Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ReSOLUTE (Research empowerment on solute carriers (ReSOLUTE))

Teaser

Metabolism is at the center of the attention of researchers worldwide as it links all cellular, organ- and organismal phenomena to physiology through energy and the flux of chemical matter that allows to build, fuel, renew and recycle the very structures that support life...

Summary

Metabolism is at the center of the attention of researchers worldwide as it links all cellular, organ- and organismal phenomena to physiology through energy and the flux of chemical matter that allows to build, fuel, renew and recycle the very structures that support life. Critical to empower access to nutrients and energy are cellular transporters that take up and distribute metabolites and chemical matter in general. The net result is the differential accumulation of solutes in the cells and in the subcellular compartments that enable cellular functions. The largest group of cellular transporters encoded in the human genome is represented by solute carrier proteins (short: 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 individual SLCs is not known. Yet creating this knowledge can rightfully be considered the bottleneck in the ability to exploit this exciting and promising group of drug targets.
RESOLUTE’s mission is to boost research on solute carriers (SLCs). RESOLUTE will accomplish this mission by providing high-quality reagents and valuable data to deorphanize and functionalize SLCs, including the poorly-studied. RESOLUTE strives to become a reference hub for SLC research and open knowledge worldwide. In the first year of the project, the RESOLUTE community has grown to more than 100 researchers distributed through 8 locations worldwide. All the researchers are highly active in performing research in one or several of the work packages. RESOLUTE researchers form a tightly-knit community that is coordinating and updating their activities on a weekly basis and through reciprocal visits, social media exchanges and larger consortial meetings. First results on reagents as well as the characterization and cell lines and tools have already been given public access to and publicized through the RESOLUTE newsletters and web site (https://re-solute.eu/).
One of the most important milestones that RESOLUTE achieved recently, was the generation of the RESOLUTE knowledgebase (https://re-solute.eu/knowledgebase). The knowledgebase is openly accessible and it features publicly available data on SLCs from multiple sources allowing SLC researchers to rapidly get an overview on the current knowledge on any human SLC transporter.
Regarding data generation by RESOLUTE, our main achievement this year was to fully characterize the transcriptome, metabolome and proteome of 6 RESOLUTE human cancer cell lines, cumulatively covering the expression of about 80% of all human SLCs. The transcriptome data is already publicly available at SRA (PRJNA545487), while proteomics and targeted metabolomics data will be released later this year. Furthermore, we have established robust experimental protocols for different omics approaches and we piloted 8 different transport assays featuring 30 SLCs.
Regarding reagent generation, RESOLUTE designed and synthesized codon-optimized ORF sequences for all 446 human SLCs in Gateway vectors. These plasmids allow fast and efficient sub- cloning into a wide range of expression vectors. These 446 entry vectors will be made available to the research community through the Addgene plasmid repository (www.addgene.org) by September 2019. Additionally, we have designed sgRNAs targeting all 446 SLCs. Then, we established an efficient strategy to knock out SLCs in polyploid cells, as well as to assess the efficiency of these knockouts in a high-throughput mode. We are currently using these plasmids to generate SLC-knockout and SLC- overexpressing cell clones using the RESOLUTE cell lines. Moreover, we are producing SLC-knockout and SLC-overexpression cell lines in HEK293 Jump-In to use as additional control.
These efforts are without a doubt the first steps to empower the research community in general, and RESOLUTE in particular, with accessible state-of-the-art SLC knowledge and reagents.

Work performed

RESOLUTE has two main objectives:

Objective 1: Create a comprehensive set of long-lasting, intensely characterized tools enabling SLC research and packages of reliable knowledge for the majority of SLCs.

To this end we have generated the following tools in the last year:

10+ custom expression vectors to sub-clone SLC cDNAs and sgRNAs

Lentiviral CRISPR/Cas9 vectors targeting 446 SLCs

446 codon-optimized sequences of SLCs – made available via Addgene

2 x 446 inducible SLC expression vectors for two cell systems

420 cell lines overexpressing tagged SLCs

At least one k.o. clone for 80 SLCs

RESOLUTE webpage with information on the mission and progress of the project

Twitter account with 89 tweets and 300+ followers to distribute news on RESOLUTE and solute carriers

SLC knowledgebase compiling information on SLCs from public resources

10+ standard operation procedures developed by RESOLUTE (i.e proteomics, ionomics, targeted and untargeted metabolomics, immunofluorescence, expression test and cell line data sheets)

Objective 2: Develop robust functional assays for at least 50 of the 72 prioritized SLCs.

To accomplish this goal, RESOLUTE has performed and developed more than 8 different kinds of transport assays for 30 SLCs. For one SLC, the assay has reached the parameters needed to operate in high throughput mode.

Final results

At month 12 since inception everything regarding assessing impact has to be considered preliminary. Time is just too short to measure parameters in a significant matter. Yet many signs conspire to make it very likely that RESOLUTE’s impact is likely to be very large and exceed the expectations formulated in the applications. The general interest in the SLC superfamily of transporters has increased and the scientific community is well aware of the efforts of the RESOLUTE IMI project. The RESOLUTE community itself has grown and has become more integrated, creating a growing ripple effect throughout the community.

Perhaps yet more interesting, judging initial signs of the RESOLUTE impact, is the scientific perspective. A few important considerations can be made that were not possible two years ago, when the final text of the RESOLUTE research plan was created. The RESOLUTE focus on de-orphanising SLC transporters through the integration of a variety of approaches has validated. The identification of protein partners in molecular complexes to which SLC transporters participate is likely to be key in identifying the molecular signalling and metabolic pathway to which the SLC participates. Moreover, it will empower structural analysis of SLCs in other IMI consortia as well as any future attempt to include a proper biochemical characterization of the target for the identification of chemical matter targeting SLCs in future IMI consortia. Indeed, so-called interaction proteomics is proving to be a particularly successful branch of the RESOLUTE research activities.

All in all, there are all reasons to expect that RESOLUTE will create a rapid and lasting impact on the scientific community. As a result, the perception of SLCs as target for therapeutics is changing and SLCs will become one of the most attractive target classes for new small chemical entities, next to GPCRs, kinase, proteases. In fact, two new biotech companies have been formed entirely dedicated to targeting SLCs: Jnana Therapeutics and Flagship 54 (still incubating) both have been inspired by RESOLUTE.

Website & more info

More info: https://re-solute.eu/.