Pharmacophore Generation from a Drug-like Core Molecule Surrounded by a Library Peptide via the 10BASEd-T on Bacteriophage T7

Abstract

Abstract:We have achieved site-specific conjugation of several haloacetamide derivatives into designated cysteines on bacteriophage T7-displayed peptides, which are fused to T7 capsid protein gp10. This easiest gp10 based-thioetherification (10BASE d -T) undergoes almost quantitatively like a click reaction without side reaction or loss of phage infectivity. The post-translational modification yield, as well as the site-specificity, is quantitatively analyzed by a fluorescent densitometric analysis after gel electrophoresis. The detailed structure of the modified peptide on phage is identified with tandem mass spectrometry. Construction of such a peptide-fused phage library possessing non-natural core structures will be useful for future drug discovery. For this aim, we propose a novel concept of pharmacophore generation from a drug-like molecule (i.e., salicylic acid) conjugated with surrounding randomized peptides. By using the hybrid library, streptavidin-specific binders are isolated through four rounds of biopanning.

Encoded libraries of chemically modified peptides

“…In a proof-of-principle study, they conjugated a penicillin derivative of iodoacetamide to an invariant cysteine in an mRNAencoded peptide library (Figure 2a), selected the library against Staphylococcus aureus penicillin binding protein 2a (PBP2a) and succeeded in identifying penicillin-peptide conjugates that bound better than penicillin alone. Similar chemistry was used to build a model pharmacophore by Taki et al (Figure 2b) [16].…”

Expanding the Chemical Diversity of Genetically Encoded Libraries

“…Fortunately, regioselectivity can often be simultaneously assessed while determining PTM percent yield. Employing fluorescent reporters to measure yield via SDS-PAGE and in-gel imaging (Figure B) can also indicate site specificity if done alongside controls lacking the reactive handle, as fluorescence should only appear on the chemically amenable fusions. ,,, For in vivo display systems, observed fluorescence in negative control gel bands can potentially be sourced back to the proteins undergoing unexpected modification. Fluorescence reporting has also been used in synergy with affinity tag-containing libraries based on the principle that affinity tag and fluorescence detection should only occur at the same site if a PTM is regioselective. , For libraries with more than one reactive handle, nuclease digestion and SDS-PAGE analysis has been used to identify the number of different products per PTM .…”

“…Cysteine alkylation or arylation with alkyl or aryl halides has been used extensively with various GELs, including phage, mRNA display, and cell-surface display libraries. This basic S N 2 chemistry has been used to fit libraries with various pharmacophores, , fluorophores, , and carbohydrate-based epitopes (Figure .1) . In a recent, novel application, a phage library was fitted with a boronic acid functionality to covalently target bacterial cell walls .…”

Dihydrofolate reductase inhibitory peptides screened from a structured designed β-loop peptide library displayed on phage

“…6 Structurally constrained peptides are particularly promising because the side chains of the amino acids essential for inhibition can be appropriately oriented by using stable backbone structures as scaffolds. [7][8][9][10] We have previously designed and constructed a b-loop peptide phage library in which a loop structure composed of four or five randomized amino acids was stabilized by antiparallel b-strands (Fig. 1).…”