Superfamily-wide portrait of serine hydrolase inhibition achieved by library-versus-library screening

Abstract

Serine hydrolases (SHs) are one of the largest and most diverse enzyme classes in mammals. They play fundamental roles in virtually all physiological processes and are targeted by drugs to treat diseases such as diabetes, obesity, and neurodegenerative disorders. Despite this, we lack biological understanding for most of the 110þ predicted mammalian metabolic SHs, in large part because of a dearth of assays to assess their biochemical activities and a lack of selective inhibitors to probe their function in living systems. We show here that the vast majority (>80%) of mammalian metabolic SHs can be labeled in proteomes by a single, active site-directed fluorophosphonate probe. We exploit this universal activitybased assay in a library-versus-library format to screen 70þ SHs against 140þ structurally diverse carbamates. Lead inhibitors were discovered for ∼40% of the screened enzymes, including many poorly characterized SHs. Global profiles identified carbamate inhibitors that discriminate among highly sequence-related SHs and, conversely, enzymes that share inhibitor sensitivity profiles despite lacking sequence homology. These findings indicate that sequence relatedness is not a strong predictor of shared pharmacology within the SH superfamily. Finally, we show that lead carbamate inhibitors can be optimized into pharmacological probes that inactivate individual SHs with high specificity in vivo.enzymology | mass spectrometry | profiling | proteomics A major challenge facing biological researchers in the 21st century is the functional characterization of the large number of unannotated gene products identified by genome sequencing efforts (1). Many proteins partly or completely uncharacterized with respect to their biochemical activities belong to expansive, sequence-related families (2). Although such membership can inform on the general mechanistic class to which a protein belongs (e.g., enzyme, receptor, or channel), it is insufficient to predict specific biochemical and physiological functions, which require knowledge of substrates, ligands, and interacting biomolecules. On the contrary, membership within a large protein family can even present a barrier to achieving these goals by frustrating the implementation of standard genetic and pharmacological methods to probe protein function. For example, targeted gene disruption of one member of a protein superfamily may result in cellular compensation from other family members.Problems are also encountered when attempting to develop specific inhibitors and/or ligands for uncharacterized members of large protein families, where at least two major experimental issues must be addressed. First, there is an intrinsic difficulty facing ligand discovery for uncharacterized proteins, which often lack the functional information required to develop high-quality assays for compound screening. Creative solutions to this problem have emerged for specific protein classes, such as G-protein coupled receptors (GPCRs) (3) and kinases (4, 5), where generic assays have been develop...

Chemical genetic strategy for targeting protein kinases based on covalent complementarity

“…This strategy may also be useful for studying the properties of pseudokinases, for which there is no good readout of active site occupancy. The use of irreversible inhibitors for poorly studied members of large families allows rapid validation of target specificity when downstream assays are not known, as has been shown in the serine hydrolase family (46). In addition, the technology reported in this study may enable multiplex inhibitor studies, such as those requiring washing steps or the use of more than one chemical genetic inhibitor.…”

Chemical Probes of Endocannabinoid Metabolism

“…ABHD6 is an ;30-kDa integral membrane serine hydrolase predicted to adopt an intracellular orientation (Blankman et al, 2007). Murine ABHD6 expression is abundant in brain and multiple peripheral tissues and cell types (Su et al, 2002;Bachovchin et al, 2010;Marrs et al, 2010). In the mouse brain, ABHD6 is highly expressed in cortical areas, where it preferentially localizes to postsynaptic dendrites that are often juxtaposed to presynaptic CB1 receptors (Marrs et al, 2010).…”

O-Hydroxyacetamide Carbamates as a Highly Potent and Selective Class of Endocannabinoid Hydrolase Inhibitors

“…CC-ABPP did not reveal any other major off-targets for SA-57yne with the exception of a faint 70 kDa protein in soluble mouse brain at doses of 6.25− 12.5 mg kg . This enzyme likely represents the carboxylesterase ES-1, a common off-target of carbamates 22,32,50,51 that originates from contaminating blood 52 in the brain proteome samples. Considering that ES-1 signals were not noticeably depleted by SA-57yne in our competitive ABPP experiments (Figure 7b), we conclude that this inhibitor only partially inactivates ES-1 at high doses.…”