António J. M. Ribeiro

Protein-Ligand Docking in the New Millennium – A Retrospective of 10 Years in the Field

Protein-ligand docking is currently an important tool in drug discovery efforts and an active area of research that has been the subject of important developments over the last decade. These are well portrayed in the rising number of available protein-ligand docking software programs, increasing level of sophistication of its most recent applications, and growing number of users. While starting by summarizing the key concepts in protein-ligand docking, this article presents an analysis of the evolution of this important field of research over the past decade. Particular attention is given to the massive range of alternatives, in terms of protein-ligand docking software programs currently available. The emerging trends in this field are the subject of special attention, while old established docking alternatives are critically revisited. Current challenges in the field of protein-ligand docking such as the treatment of protein flexibility, the presence of structural water molecules and its effect in docking, and the entropy of binding are dissected and discussed, trying to anticipate the next years in the field.

Benchmarking of DFT Functionals for the Hydrolysis of Phosphodiester Bonds.

Phosphodiester bonds are an important chemical component of biological systems, and their hydrolysis and formation reactions are involved in major steps throughout metabolic pathways of all organisms. In this work, we applied dimethylphosphate as a model for this kind of bonds and calculated the potential energy surface for its hydrolysis at the approximated CCSD(T)/CBS//B3LYP/6-311++G(2d,2p) level. By varying the nucleophile (water or hydroxide) and the medium (vacuum or aqueous implicit solvent) we obtained and described four reaction paths. These structures were then used in a DFT functional benchmarking in which we tested a total of 52 functionals. Furthermore, the performances of HF, MP2, MP3, MP4, and CCSD were also evaluated. This benchmarking showed that MPWB1K, MPW1B95, and PBE1PBE are the more accurate functionals to calculate the energies of dimethylphosphate hydrolysis as far as activation and reaction energies are concerned. If considering only the activation energies, MPWB1K, MPW1B95, and B1B95 give the lowest errors when comparing to CCSD(T). A basis set benchmarking on the same system shows that 6-311+G(2d,2p) is the best basis set concerning the relationship between computational time and accuracy. We believe that our results will be of great help to further studies on related phosphodiester systems. This includes not only pure chemical problems but also biochemical studies in which DNA, RNA, and phospholipids are required to be depicted at a quantum level.

Comparative analysis of the performance of commonly available density functionals in the determination of geometrical parameters for zinc complexes.

In this study, a set of 50 transition‐metal complexes of Cu(I) and Cu(II), were used in the evaluation of 18 density functionals in geometry determination. In addition, 14 different basis sets were considered, including four commonly used Poples all‐electron basis sets; four basis sets including popular types of effective‐core potentials: Los Alamos, Steven‐Basch‐Krauss, and Stuttgart‐Dresden; and six triple‐ζ basis sets. The results illustrate the performance of different methodological alternatives for the treatment of geometrical properties in relevant copper complexes, pointing out Double‐Hybrid (DH) and Long‐range Correction (LC) Generalized Gradient Approximation (GGA) methods as better descriptors of the geometry of the evaluated systems. These however, are associated with a computational cost several times higher than some of the other methods employed, such as the M06 functional, which has also demonstrated a comparable performance. Regarding the basis sets, 6–31+G(d) and 6–31+G(d,p) were the best performing approaches. In addition, the results show that the use of effective‐core potentials has a limited impact, in terms of the accuracy in the determination of metal‐ligand bond‐lengths and angles in our dataset of copper complexes. Hence, these could become a good alternative for the geometrical description of these systems, particularly CEP‐121G and SDD basis sets, if one is considering larger copper complexes where the computational cost could be an issue.

The catalytic mechanism of protein phosphatase 5 established by DFT calculations.

In order to elucidate the catalytic mechanism of the Mn-Mn containing serine/threonine protein phosphatase 5 (PP5), we present a density functional theory study with a cluster model approach. According to our results, the reaction occurs through an in-line concerted transition state with an energy of 15.8 kcal mol(-1) , and no intermediates are formed. The important role played by His304 and Asp274 as stabilizers of the leaving group has been shown, whereas the role played by the metal ions seems to be mostly electrostatic. The indispensable requirement of having a neutral active center has been demonstrated by testing different protonation states of the cluster model. We have shown also the importance of describing properly the electronic configuration of the Mn-Mn binuclear centers.

Application of quantum mechanics/molecular mechanics methods in the study of enzymatic reaction mechanisms

Quantum mechanics/molecular mechanics (QM/MM) methods offer a very appealing option for the computational study of enzymatic reaction mechanisms, by separating the problem into two parts that can be treated with different computational methods. Hence, in a QM/MM formalism, the part of the system in which catalysis actually occurs and that involves the active site, substrates and directly participating amino acid residues is treated at an adequate quantum mechanical level to describe the chemistry taking place. For the remaining of the enzyme, which does not participate directly in the reaction, but that typically involves a much larger number of atoms, molecular mechanics is employed, traditionally through the application of a biomolecular force field. When applied with care, QM/MM methods can be used with great advantage in comparing, at a structural and energetic level, different mechanistic proposals, discarding mechanistic alternatives and proposing new mechanistic pathways that are consistent with the available experimental data. With time, diverse flavors within the QM/MM methods have emerged, differing in a variety of technical and conceptual aspects. Hence present alternatives differ between additive and subtractive QM/MM schemes, the type of boundary schemes, and the way in which the electrostatic interactions between the two regions are accounted for. Also, single‐conformation QM/MM, multi‐PES approaches, and QM/MM Molecular Dynamics coexist today, each type with its own advantages and limitations. This review focuses on the application of QM/MM methods in the study of enzymatic reaction mechanisms, briefly presenting also the most important technical aspects involved in these calculations. Particular attention is dedicated to the application of the single‐conformation QM/MM, multi‐PES QM/MM studies, and QM/MM‐FEP methods and to the advantages and disadvantages of the different types of QM/MM. Recent breakthroughs are also introduced. A selection of hand‐picked examples is used to illustrate such features. WIREs Comput Mol Sci 2017, 7:e1281. doi: 10.1002/wcms.1281

Synthesis and Hydrolytic Studies on the Air-Stable [(4-CN-PhO)(E)P(μ-NtBu)]2 (E = O, S, and Se) Cyclodiphosphazanes

Reaction of 4-CN-PhOH with [ClP(μ-N(t)Bu)]2 (1) (2:1 ratio) in the presence of Et3N produced the functionalized cyclodiphosph(III/III)azane [(4-CN-PhO)P(μ-N(t)Bu)]2 (2). Oxidation of 2 produced cyclodiphosph(V/V)azanes [(4-CN-PhO)(E)P(μ-N(t)Bu)]2 [E = O (3), S (4), and Se (5)]. This is the first example of a series of cyclodiphosph(V/V)azane derivatives obtained from a single cyclophosph(III/III)azane precursor where all the accessible chalcogen oxidized products are air-stable over prolonged periods of time.

New insights in the catalytic mechanism of tyrosine ammonia-lyase given by QM/MM and QM cluster models

Tyrosine ammonia lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid in purple phototropic bacteria and Actinomycetales. The enzyme is used in bioengineering and has the potential to be used industrially. It belongs to a family of enzymes that uses a 4-methylidene-imidazole-5-one (MIO) cofactor to catalyze the deamination amino acids. In the present work, we used a QM/MM and a QM cluster models of TAL to explore two putative reaction paths for its catalytic mechanism. Part of the N-MIO mechanism was previously studied by computational methods. We improved on previous studies by using a larger, more complete model of the enzyme, and by describing the complete reaction path. The activation energy for this mechanism, in agreement with the previous study, is 28.5 kcal/mol. We also found another reaction path that has overall better kinetics and reaches the products in a single reaction step. The barrier for this Single-Step mechanism is 16.6 kcal/mol, which agrees very well with the experimental kcat of 16.0 kcal/mol. The geometrical parameters obtained for the cluster and QM/MM models are very similar, despite differences in the relative energies. This means that both approaches are capable of describing the correct catalytic path of TAL.

Unique Triphenylphosphonium Derivatives for Enhanced Mitochondrial Uptake and Photodynamic Therapy

In this study, unique methyl-functionalized derivatives (T*PP+) of the drug carrier triphenylphosphonium (TPP+) that exhibit significant enhancement of the accumulation of both the cation and its conjugated cargo in cell mitochondria are designed. We show that the presence of methyl group(s) at key positions within the phenyl ring results in an increase in the hydrophobicity and solvent accessible surface area of T*PP+. In particular, when the para position of the phenyl ring in T*PP+ is functionalized with a methyl group, the cation is most exposed to the surrounding environment, leading to a large decrease in water entropy and an increase in the level of van der Waals interaction with and partition into a nonpolar solvent. Therefore, stronger binding between the hydrophobic T*PP+ and mitochondrial membrane occurs. This is exemplified in a (hexachloro-fluorescein)-TPP+ conjugate system, where an ∼12 times increase in the rate of mitochondrial uptake and a 2 times increase in photodynamic therapy (PDT) efficacy against HeLa and FU97 cancer cells are achieved when TPP+ is replaced with T*PP+. Importantly, nearly all the FU97 cells treated with the (hexachloro-fluorescein)-T*PP+ conjugate are killed as compared to only half the population of cells in the case of the (hexachloro-fluorescein)-TPP+ conjugate at a similar PDT light dosage. This study thus forms a platform for the healthcare community to explore alternative TPP+ derivatives that can act as optimal drug transporters for enhanced mitochondrially targeted therapies.

Improving the Biodesulfurization of Crude Oil and Derivatives: A QM/MM Investigation of the Catalytic Mechanism of NADH-FMN Oxidoreductase (DszD)

The development of biocatalytic desulfurization strategies of petroleum and its derivatives could result in more economic alternatives than the widely used chemical desulfurization. The organism Rhodococcus erythropolis IGTS8 has been shown to metabolize organic sulfur compounds through a mechanism known as 4S pathway, which involves four enzymes (DszA, DszB, DszC, and DszD) and has been explored in biodesulfurization. Here we have applied QM/MM methods to study the catalytic mechanism of the enzyme DszD, a NADH-FMN oxidoreductase that occupies a central place on the 4S pathway by catalyzing the formation of the FMNH2 that is used by the two monooxynases in the cycle: DszA and DszC. In addition, to clarify the catalytic mechanism of this enzyme, this study analyzed in detail the role played by the active site Thr residue and of Asn and Ala enzyme mutants. The results help to explain previous experimental evidence and suggest new strategies for improving biodesulfurization through an increase in the activity of DszD.

Divalent metal ion-based catalytic mechanism of the Nudix hydrolase Orf153 (YmfB) from Escherichia coli

YmfB from Escherichia coli is the Nudix hydrolase involved in the metabolism of thiamine pyrophosphate, an important compound in primary metabolism and a cofactor of many enzymes. In addition, it hydrolyzes (d)NTPs to (d)NMPs and inorganic orthophosphates in a stepwise manner. The structures of YmfB alone and in complex with three sulfates and two manganese ions determined by X-ray crystallography, when compared with the structures of other Nudix hydrolases such as MutT, Ap4Aase and DR1025, provide insight into the unique hydrolysis mechanism of YmfB. Mass-spectrometric analysis confirmed that water attacks the terminal phosphates of GTP and GDP sequentially. Kinetic analysis of binding-site mutants showed that no individual residue is absolutely required for catalytic activity, suggesting that protein residues do not participate in the deprotonation of the attacking water. Thermodynamic integration calculations show that a hydroxyl ion bound to two divalent metal ions attacks the phosphate directly without the help of a nearby catalytic base.