2 research outputs found
Chemical Library Screening Using a SPR-Based Inhibition in Solution Assay: Simulations and Experimental Validation
We
have developed a surface plasmon resonance (SPR)-based inhibition
in solution assay (ISA) to search for inhibitors of the medium affinity
(<i>K</i><sub>D</sub> = 0.8 μM) interaction between
an E6-derived peptide (E6<sub>peptide</sub>) immobilized on the sensor
and a PDZ domain (MAGI-1 PDZ1) in the mobile phase. DZ domains are
widespread protein-protein interaction modules that recognize the
C-terminus of various partners. Simulations indicated that relatively
low compound concentrations (10 μM) and limited peptide densities
(<i>R</i><sub>max</sub> < 200 resonance units) should
allow the detection of inhibitors with a target affinity close to
100 μM, which was then demonstrated experimentally. ISA screening,
carried out on the Prestwick Chemical Library® (1120 compounds),
identified 36 compounds that inhibited the interaction by more than
5%. Concentration-dependent ISA, carried out on a subset of 19 potential
inhibitors, indicated that 13 of these indeed affected the interaction
between MAGI-1 PDZ1 and the E6<sub>peptide</sub>. No effect was observed
for 84 compounds randomly chosen among noninhibitors. One of the four
best inhibitors was a peptide binder, and three were PDZ binders with <i>K</i><sub>D</sub> in the 10–50 μM range. We propose
that a medium (μM) affinity between the target and surface-bound
partner is optimal for SPR-based ISA screening