Inhibitor focusing: direct selection of drug targets from proteomes using activity-based probes

Assay Drug Dev Technol. 2003 Feb;1(1 Pt 2):137-46. doi: 10.1089/154065803321537854.

Abstract

In the latter stages of drug discovery and development, assays that establish drug selectivity and toxicity are important when side effects, which are often due to lack of specificity, determine drug candidate viability. There has been no comprehensive or systematic methodology to measure these factors outside of whole-animal assays, and such phenomenological assays generally fail to establish the additional targets of a given small molecule, or the molecular origin of toxicity. Consequently, small-molecule development programs destined for failure often reach advanced stages of testing, and the money and time invested in such programs could be saved if information on selectivity were available early in the process. Here, we present a methodology that utilizes chemical ABPs in combination with small-molecule inhibitors to selectively label small-molecule binding sites in whole proteomic samples. In principle, the ABP and small molecule will compete for similar binding sites, such that the small molecule will protect against modification by the ABP. Thus, after removal of the small molecule, the binding site for the ABP will be revealed, and a second probe can then be used to label the small-molecule binding sites selectively. To demonstrate this experimentally, we mapped the binding sites of the DPP4 inhibitor, IT, in a number of different tissue types.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Binding, Competitive
  • Cell Line
  • Cells, Cultured
  • Dipeptidyl Peptidase 4 / metabolism
  • Drug Design
  • Fluorescent Dyes / chemistry
  • Humans
  • In Vitro Techniques
  • Isoleucine / analogs & derivatives*
  • Isoleucine / metabolism
  • Kidney / chemistry
  • Kidney / metabolism
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism
  • Mice
  • Organophosphonates / chemistry
  • Pharmaceutical Preparations / chemistry*
  • Pharmaceutical Preparations / metabolism
  • Protease Inhibitors / metabolism
  • Proteins / chemistry*
  • Proteins / metabolism
  • Proteome / chemistry*
  • Structure-Activity Relationship
  • Thiazoles / metabolism

Substances

  • Fluorescent Dyes
  • Membrane Proteins
  • Organophosphonates
  • Pharmaceutical Preparations
  • Protease Inhibitors
  • Proteins
  • Proteome
  • Thiazoles
  • isoleucyl-thiazolidide
  • Isoleucine
  • Dipeptidyl Peptidase 4