Development of a Novel Multiplex Screening Approach and its Applications

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Binding, the non-covalent interaction between biomolecules or the interaction between ligands and receptors can either enhance or inhibit biological functions. We can virtually modify compound screening and predict the best hits with the fixed peptide sequence. More and more ligands have been discovered and some of them have been developed as therapeutic agents. Besides, several protein tags and their corresponding ligands, such as GST and glutathione, have been utilized for affinity purification, immobilization of tagged proteins, or the labeling of the tagged proteins with fluorophore. While the demand of exploring novel therapeutic targets, therapeutic agents, and protein tagging systems is increasing, instead of screening either OBOC or PHD libraries against a single target, my study is focusing on the development of a approach of screening PHD libraries against OBOC small molecule library.

This novel multiplex screening method is employed in two applications in my studies. One is to screen the OBOC small molecule library against phage display human tumor proteome library to identify novel drugable targets and their corresponding therapeutics at the same time. A small molecule inhibitor against Human translation initiation factor 5B, EIF5B, was discovered and can be a valuable tool to dissect the effect of translation regulation. The other one is to screen the same OBOC small molecule library against a phage display random peptide library.

A peptide-small molecule binding partner was discovered through the screening and affinity optimization. The peptide can be used to tag the protein of interest and the small molecule can be used to probe the tagged protein. As more peptide-small molecule binding pairs will be discovered through this novel screening strategy, these binding pairs can be utilized to develop multi-color probing system to visualize in vivo protein colocalization in cells.

In this study, we only modified the peptide sequences to optimize the peptide binding affinity to the small molecule. On the other hand, we can also modify the structure of the small molecule to improve the binding affinity and specificity. The critical binding sites on the molecules can be predicted using molecular docking. We can synthesize the predicted small molecules and perform the binding assay for confirmation. The binding affinities between peptides and small molecules can also be quantified by using SPR analysis. There are apparently many details we can adjust to refine the multiplex probing and tagging system. None of the sequences of the peptides which were confirmed to bind to the small molecules was found to match to any human protein sequences when a BLAST search32 was performed. Although the chances of the small molecules binding to these human proteins are relatively low considering the low similarity between those peptide tags and the human proteins, we will further modify the peptide sequences so that it will have the lowest similarity to the endogenous proteins and highest binding affinity and specificity to its corresponding small molecules. We checked the parent peptide APNTKNISC which has the highest binding affinity to compound screening and did not find any human proteins which had a sequence 100% identical to APNTKNISC.