Robust and Systematic Drug Screening Method Using Chemical Arrays and the Protein Library: Identification of Novel Inhibitors of Carbonic Anhydrase II

Abstract

The identification of specific interactions between small molecules and human proteins of interest is a fundamental step in chemical biology and drug development. Here we describe an efficient method to obtain novel binding ligands of human proteins by a chemical array approach. Our method includes large-scale ligand screening with two libraries, proteins and chemicals, the use of cell lysates that express proteins of interest fused with red fluorescent protein, and high-throughput screening by merged display analysis, which removes false positive signals from array experiments. Using our systematic platform, we detected novel inhibitors of carbonic anhydrase II. It is suggested that our systematic platform is a rapid and robust approach to screen novel ligands for human proteins of interest.

Crystal structure of the predicted phospholipase LYPLAL1 reveals unexpected functional plasticity despite close relationship to acyl protein thioesterases

“…The slides of Natural Products Depository (NPDepo ) ( 15 ) were prepared and analyzed as reported before (16)(17)(18). The microarray screening was performed using the following buffer: 20 mM Hepes, 150 mM NaCl, pH 7.53-7.55.…”

Discovery of Novel Antiviral Agents Directed Against the Influenza A Virus Nucleoprotein

“…Chemical arrays represent one of the most promising and high-throughput approaches for screening ligands against proteins of interest (Kanoh et al, 2006), and several successful results from chemical arrays have been reported (Koehler et al, 2003;Kuruvilla et al, 2002;Miyazaki et al, 2008). The screening protocol using chemical array was shown in Fig.…”

Introduction of New Tools for Chemical Biology Research on Microbial Metabolites

“…The platform consists of a chemical array that bears compounds deposited in the Natural Product Depository (NPDepo) 20) and the protein library generated from the Gene Library of the Osada Laboratory of RIKEN for chemical array analysis (GLORIA). 21) To use GLORIA for our chemical array system, we cloned 100 genes and engineered them to express a fusion between red fluorescent protein (RFP) 22) and the protein of interest. HEK293T cells were transfected with vectors that encoded RFP-fused proteins or RFP alone, and cell lysates were prepared.…”

“…After merged display analysis, we identified two hit compounds. 21) To test the specificity of 1 and 2 for CAII, we prepared cell lysates from HEK293T cells that expressed 33 RFPfused proteins, including CAII, from GLORIA. Among the 33 proteins tested, only the cell lysate that expressed RFP-fused CAII interacted with 1 and 2, suggesting that 1 and 2 are selective inhibitors of CAII.…”

“…Based on these observations, several derivatives were synthesized, and a new derivative, 4, showed the most potent inhibitory activity against CAII. 21) In summary, our screening method makes possible large-scale chemical array screening with a combination of a gene library (GLORIA) and a chemical library in NPDepo. To identify useful ligands against human proteins, we have increased the numbers of genes in GLORIA and the compounds in NPDepo, which should dissolve one of the bottlenecks in our quest for useful tools for modern chemical biology studies and drug discovery research.…”