"Inject-mix-react-separate-and-quantitate" (IMReSQ) method for screening enzyme inhibitors.
Many regulatory enzymes are considered attractive therapeutic targets, and their inhibitors are potential drug candidates. Screening combinatorial libraries for enzyme inhibitors is pivotal to identifying hit compounds for the development of drugs targeting regulatory enzymes. Here, we introduce the first inhibitor screening method that consumes only nanoliters of the reactant solutions and is applicable to regulatory enzymes. The method is termed inject-mix-react-separate-and-quantitate (IMReSQ) and includes five steps. First, nanoliter volumes of substrate, candidate inhibitor, and enzyme solutions are injected by pressure into a capillary as separate plugs. Second, the plugs are mixed inside this capillary microreactor by transverse diffusion of laminar flow profiles. Third, the reaction mixture is incubated to form the enzymatic product. Fourth, the product is separated from the substrate inside the capillary by electrophoresis. Fifth, the amounts of the product and substrate are quantitated. In this proof-of-principle work, we applied IMReSQ to study inhibition of recently cloned protein farnesyltransferase from parasite Entamoeba histolytica. This enzyme is a potential therapeutic target for antiparasitic drugs. We identified three previously unknown inhibitors of this enzyme and proved that IMReSQ could be used for quantitatively ranking the potencies of inhibitors.
Wong
Edmund
E
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
Okhonin
Victor
V
Berezovski
Maxim V
MV
Nozaki
Tomoyoshi
T
Waldmann
Herbert
H
Alexandrov
Kirill
K
Krylov
Sergey N
SN
D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D051231:Farnesyltranstransferase / Q000037:antagonists & inhibitors* / Q000737:chemistry
D004791:Enzyme Inhibitors; D051231:Farnesyltranstransferase