ABSTRACT
The binding profile of a known inhibitor, benzenesulfonamide, against a family of carbonic anhydrase isozymes was efficiently enhanced via high-throughput screening of customized combinatorial one-bead-one-compound peptide libraries modified with the inhibitor molecule. The screening of the conjugate libraries recognized subtle variations in the microenvironments of the target enzyme and thus facilitated the identification of short peptide sequences that bind selectively to a close proximity of the active site. The identified peptide portions contributed significantly to the overall binding of the conjugate peptides with greatly enhanced affinity as well as improved specificity towards the target isozyme. The interactions between the inhibitors and the isozymes were validated by surface plasmon resonance (SPR), pull-down assay and enzymatic activity measurement. This high-throughput approach proved useful and efficient to enhance the binding profile of known inhibitors and may apply to developing effective inhibitors for a wide range of isozyme families.
Subject(s)
Carbonic Anhydrase Inhibitors/chemistry , Carbonic Anhydrase Inhibitors/pharmacology , Carbonic Anhydrases/metabolism , Peptides/chemistry , Peptides/pharmacology , Sulfonamides/chemistry , Sulfonamides/pharmacology , High-Throughput Screening Assays/methods , Humans , Isoenzymes/metabolism , Molecular Docking Simulation , Peptide Library , Protein Binding , BenzenesulfonamidesABSTRACT
Screening of bead-based peptide libraries against fluorescent dye-labeled target proteins was found to be significantly influenced by the dye characteristics. Commercially available red fluorescent dyes with net negative charges adversely showed strong interactions with library beads. The introduction of zwitterionic dyes significantly reduced the unwanted interactions, which sheds light upon using the right fluorescent probe for acquisition of reliable results in various fluorescence-assisted applications.
Subject(s)
Fluorescent Dyes/chemistry , Carbocyanines/chemistry , Carbonic Anhydrase II/chemistry , Humans , Peptide Library , Peptides/chemistryABSTRACT
With an aim to develop peptide-based protein capture agents that can replace antibodies for in vitro diagnosis, an ultra-high-throughput screening strategy has been investigated by automating labor-intensive, time-consuming processes that are the construction of peptide libraries, sorting of positive beads, and peptide sequencing through analysis of tandem mass spectrometry data. Although instruments for automation, such as peptide synthesizers and automatic bead sorters, have been used in some groups, the overall process has not been well optimized to minimize time, cost, and efforts, as well as to maximize product quality and performance. Herein we suggest and explore several solutions to the existing problems with the automation of the key processes. The overall process optimization has been done successfully in orchestration with the technologies such as rapid cleavage of peptides from beads and semiautomatic peptide sequencing that we have developed previously. This optimization allowed one-round screening, from peptide library construction to peptide sequencing, to be completed within 4 to 5 days. We also successfully identified a 6-mer ligand for carcinoembryonic antigen-cell adhesion molecule 5 (CEACAM 5) through three-round screenings, including one-round screening of a focused library.
