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Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkages that form the backbone of the RNA strand in RNA x DNA hybrids. This enzyme is implicated in replication initiation and DNA topology restoration and represents a very promising target for anti-HIV drug design. Structural information has been provided by high-resolution crystal structures of the complex RNase H/RNA x DNA from Bacillus halodurans (Bh), which reveals that two metal ions are required for formation of a catalytic active complex. Here, we use classical force field-based and quantum mechanics/molecular mechanics calculations for modeling the nucleotidyl transfer reaction in RNase H, clarifying the role of the metal ions and the nature of the nucleophile (water versus hydroxide ion). During the catalysis, the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both transition state and leaving group. Importantly, the two Mg(2+) metals also support the formation of a meta-stable phosphorane intermediate along the reaction, which resembles the phosphorane intermediate structure obtained only in the debated beta-phosphoglucomutase crystal (Lahiri, S. D.; et al. Science 2003, 299 (5615), 2067-2071). The nucleophile formation (i.e., water deprotonation) can be achieved in situ, after migration of one proton from the water to the scissile phosphate in the transition state. This proton transfer is actually mediated by solvation water molecules. Due to the highly conserved nature of the enzymatic bimetal motif, these results might also be relevant for structurally similar enzymes belonging to the NT superfamily.

Catalytic Metal Ions and Enzymatic Processing of DNA and RNA

CONSPECTUS: Two-metal-ion-dependent nucleases cleave the phosphodiester bonds of nucleic acids via the two-metal-ion (2M) mechanism. Several high-resolution X-ray structures portraying the two-metal-aided catalytic site, together with mutagenesis and kinetics studies, have demonstrated a functional role of the ions for catalysis in numerous metallonucleases. Overall, the experimental data confirm the general mechanistic hypothesis for 2M-aided phosphoryl transfer originally reported by Steitz and Steitz ( Proc. Natl. Acad. Sci. U.S.A. 1993 , 90 ( 14 ), 6498 - 6502 ). This seminal paper proposed that one metal ion favors the formation of the nucleophile, while the nearby second metal ion facilitates leaving group departure during RNA hydrolysis. Both metals were suggested to stabilize the enzymatic transition state. Nevertheless, static X-ray structures alone cannot exhaustively unravel how the two ions execute their functional role along the enzymatic reaction during processing of DNA or RNA strands when moving from reactants to products, passing through metastable intermediates and high-energy transition states. In this Account, we discuss the role of multiscale molecular simulations in further disclosing mechanistic insights of 2M-aided catalysis for two prototypical enzymatic targets for drug discovery, namely, ribonuclease H (RNase H) and type II topoisomerase (topoII). In both examples, first-principles molecular simulations, integrated with structural data, emphasize a cooperative motion of the bimetal motif during catalysis. The coordinated motion of both ions is crucial for maintaining a flexible metal-centered structural architecture exquisitely tailored to accommodate the DNA or RNA sugar-phosphate backbone during phosphodiester bond cleavage. Furthermore, our analysis of RNase H and the N-terminal domain (PAN) of influenza polymerase shows that classical molecular dynamics simulations coupled with enhanced sampling techniques have contributed to describe the modulatory effect of metal ion concentration and metal uptake on the 2M mechanism and efficiency. These aspects all point to the emerging and intriguing role of additional adjacent ions potentially involved in the modulation of phosphoryl transfer reactions and enzymatic turnover in 2M-catalysis, as recently observed experimentally in polymerase η and homing endonuclease I-DmoI. These computational results, integrated with experimental findings, describe and reinforce the nascent concept of a functional and cooperative dynamics of the catalytic metal ions during the 2M-dependent enzymatic processing of DNA and RNA. Encouraged by the insights provided by computational approaches, we foresee further experiments that will feature the functional and joint dynamics of the catalytic metal ions for nucleic acid processing. This could impact the de novo design of artificial metallonucleases and the rational design of potent metal-chelating inhibitors of pharmaceutically relevant enzymes.

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.

Identification and Characterization of Carprofen as a Multitarget Fatty Acid Amide Hydrolase/Cyclooxygenase Inhibitor

Pain and inflammation are major therapeutic areas for drug discovery. Current drugs for these pathologies have limited efficacy, however, and often cause a number of unwanted side effects. In the present study, we identify the nonsteroidal anti-inflammatory drug carprofen as a multitarget-directed ligand that simultaneously inhibits cyclooxygenase-1 (COX-1), COX-2, and fatty acid amide hydrolase (FAAH). Additionally, we synthesized and tested several derivatives of carprofen, sharing this multitarget activity. This may result in improved analgesic efficacy and reduced side effects (Naidu et al. J. Pharmacol. Exp. Ther.2009, 329, 48-56; Fowler, C. J.; et al. J. Enzyme Inhib. Med. Chem.2012, in press; Sasso et al. Pharmacol. Res.2012, 65, 553). The new compounds are among the most potent multitarget FAAH/COX inhibitors reported so far in the literature and thus may represent promising starting points for the discovery of new analgesic and anti-inflammatory drugs.

Understanding the Effect of Magnesium Ion Concentration on the Catalytic Activity of Ribonuclease H through Computation: Does a Third Metal Binding Site Modulate Endonuclease Catalysis?

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase superfamily and hydrolyzes the phosphodiester linkage on the RNA strand of a DNA/RNA hybrid duplex. Due to its activity in HIV reverse transcription, it represents a promising target for anti-HIV drug design. While crystallographic data have located two ions in the catalytic site, there is ongoing debate concerning just how many metal ions bound at the active site are optimal for catalysis. Indeed, experiments have shown a dependency of the catalytic activity on the Mg(2+) concentration. Moreover, in RNase H, the glutamate residue E188 has been shown to be essential for full enzymatic activation, regardless of the Mg(2+) concentration. The catalytic center is known to contain two Mg(2+) ions, and E188 is not one of the primary metal ligands. Herein, classical molecular dynamics (MD) simulations are employed to study the metal-ligand coordination in RNase H at different concentration of Mg(2+). Importantly, the presence of a third Mg(2+) ion, bound to the second-shell ligand E188, is a persistent feature of the MD simulations. Free energy calculations have identified two distinct conformations, depending on the concentration of Mg(2+). At standard concentration, a third Mg(2+) is found in the catalytic pocket, but it does not perturb the optimal RNase H active conformation. However, at higher concentration, the third Mg(2+) ion heavily perturbs the nucleophilic water and thereby influences the catalytic efficiency of RNase H. In addition, the E188A mutant shows no ability to engage additional Mg(2+) ions near the catalytic pocket. This finding likely explains the decrease in catalytic activity of E188A and also supports the key role of E188 in localizing the third Mg(2+) ion at the active site. Glutamate residues are commonly found surrounding the metal center in the endonuclease family, which suggests that this structural motif may be an important feature to enhance catalytic activity. The present MD calculations support the hypothesis that RNase H can accommodate three divalent metal ions in its catalytic pocket and provide an in-depth understanding of their dynamic role for catalysis.

Molecular Simulations Highlight the Role of Metals in Catalysis and Inhibition of Type II Topoisomerase

Type II topoisomerase (topoII) is a metalloenzyme targeted by clinical antibiotics and anticancer agents. Here, we integrate existing structural data with molecular simulation and propose a model for the yet uncharacterized structure of the reactant state of topoII. This model describes a canonical two-metal-ion mechanism and suggests how the metals could rearrange at the catalytic pocket during enzymatic turnover, explaining also experimental evidence for topoII inhibition. These results call for further experimental validation.

A Binding Site for Nonsteroidal Anti-inflammatory Drugs in Fatty Acid Amide Hydrolase.

In addition to inhibiting the cyclooxygenase (COX)-mediated biosynthesis of prostanoids, various widely used nonsteroidal anti-inflammatory drugs (NSAIDs) enhance endocannabinoid signaling by blocking the anandamide-degrading membrane enzyme fatty acid amide hydrolase (FAAH). The X-ray structure of FAAH in complex with the NSAID carprofen, along with site-directed mutagenesis, enzyme activity assays, and NMR analysis, has revealed the molecular details of this interaction, providing information that may guide the design of dual FAAH-COX inhibitors with superior analgesic efficacy.

The increasing role of QM/MM in drug discovery.

Since its first appearance in 1976, the quantum mechanics/molecular mechanics (QM/MM) approach has mostly been used to study the chemical reactions of enzymes, which are frequently the target of drug discovery programs. In principle, a detailed understanding of the enzymatic mechanism should help researchers to design a potent enzyme inhibitor or new drug. However, QM/MM has not yet had a widespread impact on structure-based drug design. This is mostly due to its high computational cost. We expect this to change with the recent and extraordinary increases in computational power, and with the availability of more efficient algorithms for QM/MM calculations. Here, we report on some representative examples of QM/MM studies, including our own research, of pharmaceutically relevant enzymes, such as ribonuclease H and fatty acid amide hydrolase (FAAH). We aim to show how QM/MM has traditionally been used to study enzymatic catalysis. In this regard, we discuss its potential to become a routinely used drug design tool. To support this, we also discuss selected computational studies where QM/MM insights have been helpful in improving the potency of covalent inhibitors of FAAH.

Covalent Inhibitors of Fatty Acid Amide Hydrolase: A Rationale for the Activity of Piperidine and Piperazine Aryl Ureas

Recently, covalent drugs have attracted great interest in the drug discovery community, with successful examples that have demonstrated their therapeutic effects. Here, we focus on the covalent inhibition of the fatty acid amide hydrolase (FAAH), which is a promising strategy in the treatment of pain and inflammation. Among the most recent and potent FAAH inhibitors (FAAHi), there are the cyclic piperidine and piperazine aryl ureas. FAAH hydrolyzes efficiently the amide bond of these compounds, forming a covalent enzyme-inhibitor adduct. To rationalize this experimental evidence, we performed an extensive computational analysis centered on piperidine-based PF750 (1) and piperazine-based JNJ1661010 (2), two potent lead compounds used to generate covalent inhibitors as clinical candidates. We found that FAAH induces a distortion of the amide bond of the piperidine and piperazine aryl ureas. Quantum mechanics/molecular mechanics ΔE(LUMO-HOMO) energies indicate that the observed enzyme-induced distortion of the amide bond favors the formation of a covalent FAAH-inhibitor adduct. These findings could help in the rational structure-based design of novel covalent FAAHi.

A theoretical DFT investigation of the lysozyme mechanism: Computational evidence for a covalent intermediate pathway

A theoretical DFT(B3LYP) investigation of the catalytic cycle of lysozyme has provided further evidence for a mechanism involving a glycosil‐enzyme covalent intermediate, in agreement with recent experimental data. This type of intermediate has been located along two different pathways. Along the favored path the retention of the anomeric configuration of the peptidoglycan NAM unit involved in the reaction, is the result of two subsequent inversions at the C1 carbon. The other path involves the opening of the pyranose ring and a nucleophilic attack on the prochiral carbonyl group of the open aldehyde, restoring the original anomeric configuration. No evidence has been found for a pathway characterized by the formation of an oxocarbenium ion (stabilized by resonance and electrostatic interactions), as suggested in the most popular mechanistic schemes. Proteins 2005.