- 48 posts in the “news” section of the RESOLUTE web portal during this reporting period (Approx. 5,000 unique visits to the web portal).
- 11 original videos produced by RESOLUTE.
Objective 2: Develop robust functional assays for at least 50 of the 72 prioritized SLCs:
To accomplish this goal, RESOLUTE has performed and developed 25 different MTS/HTS and 11 validated proof-of-principle transport assays for 25 priority SLCs.
In the fourth year, RESOLUTE made substantial progress on multiple levels towards achieving its goal of triggering an escalation of SLC research. First, we made publicly available plasmids, cell lines, assays, datasets and dashboards focused on SLCs. Furthermore, the progress on sample processing in the remaining ‘omics approaches is reaching the finish line and the datasets will be released next year. Second, with our dissemination activities and scientific publications, we are creating awareness of the RESOLUTE project and the exciting potential of SLCs as drug targets. The number of requested RESOLUTE resources keeps increasing and hence they are empowering basic research on SLCs. Third, we developed assays to test the effect of hundreds of chemical compounds on the individual SLC function for future use drug discovery by the pharmaceutical industry and small/medium biotech companies. We shared protocols and first results in the open access repository PubChem (https://pubchem.ncbi.nlm.nih.gov/source/24749). Based on these key performance indicators, as well as the fact that external laboratories, companies and patient organizations keep reaching out for collaboration, we expect that RESOLUTE will create a lasting impact on the scientific community. As a result, the perception of SLCs as targets for therapeutics is changing and SLCs are becoming an attractive target class for new small chemical entities, next to GPCRs, kinase, proteases.