eTOX ALLIES:an automated pipeLine for linear interaction energy-based simulations

Abstract Background Computational methods to predict binding affinities of small ligands toward relevant biological (off-)targets are helpful in prioritizing the screening and synthesis of new drug candidates, thereby speeding up the drug discovery process. However, use of ligand-based approaches can lead to erroneous predictions when structural and dynamic features of the target substantially affect ligand binding. Free energy methods for affinity computation can include steric and electrostatic protein–ligand interactions, solvent effects, and thermal fluctuations, but often they are computationally demanding and require a high level of supervision. As a result their application is typically limited to the screening of small sets of compounds by experts in molecular modeling. Results We have developed eTOX ALLIES, an open source framework that allows the automated prediction of ligand-binding free energies requiring the ligand structure as only input. eTOX ALLIES is based on the linear interaction energy approach, an efficient end-point free energy method derived from Free Energy Perturbation theory. Upon submission of a ligand or dataset of compounds, the tool performs the multiple steps required for binding free-energy prediction (docking, ligand topology creation, molecular dynamics simulations, data analysis), making use of external open source software where necessary. Moreover, functionalities are also available to enable and assist the creation and calibration of new models. In addition, a web graphical user interface has been developed to allow use of free-energy based models to users that are not an expert in molecular modeling. Conclusions Because of the user-friendliness, efficiency and free-software licensing, eTOX ALLIES represents a novel extension of the toolbox for computational chemists, pharmaceutical scientists and toxicologists, who are interested in fast affinity predictions of small molecules toward biological (off-)targets for which protein flexibility, solvent and binding site interactions directly affect the strength of ligand-protein binding

A Catalytic Mechanism for Cysteine N-Terminal Nucleophile Hydrolases, as Revealed by Free Energy Simulations

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors

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

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

Quantum mechanics/molecular mechanics modeling of covalent addition between EGFR-cysteine 797 and N -(4-Anilinoquinazolin-6-yl) acrylamide

Irreversible epidermal growth factor receptor (EGFR) inhibitors can circumvent resistance to first-generation ATP-competitive inhibitors in the treatment of nonsmall-cell lung cancer. They covalently bind a noncatalytic cysteine (Cys797) at the surface of EGFR active site by an acrylamide warhead. Herein, we used a hybrid quantum mechanics/molecular mechanics (QM/MM) potential in combination with umbrella sampling in the path-collective variable space to investigate the mechanism of alkylation of Cys797 by the prototypical covalent inhibitor N-(4-anilinoquinazolin-6-yl) acrylamide. Calculations show that Cys797 reacts with the acrylamide group of the inhibitor through a direct addition mechanism, with Asp800 acting as a general base/general acid in distinct steps of the reaction. The obtained reaction free energy is negative (Delta A = -12 kcal/mol) consistent with the spontaneous and irreversible alkylation of Cys797 by N-(4-anilinoquinazolin-6-yl) acrylamide. Our calculations identify desolvation of Cys797 thiolate anion as a key step of the alkylation process, indicating that changes in the intrinsic reactivity of the acrylamide would have only a minor impact on the inhibitor potency

Quantum mechanics/molecular mechanics modeling of fatty acid amide hydrolase reactivation distinguishes substrate from irreversible covalent inhibitors.

Carbamate and urea derivatives are important classes of fatty acid amide hydrolase (FAAH) inhibitors that carbamoylate the active-site nucleophile Ser241. In the present work, the reactivation mechanism of carbamoylated FAAH is investigated by means of a quantum mechanics/molecular mechanics (QM/MM) approach. The potential energy surfaces for decarbamoylation of FAAH covalent adducts, derived from the O-aryl carbamate URB597 and from the N-piperazinylurea JNJ1661610, were calculated and compared to that for deacylation of FAAH acylated by the substrate oleamide. Calculations show that a carbamic group bound to Ser241 prevents efficient stabilization of transition states of hydrolysis, leading to large increments in the activation barrier. Moreover, the energy barrier for the piperazine carboxylate was significantly lower than that for the cyclohexyl carbamate derived from URB597. This is consistent with experimental data showing slowly reversible FAAH inhibition for the N-piperazinylurea inhibitor and irreversible inhibition for URB597

Quantum Mechanics/Molecular Mechanics Modeling of Covalent Addition between EGFR–Cysteine 797 and <i>N</i>‑(4-Anilinoquinazolin-6-yl) Acrylamide

Irreversible epidermal growth factor receptor (EGFR) inhibitors can circumvent resistance to first-generation ATP-competitive inhibitors in the treatment of nonsmall-cell lung cancer. They covalently bind a noncatalytic cysteine (Cys797) at the surface of EGFR active site by an acrylamide warhead. Herein, we used a hybrid quantum mechanics/molecular mechanics (QM/MM) potential in combination with umbrella sampling in the path-collective variable space to investigate the mechanism of alkylation of Cys797 by the prototypical covalent inhibitor <i>N</i>-(4-anilinoquinazolin-6-yl) acrylamide. Calculations show that Cys797 reacts with the acrylamide group of the inhibitor through a direct addition mechanism, with Asp800 acting as a general base/general acid in distinct steps of the reaction. The obtained reaction free energy is negative (Δ<i>A</i> = −12 kcal/mol) consistent with the spontaneous and irreversible alkylation of Cys797 by <i>N</i>-(4-anilinoquinazolin-6-yl) acrylamide. Our calculations identify desolvation of Cys797 thiolate anion as a key step of the alkylation process, indicating that changes in the intrinsic reactivity of the acrylamide would have only a minor impact on the inhibitor potency

A Catalytic Mechanism for Cysteine N-Terminal Nucleophile Hydrolases, as Revealed by Free Energy Simulations

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors

Quantum mechanics/molecular mechanics modeling of fatty acid amide hydrolase reactivation distinguishes substrate from irreversible covalent inhibitors

Carbamate and urea derivatives are important classes of fatty acid amide hydrolase (FAAH) inhibitors that carbamoylate the active-site nucleophile Ser241. In the present work, the reactivation mechanism of carbamoylated FAAH is investigated by means of a quantum mechanics/molecular mechanics (QM/MM) approach. The potential energy surfaces for decarbamoylation of FAAH covalent adducts, deriving from the O-aryl carbamate URB597 and from the N-piperazinylurea JNJ1661610, were calculated and compared to that for deacylation of FAAH acylated by the substrate oleamide. Calculations show that a carbamic group bound to Ser241 prevents efficient stabilization of transition states of hydrolysis, leading to large increments in the activation barrier. Moreover, the energy barrier for the piperazine carboxylate was significantly lower than that for the ciclohexyl carbamate derived from URB597. This is consistent with experimental data showing slowly reversible FAAH inhibition for the N-piperazinylurea inhibitor and irreversible inhibition for URB597