Abstract by Salvia Sajid

Proton-coupled oligopeptide transporters (POTs) transport a variety of substrates, including peptidomimetics and antibiotics, and thus, are promising candidates for drug delivery. POTs are ubiquitous across the phylogeny with bacterial POTs sharing substantial structural and substrate similarities with human peptide transporters making them conspicuous for drug transport research. For instance, di- and tripeptide permeases from E. coli (YdgR, YjdL) closely resemble the human di- and tripeptide permeases (hPepT1, hPepT2). Therefore, it is relevant to investigate the mechanism of bacterial POTs to understand human peptide transporters better. This thesis focuses on the bacterial transporter YdgR from E. coli as a model system to explore its potential in drug delivery.

In this research, YdgR from E. coli was used for three fundamental objectives: Firstly, to explore the promiscuity of the substrate space by comparative analysis across POT orthologues for predicting novel POT substrates. Based on this objective, Tanimoto similarity cheminformatic analysis was conducted to screen known substrates of POTs against β-Ala-Lys dipeptide. Based on their structural diversity, selected ligands were then assessed for YdgR-mediated transport (Appendix I). Secondly, to investigate the specificity of the substrate binding site within YdgR particularly its preference for certain substrates such as β-Ala-Lys(AMCA). For such purpose, several dipeptide-fluorophore conjugates were synthesized in-house that can be potentially used as carrier moieties. These conjugates were tested for their ability to be transported through YdgR (Appendix II). Lastly, to evaluate the effects of YdgR overexpression the phenotypic changes at the cellular level were assessed. This involved overexpressing YdgR in its functional and inactive mutant states and analyzing the cell’s omics profiles to understand the comprehensive physiological consequences (Appendix III).

The results indicate that computer-aided substrate design based on molecular similarity may not be reliable, as the transporters are promiscuous yet exhibit unexpected specificity towards their substrates. Even minor structural modifications in known substrates could significantly affect substrate transport efficiency as incase of β-Ala-Lys(AMCA). Additionally, cells with induced transporter mutations showed considerable changes in cellular biochemistry, highlighting the complexity of transporter function. Similarly, the outcomes also revealed the presence of several uncharacterized genes and proteins (known as the y-ome) as a repercussion of transporter over-expression (Appendix IV). The insights from this research could help to use bacterial transporters as a model system for human transporter homologs and to design drugs targeting bacteria and actively utilize them to benefit the host.