Periodic Reporting for period 4 - CHEMMINE (Chemical proteome mining for functional annotation of disease relevant proteins)
Período documentado: 2021-09-01 hasta 2022-08-31
When the human genome was sequenced in 2001 about 50 % of genes and corresponding proteins did not have an assigned function. About 20 years later the knowledge has only marginally increased and novel methods to filter the uncharacterized proteome for a streamlined assignment are needed. Given the limited number of drug targets, increasing the scope for drug development especially in the fields of cancer and bacterial infections is an important task. For this purpose, enzymes containing cofactors are prime candidates as they feature conserved chemical moieties and may thus be a convenient starting point for a functional assignment. For example, pyridoxal phosphate dependent enzymes (PLP-DEs) represent a large cofactor class with over 200 reported catalytic activities. The consolidated study of these enzymes bears a unique potential not only to identify novel enzyme members but also select essential proteins as drug targets. This major objectives of our CHEMMINE project is to develop pyridoxal probes which are able to infiltrate bacterial and human cells, get metabolized and finally incorporated in cognate PLP-DEs. These tools are equipped with a bio-orthogonal handle which allows protein enrichment and identification via mass-spectrometry. We seek to utilize this methodology for profiling pathogenic bacteria and cancer cells, report the PLP-ome, select uncharacterized enzymes, unravel their function, monitor the selectivity of pyridoxal-based drugs and design inhibitors against essential proteins. Once successfully established we expand this strategy towards other cofactors and post-translational modifications.
In order to tackle the challenge of identifying new drug targets we devised a chemical proteomic strategy based on the modification of the pyridoxal (vitamin B6) cofactor with a bio-orthogonal tag that allows for the selective reporting of all pyridoxal phosphate binding proteins in living cells. The functionalized pyridoxal scaffold was utilized for profiling of bacterial as well as cancer cells and revealed a wealth of known PLP-DEs with an overall coverage of 75%. In addition, several uncharacterized proteins were identified and selected for in-depth studies. For example, we were able to assign one protein to catalyze the final step of para-aminobenzoic acid biosynthesis, an essential pathway for bacteria. Moreover, we utilized the methodology to determine undesired off-targets of drugs such as D-cycloserine. Contrary to previous believe we could demonstrate that alanine racemase is not a target of the drug but several other PLP-DEs involved in cell wall biosynthesis. Functionalization of the pyridoxal scaffold with different side chains demonstrated selectivity for certain enzymes and paves the way for inhibitor development.
In addition, we demonstrated that the workflow also works in human cells. Although the metabolic machinery is more selective one of our pyridoxal probes worked well and was also utilized in the characterization of enzymes with unknown function as well as off-target screening.
In addition, we demonstrated that the workflow also works in human cells. Although the metabolic machinery is more selective one of our pyridoxal probes worked well and was also utilized in the characterization of enzymes with unknown function as well as off-target screening.
Mining the cellular inventory for a conserved chemical feature in enzymes is a novel approach which already demonstrated its power in the functional assignment of proteins as well as in off-target screening of marketed drugs. We seek to further expand the methodology and in a next step develop customized inhibitors against essential cofactor dependent proteins. This will push the project into a next level and provide new lead compounds for testing their activity against devastating diseases such as cancer and bacterial infections.