Vittorio Limongelli

Funnel metadynamics as accurate binding free-energy method

A detailed description of the events ruling ligand/protein interaction and an accurate estimation of the drug affinity to its target is of great help in speeding drug discovery strategies. We have developed a metadynamics-based approach, named funnel metadynamics, that allows the ligand to enhance the sampling of the target binding sites and its solvated states. This method leads to an efficient characterization of the binding free-energy surface and an accurate calculation of the absolute protein–ligand binding free energy. We illustrate our protocol in two systems, benzamidine/trypsin and SC-558/cyclooxygenase 2. In both cases, the X-ray conformation has been found as the lowest free-energy pose, and the computed protein–ligand binding free energy in good agreement with experiments. Furthermore, funnel metadynamics unveils important information about the binding process, such as the presence of alternative binding modes and the role of waters. The results achieved at an affordable computational cost make funnel metadynamics a valuable method for drug discovery and for dealing with a variety of problems in chemistry, physics, and material science.

Molecular basis of cyclooxygenase enzymes (COXs) selective inhibition

The widely used nonsteroidal anti-inflammatory drugs block the cyclooxygenase enzymes (COXs) and are clinically used for the treatment of inflammation, pain, and cancers. A selective inhibition of the different isoforms, particularly COX-2, is desirable, and consequently a deeper understanding of the molecular basis of selective inhibition is of great demand. Using an advanced computational technique we have simulated the full dissociation process of a highly potent and selective inhibitor, SC-558, in both COX-1 and COX-2. We have found a previously unreported alternative binding mode in COX-2 explaining the time-dependent inhibition exhibited by this class of inhibitors and consequently their long residence time inside this isoform. Our metadynamics-based approach allows us to illuminate the highly dynamical character of the ligand/protein recognition process, thus explaining a wealth of experimental data and paving the way to an innovative strategy for designing new COX inhibitors with tuned selectivity.

Kinetics of protein–ligand unbinding: Predicting pathways, rates, and rate-limiting steps

Significance A crucial factor for drug efficacy is not just the binding affinity, but also the mean residence time in the binding pocket, usually quantified by its inverse, koff. This is an important parameter that regulates the time during which the drug is active. Whereas the calculation of the binding affinity is by now routine, the calculation of koff has proven more challenging because the timescales involved far exceed the limits of standard molecular dynamics simulation. We propose a metadynamics-based strategy that allows reaching timescales of seconds, and estimate koff along with unbinding pathways and associated dynamical bottlenecks. The protocol is exemplified for trypsin–benzamidine unbinding. This work is a step towards a more effective computer-based drug design. The ability to predict the mechanisms and the associated rate constants of protein–ligand unbinding is of great practical importance in drug design. In this work we demonstrate how a recently introduced metadynamics-based approach allows exploration of the unbinding pathways, estimation of the rates, and determination of the rate-limiting steps in the paradigmatic case of the trypsin–benzamidine system. Protein, ligand, and solvent are described with full atomic resolution. Using metadynamics, multiple unbinding trajectories that start with the ligand in the crystallographic binding pose and end with the ligand in the fully solvated state are generated. The unbinding rate koff is computed from the mean residence time of the ligand. Using our previously computed binding affinity we also obtain the binding rate kon. Both rates are in agreement with reported experimental values. We uncover the complex pathways of unbinding trajectories and describe the critical rate-limiting steps with unprecedented detail. Our findings illuminate the role played by the coupling between subtle protein backbone fluctuations and the solvation by water molecules that enter the binding pocket and assist in the breaking of the shielded hydrogen bonds. We expect our approach to be useful in calculating rates for general protein–ligand systems and a valid support for drug design.

Sampling protein motion and solvent effect during ligand binding

An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one.

Design, synthesis, and biological evaluation of novel aminobisphosphonates possessing an in vivo antitumor activity through a gammadelta-T lymphocytes-mediated activation mechanism.

A small series of aminobisphosphonates (N-BPs) structurally related to zoledronic acid was synthesized with the aim of improving activity toward activation of human gammadelta T cells and in turn their in vivo antitumor activity. The absence of the 1-OH moiety, together with the position and the different basicity of the nitrogen, appears crucial for antitumor activity. In comparison to zoledronic acid, compound 6a shows a greater ability to activate gammadelta T cells expression (100 times more) and a proapoptotic effect that is better than zoledronic acid. The potent activation of gammadelta T cells, in addition to evidence of the in vivo antitumor activity of 6a, suggests it may be a new potential drug candidate for cancer treatment.

Investigating the Mechanism of Substrate Uptake and Release in the Glutamate Transporter Homologue GltPh through Metadynamics Simulations

A homeostatic concentration of glutamate in the synaptic cleft ensures a correct signal transduction along the neuronal network. An unbalance in this concentration can lead to neuronal death and to severe neurodegenerative diseases such as Alzheimers or Parkinsons. Glutamate transporters play a crucial role in this respect because they are responsible for the reuptake of the neurotransmitter from the synaptic cleft, thus controlling the glutamate concentration. Understanding the molecular mechanism of this transporter can provide the possibility of an exogenous control. Structural studies have shown that this transporter can assume at least three conformations, thus suggesting a pronounced dynamical behavior. However, some intermediate states that lead to the substrate internalization have not been characterized and many aspects of the transporter mechanism still remain unclear. Here, using metadynamics simulations, we investigate the substrate uptake from the synaptic cleft and its release in the intracellular medium. In addition, we focus on the role of ions and substrate during these processes and on the stability of the different conformations assumed by the transporter. The present dynamical results can complement available X-ray data and provide a thorough description of the entire process of substrate uptake, internalization, and release.

Design, Synthesis and Biological Evaluation of Carboxy Analogues of Arginine Methyltransferase Inhibitor 1 (AMI-1)

Here we report the synthesis of a number of compounds structurally related to arginine methyltransferase inhibitor 1 (AMI‐1). The structural alterations that we made included: 1) the substitution of the sulfonic groups with the bioisosteric carboxylic groups; 2) the replacement of the ureidic function with a bis‐amidic moiety; 3) the introduction of a N‐containing basic moiety; and 4) the positional isomerization of the aminohydroxynaphthoic moiety. We have assessed the biological activity of these compounds against a panel of arginine methyltransferases (fungal RmtA, hPRMT1, hCARM1, hPRMT3, hPRMT6) and a lysine methyltransferase (SET7/9) using histone and nonhistone proteins as substrates. Molecular modeling studies for a deep binding‐mode analysis of test compounds were also performed. The bis‐carboxylic acid derivatives 1 b and 7 b emerged as the most effective PRMT inhibitors, both in vitro and in vivo, being comparable or even better than the reference compound (AMI‐1) and practically inactive against the lysine methyltransferase SET7/9.

Modeling of Cdc25B dual specifity protein phosphatase inhibitors: docking of ligands and enzymatic inhibition mechanism.

The Cdc25 dual specificity phosphatases have central roles in coordinating cellular signalling processes and cell proliferation. It has been reported that an improper amplification or activation of these enzymes is a distinctive feature of a number of human cancers, including breast cancers. Thus, the inhibition of Cdc25 phosphatases might provide a novel approach for the discovery of new and selective antitumor agents. By using the crystal structure of the catalytic domain of Cdc25B, structural models for the interaction of various Cdc25B inhibitors (1–13) with the enzyme were generated by computational docking. The parallel use of two efficient and predictive docking programs, AutoDock and GOLD, allowed mutual validation of the predicted binding poses. To evaluate their quality, the models were validated with known structure–activity relationships and site‐directed mutagenesis data. The results provide an improved basis for structure‐based ligand design and suggest a possible explanation for the inhibition mechanism of the examined Cdc25B ligands. We suggest that the recurring motif of a tight interaction between the inhibitor and the two arginine residues, 482 and 544, is of prime importance for reversible enzyme inhibition. In contrast, the irreversible inhibition mechanism of 1–4 seems to be associated with the close vicinity of the quinone ring and the Cys473 catalytic thiolate. We believe that this extensive study might provide useful hints to guide the development of new potent Cdc25B inhibitors as novel anticancer drugs.

Mechanistic insight into ligand binding to G-quadruplex DNA

Specific guanine-rich regions in human genome can form higher-order DNA structures called G-quadruplexes, which regulate many relevant biological processes. For instance, the formation of G-quadruplex at telomeres can alter cellular functions, inducing apoptosis. Thus, developing small molecules that are able to bind and stabilize the telomeric G-quadruplexes represents an attractive strategy for antitumor therapy. An example is 3-(benzo[d]thiazol-2-yl)-7-hydroxy-8-((4-(2-hydroxyethyl)piperazin-1-yl)methyl)-2H-chromen-2-one (compound 1), recently identified as potent ligand of the G-quadruplex [d(TGGGGT)]4 with promising in vitro antitumor activity. The experimental observations are suggestive of a complex binding mechanism that, despite efforts, has defied full characterization. Here, we provide through metadynamics simulations a comprehensive understanding of the binding mechanism of 1 to the G-quadruplex [d(TGGGGT)]4. In our calculations, the ligand explores all the available binding sites on the DNA structure and the free-energy landscape of the whole binding process is computed. We have thus disclosed a peculiar hopping binding mechanism whereas 1 is able to bind both to the groove and to the 3’ end of the G-quadruplex. Our results fully explain the available experimental data, rendering our approach of great value for further ligand/DNA studies.

Acetic Acid Aldose Reductase Inhibitors Bearing a Five-Membered Heterocyclic Core with Potent Topical Activity in a Visual Impairment Rat Model

A number of 1,2,4-oxadiazol-5-yl-acetic acids and oxazol-4-yl-acetic acids were synthesized and tested for their ability to inhibit aldose reductase (ALR2). The oxadiazole derivatives, 7c, 7f, 7i, and 8h, 8i, proved to be the most active compounds, exhibiting inhibitory levels in the submicromolar range. In this series, the phenyl group turned out to be the preferred substitution pattern, as its lengthening to a benzyl moiety determined a general reduction of the inhibitory potency. The lead compound, 2-[3-(4-methoxyphenyl)-1,2,4-oxadiazol-5-yl]acetic acid, 7c, showed an excellent in vivo activity, proving to prevent cataract development in severely galactosemic rats when administered as an eye-drop solution in the precorneal region of the animals. Computational studies on the ALR2 inhibitors were performed to rationalize the structure-activity relationships observed and to provide the basis for further structure-guided design of novel ALR2 inhibitors.