Confirming target engagement for reversible inhibitors in vivo by kinetically tuned activity-based probes.
The development of small-molecule inhibitors for perturbing enzyme function requires assays to confirm that the inhibitors interact with their enzymatic targets in vivo. Determining target engagement in vivo can be particularly challenging for poorly characterized enzymes that lack known biomarkers (e.g., endogenous substrates and products) to report on their inhibition. Here, we describe a competitive activity-based protein profiling (ABPP) method for measuring the binding of reversible inhibitors to enzymes in animal models. Key to the success of this approach is the use of activity-based probes that show tempered rates of reactivity with enzymes, such that competition for target engagement with reversible inhibitors can be measured in vivo. We apply the competitive ABPP strategy to evaluate a newly described class of piperazine amide reversible inhibitors for the serine hydrolases LYPLA1 and LYPLA2, two enzymes for which selective, in vivo active inhibitors are lacking. Competitive ABPP identified individual piperazine amides that selectively inhibit LYPLA1 or LYPLA2 in mice. In summary, competitive ABPP adapted to operate with moderately reactive probes can assess the target engagement of reversible inhibitors in animal models to facilitate the discovery of small-molecule probes for characterizing enzyme function in vivo.
Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Alexander
A
Adibekian
Brent R
BR
Martin
Jae Won
JW
Chang
Ku-Lung
KL
Hsu
Katsunori
K
Tsuboi
Daniel A
DA
Bachovchin
Anna E
AE
Speers
Steven J
SJ
Brown
Timothy
T
Spicer
Virneliz
V
Fernandez-Vega
Jill
J
Ferguson
Peter S
PS
Hodder
Hugh
H
Rosen
Benjamin F
BF
Cravatt
D000577:Amides / Q000737:chemistry*; D000818:Animals; D001667:Binding, Competitive; D002478:Cells, Cultured; D016503:Drug Delivery Systems; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D051379:Mice; D015394:Molecular Structure; D010880:Piperidines / Q000737:chemistry*; D011485:Protein Binding / Q000187:drug effects; D054852:Small Molecule Libraries / Q000737:chemistry* / Q000494:pharmacology; D013329:Structure-Activity Relationship
D000577:Amides; D004791:Enzyme Inhibitors; D010880:Piperidines; D054852:Small Molecule Libraries