Team hopes easily modified compounds will provide new insights into function of serine hydrolase enzymes.

Scientists have developed a class of compounds they claim can be easily modified to selectively inhibit a substantial number of serine hydrolases. The Scripps Research Institute team based the work on their own and independent research suggesting that tetrazole urea and other N-heterocyclic ureas inhibited numerous serine hydrolases. Homing in on 1,2,3-triazole ureas from this starting point, the team then tweaked the basic triazole urea scaffold using a two-step click chemistry technique developed at Scripps, to generate individual compounds that blocked specific serine hydrolases both in vitro and in vivo. The research is described in Nature Chemical Biology, in a paper titled “Click-generated triazole ureas as ultrapotent in vivo–active serine hydrolase inhibitors.”

Serine hydrolases are one of the largest and most diverse enzyme classes in eukaryotic and prokaryotic proteomes, representing about 1% of all human proteins, reports Benjamin F. Cravatt III, Ph.D., and colleagues. Drugs that target specific serine hydrolase enzymes have already been developed for disease including obesity, diabetes, microbial infections, and Alzheimer disease. However, a lack of specific inhibitors for the vast majority of the 200 or so serine hydrolases identified means that, to date, the biological functions of most of these enzymes remain poorly characterized.  

Starting with the triazole urea scaffold, Dr. Cravatt’s team used a two-step synthetic strategy based on click chemistry, coupled with a quantitative platform for competitive activity-based protein profiling (ABPP), to generate selective inhibitors for individ­ual enzymes from diverse branches of the serine hydrolase class, including peptidases (APEH), lipases (PAFAH2), and uncharac­terized enzymes (ABHD11). Selective activity of the inhibitors was verified in cells in vitro, and one inhibitor, designated AA74-1, was tested in vivo and found to block the activity of APEH in mice, without impacting on other enzymes.

Although APEH has for many years been postulated to serve as a key regulator of N-terminally acetylated proteins, “very few endogenous substrates have been identified for APEH, nor have the biological effects of disrupting this enzyme been examined,” the researchers note. Surprisingly, studies in mice with the newly identified inhibitor AA74-1 found that dozens of proteins were affected by blocking APEH. Considering the majority of the proteins affected are known to exist as N-terminally acetylated molecules, the findings suggest that APEH plays a truly constitutive role in regulating stability of this modification, they claim.

The finding was “unexpected and unusual,” professor Cravatt states. “But it’s what one wants to see with these compounds – strong enzyme-inhibiting activity in different tissues, at a low dose. And it’s the first time this kind of evaluation has been done in both cultured cells and animal tissues.”

Interestingly, proteomic changes caused by AA74-1 were also accompanied by increased T-cell proliferation. The Scripps team says that although they don’t yet understand the mechanism underlying the pro-proliferative effect of APEH inhibition, some of the identified substrates for the enzyme have been suggested to promote cellular proliferation, and it’s possible that changes in N-acetylation may affect their biological activity. Moreover, they add, the APEH gene is known to be deleted in certain cancers and has been proposed to serve as a potential tumor suppressor.

The team concludes the combined findings of their research “bolster our confidence that the 1,2,3-triazole urea will serve as a highly versatile scaffold for the future development of potent and selective inhibitors for the numerous serine hydrolases that populate eukaryotic and prokary­otic proteomes.  The strong activity shown by 1,2,3-triazole ureas in cells and animals suggests that in vitro potency should translate into in vivo activity for most compounds of this class.”

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