Katerina Otrubova

The discovery and development of inhibitors of fatty acid amide hydrolase (FAAH).

A summary of the discovery and advancement of inhibitors of fatty acid amide hydrolase (FAAH) is presented.

Fatty acid amide signaling molecules.

Key studies leading to the discovery and definition of the role of endogenous fatty acid amide signaling molecules are summarized.

Comprehensive Study of Sansalvamide A Derivatives and their Structure–Activity Relationships against Drug-Resistant Colon Cancer Cell Lines

We report an extensive structure-activity relationship (SAR) of 62 compounds active against two drug-resistant colon cancer cell lines. Our comprehensive evaluation of two generations of compounds utilizes SAR, NMR, and molecular modeling to evaluate the key 3D features of potent compounds. Of the seven most potent compounds reported here, five are second-generation, emphasizing our ability to incorporate potent features found in the first generation and utilize their structures to design potency into the second generation. These analogs share no structural homology to current colon cancer drugs, are cytotoxic at levels on par with existing drugs treating other cancers, and demonstrate selectivity for drug-resistant colon cancer cell lines over noncancerous cell lines. Thus, we have established sansalvamide A as an excellent lead for treating multiple drug-resistant colon cancers.

Rational design of fatty acid amide hydrolase inhibitors that act by covalently bonding to two active site residues.

The design and characterization of α-ketoheterocycle fatty acid amide hydrolase (FAAH) inhibitors are disclosed that additionally and irreversibly target a cysteine (Cys269) found in the enzyme cytosolic port while maintaining the reversible covalent Ser241 attachment responsible for their rapid and initially reversible enzyme inhibition. Two α-ketooxazoles (3 and 4) containing strategically placed electrophiles at the C5 position of the pyridyl substituent of 2 (OL-135) were prepared and examined as inhibitors of FAAH. Consistent with the observed time-dependent noncompetitive inhibition, the cocrystal X-ray structure of 3 bound to a humanized variant of rat FAAH revealed that 3 was not only covalently bound to the active site catalytic nucleophile Ser241 as a deprotonated hemiketal, but also to Cys269 through the pyridyl C5-substituent, thus providing an inhibitor with dual covalent attachment in the enzyme active site. In vivo characterization of the prototypical inhibitors in mice demonstrates that they raise endogenous brain levels of FAAH substrates to a greater extent and for a much longer duration (>6 h) than the reversible inhibitor 2, indicating that the inhibitors accumulate and persist in the brain to completely inhibit FAAH for a prolonged period. Consistent with this behavior and the targeted irreversible enzyme inhibition, 3 reversed cold allodynia in the chronic constriction injury model of neuropathic pain in mice for a sustained period (>6 h) beyond that observed with the reversible inhibitor 2, providing effects that were unchanged over the 1-6 h time course monitored.

Design, synthesis, and characterization of α-ketoheterocycles that additionally target the cytosolic port Cys269 of fatty acid amide hydrolase.

A series of α-ketooxazoles incorporating electrophiles at the C5 position of the pyridyl ring of 2 (OL-135) and related compounds were prepared and examined as inhibitors of fatty acid amide hydrolase (FAAH) that additionally target the cytosolic port Cys269. From this series, a subset of the candidate inhibitors exhibited time-dependent FAAH inhibition and noncompetitive irreversible inactivation of the enzyme, consistent with the targeted Cys269 covalent alkylation or addition, and maintained or enhanced the intrinsic selectivity for FAAH versus other serine hydrolases. A preliminary in vivo assessment demonstrates that these inhibitors raise endogenous brain levels of anandamide and other FAAH substrates upon intraperitoneal (i.p.) administration to mice, with peak levels achieved within 1.5–3 h, and that the elevations of the signaling lipids were maintained >6 h, indicating that the inhibitors effectively reach and remain active in the brain, inhibiting FAAH for a sustained period.

Discovery libraries targeting the major enzyme classes: the serine hydrolases.

Two libraries of modestly reactive ureas containing either electron-deficient acyl anilines or acyl pyrazoles were prepared and are reported as screening libraries for candidate serine hydrolase inhibitors. Within each library is a small but powerful subset of compounds that serve as a chemotype fragment screening library capable of subsequent structural diversification. Elaboration of the pyrazole-based ureas provided remarkably potent irreversible inhibitors of fatty acid amide hydrolase (FAAH, apparent Ki=100-200 pM) complementary to those previously disclosed enlisting electron-deficient aniline-based ureas.

α-Ketoheterocycle inhibitors of fatty acid amide hydrolase: exploration of conformational constraints in the acyl side chain.

A series of α-ketooxazoles containing heteroatoms embedded within conformational constraints in the C2 acyl side chain of 2 (OL-135) were synthesized and evaluated as inhibitors of fatty acid amide hydrolase (FAAH). The studies reveal that the installation of a heteroatom (O) in the conformational constraint is achievable, although the potency of these novel derivatives is reduced slightly relative to 2 and the analogous 1,2,3,4-tetrahydronaphthalene series. Interestingly, both enantiomers (R and S) of the candidate inhibitors bearing a chiral center adjacent to the electrophilic carbonyl were found to effectively inhibit FAAH.