Application of Fragment-Based Drug Discovery to Membrane Proteins: Identification of Ligands of the Integral Membrane Enzyme DsbB

Abstract

Summary Membrane proteins are important pharmaceutical targets, but they pose significant challenges for fragment based drug discovery approaches. Here we present the first successful use of biophysical methods to screen for fragment ligands to an integral membrane protein. The E. coli inner membrane protein DsbB was solubilized in detergent micelles and lipid bilayer nanodiscs. The solubilized protein was immobilized with retention of functionality and used to screen 1,071 drug fragments for binding using Target Immobilized NMR Screening. Biochemical and biophysical validation of the 8 most potent hits revealed an IC50 range of 7 to 200 μM. The ability to insert a broad array of membrane proteins into nanodiscs, combined with the efficiency of TINS, demonstrates the feasibility of finding fragments targeting membrane proteins.

Binding Interaction Between Prazosin and Immobilized Receptor by Frontal Analysis

“…Classic strategies using animal model [4], cell or enzyme-based assays [5,6] and fractionation approach [7] have been successfully applied in screening bioactive compounds from traditional Chinese medicine and have made great contribution to drug discovery. Technologies are continuously in demand for rapidly identifying new leads and reducing the number of potential candidates, to counter the disadvantages of timeconsuming, labor-intensive and environment unfriendly traditional assays [8][9][10][11].…”

Targeting virulence not viability in the search for future antibacterials

“…Nevertheless, there are an increasing number of examples of small molecules designed to block proteinprotein interactions against other targets [77]. Furthermore, screening of small molecule 'fragments' identified compounds that interacted with DsbB and led to a series of compounds capable of inhibiting DsbB in vitro [78]. If we can develop inhibitors of DSB-mediated oxidative protein folding, these would have enormous value as antivirulence agents by potentially blocking the assembly of multiple bacterial virulence factors.…”

From Biology to Biotechnology: Disulfide Bond Formation in <i>Escherichia coli</i>

“…Promising small molecule inhibitors of bacterial Dsb proteins have been identified using fragment-based lead discovery (FBLD) [124]. FBLD identifies small molecule fragments that weakly bind to a target of interest.…”

“…Using a detergent-solubilized EcDsbB immobilized onto sepharose resin and 1 H NMR, 1071 fragments were tested for both binding to and inhibition of EcDsbB, yielding eight fragments exhibiting IC 50 values of 7-170 μM. The eight fragments were divided into two groups based on their molecular scaffolds and hypothesized mechanisms of inhibition: blocking of quinone binding and blocking of both quinone and EcDsbA binding to DsbB [124]. A further study improved the IC 50 value of a candidate molecule to 1.1 μM through additional rounds of FBLD.…”