Molecular dynamics (MD) and related methods are close to becoming routine computational tools for drug discovery. Their main advantage is in explicitly treating structural flexibility and entropic effects. This allows a more accurate estimate of the thermodynamics and kinetics associated with drug-target recognition and binding, as better algorithms and hardware architectures increase their use. Here, we review the theoretical background of MD and enhanced sampling methods, focusing on free-energy perturbation, metadynamics, steered MD, and other methods most consistently used to study drug-target binding. We discuss unbiased MD simulations that nowadays allow the observation of unsupervised ligand-target binding, assessing how these approaches help optimizing target affinity and drug residence time toward improved drug efficacy. Further issues discussed include allosteric modulation and the role of water molecules in ligand binding and optimization. We conclude by calling for more prospective studies to attest to these methods' utility in discovering novel drug candidates.

The metadynamics or hills method is a relatively new molecular dynamics technique aimed to enhance the sampling of separated regions in phase space and map out the underlying free energy landscape as a function of a small number of order parameters or collective variables. The high efficiency allows for the application of metadynamics in combination with first principles dynamics methods, in particular with Car-Parrinello molecular dynamics, to study processes in which changes in the electronic structure play a dominant role, such as chemical reactions. The option to choose several independent collective variables is important to tackle complex and concerted transformations that lack an obvious a priori choice for a single reaction coordinate. In this Account, we discuss the role of metadynamics in the search of transition states, local minima, reaction paths, free energy profiles, and reaction coordinates among a growing list of alternative methods.

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·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 highresolution crystal structures of the complex RNase H/RNA·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 β-phosphoglucomutase crystal. 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.

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.

No abstract

The enzymatic polymerization of DNA and RNA is the basis for genetic inheritance for all living organisms. It is catalyzed by the DNA/RNA polymerase (Pol) superfamily. Here, bioinformatics analysis reveals that the incoming nucleotide substrate always forms an H-bond between its 3'-OH and β-phosphate moieties upon formation of the Michaelis complex. This previously unrecognized H-bond implies a novel self-activated mechanism (SAM), which synergistically connects the in situ nucleophile formation with subsequent nucleotide addition and, importantly, nucleic acid translocation. Thus, SAM allows an elegant and efficient closed-loop sequence of chemical and physical steps for Pol catalysis. This is markedly different from previous mechanistic hypotheses. Our proposed mechanism is corroborated via ab initio QM/MM simulations on a specific Pol, the human DNA polymerase-η, an enzyme involved in repairing damaged DNA. The structural conservation of DNA and RNA Pols supports the possible extension of SAM to Pol enzymes from the three domains of life.

Summary Aberrant expression ratio of Cl − transporters, NKCC1 and KCC2, is implicated in several brain conditions. NKCC1 inhibition by the FDA-approved diuretic drug, bumetanide, rescues core symptoms in rodent models and/or clinical trials with patients. However, bumetanide has a strong diuretic effect due to inhibition of the kidney Cl − transporter NKCC2, creating critical drug compliance issues and health concerns. Here, we report the discovery of a new chemical class of selective NKCC1 inhibitors and the lead drug candidate ARN23746. ARN23746 restores the physiological intracellular Cl − in murine Down syndrome neuronal cultures, has excellent solubility and metabolic stability, and displays no issues with off-target activity in vitro . ARN23746 recovers core symptoms in mouse models of Down syndrome and autism, with no diuretic effect, nor overt toxicity upon chronic treatment in adulthood. ARN23746 is ready for advanced preclinical/manufacturing studies toward the first sustainable therapeutics for the neurological conditions characterized by impaired Cl − homeostasis.

Trans-lesion synthesis polymerases, like DNA Polymerase-η (Pol-η), are essential for cell survival. Pol-η bypasses ultraviolet-induced DNA damages via a two-metal-ion mechanism that assures DNA strand elongation, with formation of the leaving group pyrophosphate (PPi). Recent structural and kinetics studies have shown that Pol-η function depends on the highly flexible and conserved Arg61 and, intriguingly, on a transient third ion resolved at the catalytic site, as lately observed in other nucleic acid-processing metalloenzymes. How these conserved structural features facilitate DNA replication, however, is still poorly understood. Through extended molecular dynamics and free energy simulations, we unravel a highly cooperative and dynamic mechanism for DNA elongation and repair, which is here described by an equilibrium ensemble of structures that connect the reactants to the products in Pol-η catalysis. We reveal that specific conformations of Arg61 help facilitate the recruitment of the incoming base and favor the proper formation of a pre-reactive complex in Pol-η for efficient DNA editing. Also, we show that a third transient metal ion, which acts concertedly with Arg61, serves as an exit shuttle for the leaving PPi. Finally, we discuss how this effective and cooperative mechanism for DNA repair may be shared by other DNA-repairing polymerases.