Structure-based drug design of molecules influencing fatty acid derivatives signal pathways

Abstract

For two decades anandamide (AEA) has been known to be an endogenous agonist for the cannabinoids receptors CB1 and CB2. It is synthesized on demand from the membrane lipid precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) by a phospholipase D and it diffuses to its targets, mediating a variety of biological effects. Fatty acid amide hydrolase (FAAH) terminates the signal brought by the AEA, catalyzing its hydrolysis to arachidonic acid and ethanolamine. FAAH is also involved in the degradation of other fatty acid ethanolamides (FAEs), including the anti-inflammatory agent palmitoylethanolamide (PEA) and the satiety factor oleylethanolamide (OEA). Recent investigations on FAAH have indicated that this enzyme is a potential target for the treatment of chronic pain, inflammation, immunological diseases, psychiatric conditions, metabolic and cardiovascular diseases. Furthermore, it has been demonstrated that the inhibition of FAAH leads to an augmentation of FAE endogenous levels in animal model. Notably, the corresponding increment of FAE-dependent neurosignals induces therapeutic effects avoiding the typical signs of cannabinoid intoxication e.g. hypothermia, and hypomotility. FAAH is an integral-membrane serine hydrolase belonging to the amidase signature (AS) family. The mechanism of hydrolysis catalyzed by FAAH is widely accepted, with Lys142 serving as key acid and base in distinct steps of the catalytic cycle. As a base, Lys142 activates the Ser241 nucleophile for attack on the substrate amide carbonyl; as an acid, Lys142 protonates the substrate leaving group leading to its expulsion. The effect of Lys142 on Ser241 is mediated indirectly by Ser217, which acts as proton shuttle. Cyclohexyl carbamic acid biphenyl-3yl esters were developed at Universities of Parma, Urbino and Irvine and represent the first class of covalent FAAH inhibitors effective in animal models. This class of compounds, exemplified by URB597, inhibits FAAH by carbamoylating the nucleophile Ser241. FAAH inhibition by URB597 induces anxiolityc and antidepressant-like effects as well as reduction of inflammation in animal models. In an effort to discover drug candidates with limited toxicity, pharmaceutical companies have predominantly developed compounds that act through non-covalent interactions with their biological targets. Alternatively, there are instances where controlled, target-specific covalent modifications have proven useful: a variety of examples of drugs that act covalently can be found (i.e. Aspirin, penicillins, proton pump inhibitors). In general, the potency of a covalent inhibitor is influenced by three main properties: i) the "non covalent" affinity for the target; ii) the intrinsic reactivity of the compound; iii) the stability of the covalent adduct (i.e. the reversibility of the covalent interaction). Through a careful modulation of these properties is possible to obtain compounds not only potent in vivo, but also endowed with a limited ability to interact with off-targets. For the class of cyclohexylcarbamic acid biphenyl-3-yl esters, a recent structure-activity relationship (SAR) investigation has demonstrated that is possible to modulate the reactivity of the inhibitor preserving the binding affinity for the target. In particular, this study has shown that conjugated electron-donor groups on the biphenyl scaffold (which increase electron density around the carbamate carbon) reduce the interaction with plasma and liver carboxylesterases without affecting FAAH inhibitor potency in vitro. The overall effect of this chemical modification is thus an enhanced in vivo potency and a reduced inhibition of off-targets. The time-dependency is another tunable aspect of the enzyme-inhibition, that controls in vivo potency, efficacy, and pharmacokinetic properties. While the reference inhibitor URB597, inhibits FAAH through an irreversible modification of Ser241, FAAH inhibitors belonging to the piperazinyl urea class, i.e. JNJ1661010, carbamoylate Ser241 in reversible-manner. With this in mind, the first aim of this thesis is to study the mechanism of inhibition of FAAH by cyclohexyl carbamic acid biphenyl-3yl esters to discover the chemical features that determine the biological properties of this class of compound. The obtained knowledge could be exploited to design more potent compounds characterized by improved pharmacokinetic and safety profiles. Secondarily, a validated QM/MM approach will be applied to model the carbamoylation reaction of FAAH in presence of the reference inhibitor URB524 and by its p-OH and p-NH2 derivatives to rationalized why this modification does not affect the potency on FAAH in vitro. Furthermore, the decarbamoylation reaction of FAAH in presence of i) the irreversible carbamate inhibitor (URB597), ii) the prototypical reversible urea (JNJ1661010) and iii) the oleoylamide substrate will be modeled by applying the same QM/MM approach to identify the chemical determinant responsible for the irreversibility/reversibility of the inhibition.