Citrullination, catalyzed by protein arginine deiminase enzymes, involves the conversion of peptidyl-arginine to peptidyl-citrulline, disrupting protein interactions and leading to functional alterations. Despite the experimental studies on PAD2 indicating calcium dependence and substrate specificity, the catalytic mechanism remains contentious, with conflicting evidence regarding the roles of active site residues such as Cys647 and His471. The present study is an expansion of prior molecular dynamics simulations that investigated the dynamics of the enzyme PAD2, which indicated that Asp473 may function as a general acid/base, thereby challenging the experimentally proposed pathways. To further elucidate this controversial issue, quantum mechanical methods were employed to examine the protonation states of key residues and their roles in catalysis. Herein, three different pathways have been studied for the substrate-assisted citrullination mechanism of PAD2 isozyme using a model structure that includes the active site residues Asp351, His471, Val472, Asp473, and Cys647 and a water molecule. The highest barriers for two of the designed mechanisms, RM1 and RM3 are comparable: the choice of a single mechanism is not possible since the differences in barriers fall within the error margins in DFT calculations. These findings offer insights into PAD2's enzymatic activity, thereby advancing our understanding of its biological significance.

As weak acids or bases, in solution, drug molecules are in either their ionized or nonionized states. A high degree of ionization is essential for good water solubility of a drug molecule and is required for drug-receptor interactions, whereas the nonionized form improves a drug's lipophilicity, allowing the ligand to cross the cell membrane. The penetration of a drug ligand through cell membranes is mainly governed by the pKa of the drug molecule and the membrane environment. In this study, with the aim of predicting the acetonitrile pKa's (pKa(MeCN)) of eight drug-like thiazol-2-imine derivatives, we propose a very accurate and computationally affordable protocol by using several quantum mechanical approaches. Benchmark studies were conducted on a set of training molecules, which were selected from the literature with known pKa(water) and pKa(MeCN). Highly well-correlated pKa values were obtained when the calculations were performed with the isodesmic method at the M062X/6-31G** level of theory in conjunction with SMD solvation model for nitrogen-containing heterocycles. Finally, experimentally unknown pKa(MeCN) values of eight thiazol-2-imine structures, which were previously synthesized by some of us, are proposed.

By exploring an efficient and versatile method for the 6-functionalization of its scaffold, we investigated the opening of a new chemical space around benzylidenethiazolidine-2,4-dione (BTZD). The 6-chloro- and 6-formyl BTZD obtained in two steps starting from 5-lithioTZD were selected as key intermediates and involved in a Pd-catalyzed cross-coupling or Wittig olefination. A variety of aryl, heteroaryl, or alkenyl substituents was successfully introduced on the vinylic position of BTZD, and particular attention was paid to elucidate the stereochemistry of the benzylidene derivatives by using a combined DFT/NMR study.

Bcl-xL is an anti-apoptotic mitochondrial trans-membrane protein, known to play a crucial role in the survival of tumor cells. The deamidation of Bcl-xL is a pivotal switch that regulates its biological function. The potential impact of deamidation on the structure and dynamics of Bcl-xL is directly linked to the intrinsically disordered region (IDR), which is the main site for post-translational modifications (PTMs). In this study, we explored deamidation-induced conformational changes in Bcl- xL to gain insight into its loss of function by performing microsecond-long molecular dynamics (MD) simulations. MD simulation outcomes showed that the IDR motion and interaction patterns have changed notably upon deamidation. Principal component analysis (PCA) demonstrates significant differences between wild type and deamidated Bcl-xL and suggests that deamidation affects the structure and dynamics of Bcl-xL. Differences in contact patterns and essential dynamics in the binding groove (BG) are clear indications of deamidation-induced allosteric affects.

Peptidylarginine deiminase 2 (PAD2) is a Ca2+-dependent enzyme that catalyzes the conversion of protein arginine residues to citrulline. This kind of structural modification in histone molecules may affect gene regulation, leading to effects that may trigger several diseases, including breast cancer, which makes PAD2 an attractive target for anticancer drug development. To design new effective inhibitors to control activation of PAD2, improving our understanding of the molecular mechanisms of PAD2 using up-to-date computational techniques is essential. We have designed five different PAD2-substrate complex systems based on varying protonation states of the active site residues. To search the conformational space broadly, multiple independent molecular dynamics simulations of the complexes have been performed. In total, 50 replica simulations have been performed, each of 1 μs, yielding a total simulation time of 50 μs. Our findings identify that the protonation states of Cys647, Asp473, and His471 are critical for the binding and localization of the N-α-benzoyl-l-arginine ethyl ester substrate within the active site. A novel mechanism for enzyme activation is proposed according to near attack conformers. This represents an important step in understanding the mechanism of citrullination and developing PAD2-inhibiting drugs for the treatment of breast cancer.

In this study, we present an accurate protocol for the fast prediction of pKa's of carboxylic acids based on the linear relationship between computed atomic charges of the anionic form of the carboxylate fragment and their experimental pKa values. Five charge descriptors, three charge models, three solvent models, gas phase calculations and several DFT methods (combination of eight DFT functionals and fifteen basis sets) were tested. Among those, the best combination to reproduce experimental pKa's is to compute NPA atomic charge using the SMD model at the M06L/6-311G(d,p) level of theory and selecting the maximum atomic charge on carboxylic oxygen atoms (R^2 = 0.955). The applicability of the suggested protocol and its stability along geometrical changes are verified by molecular dynamics simulations performed for a set of aspartate, glutamate and alanine peptides. By reporting the calculated atomic charge of the carboxylate form into the linear relationship derived in this work, it will be possible to estimate accurately the amino acid’s pKa's in protein environment.

Recently, Sarigul and Dogan have synthesized a number of enantiomerically enriched axially chiral atropoisomeric 2-thiohydantoins by the reaction of L-amino acid ester salts and o-aryl isothiocyanates in the presence of triethyl amine (TEA) in dichloromethane. The nonaxially chiral derivative 5-methyl-3-phenyl-2-thiohydantoin gave a racemic product whereas the axially chiral 5-methyl-3-o-bromophenyl-2-thiohydantoin was less prone to racemize at C5 of the heterocyclic ring. In this study, we present a computational study (M06-2X/6-311+G(d,p) for C, H, O, N and S; M06-2X/6-311++G(3df,3pd) for Br) in order to propose plausible mechanisms for the racemization and cyclization steps for 2-thiohydantoin derivatives. The study includes rationalization based on steric as well as the electrostatic effects to elucidate the epimerization differences at C5.

By using a combination of experimental and computational experiments, we demonstrated that a second-generation dendrigraft of poly-l-lysine neutralizes the anticoagulant activity of unfractionated heparin, low-molecular-weight heparin, and fondaparinux more efficiently than protamine does in human plasma, making this synthetic polymer a promising surrogate of this problematic protein in clinical settings.

Because of the complex biological networks, many pathologic disorders fail to be treated with a molecule directed towards a single target. Thus, combination therapies are often necessary, but they have many drawbacks. An alternative consists in building molecules intended to interact with multiple targets, called designed multiple ligands. We followed such a strategy in order to treat metabolic syndrome, by setting up molecules directed towards both type 1 angiotensin II (AT1) receptor and peroxisome proliferator-activated receptor-γ (PPAR-γ). For this purpose, many molecules were prepared by merging both pharmacophores following three different strategies. Their ability to activate PPAR-γ and to block AT1 receptors were evaluated in vitro. This strategy led to the preparation of many new PPAR-γ activating and AT1 blocking molecules. Among them, some exhibited both activities, highlighting the convenience of this approach.

15-Deoxy-∆12,14-prostaglandin J2 (15d‑PGJ2) is a natural agonist of peroxisome proliferator-activated receptor γ (PPARγ) that displays anticancer activity. Various studies have indicated that the effects of 15d‑PGJ2 are due to both PPARγ-dependent and -independent mechanisms. In the present study, we examined the effects of a biotinylated form of 15d‑PGJ2 (b‑15d‑PGJ2) on hormone-dependent MCF‑7 and triple‑negative MDA‑MB‑231 breast cancer cell lines. b‑15d‑PGJ2 inhibited cell proliferation more efficiently than 15d‑PGJ2 or the synthetic PPARγ agonist, efatutazone. b‑15d‑PGJ2 was also more potent than its non-biotinylated counterpart in inducing apoptosis. We then analyzed the mechanisms underlying this improved efficiency. It was found not to be the result of biotin receptor-mediated increased incorporation, since free biotin in the culture medium did not decrease the anti-proliferative activity of b‑15d‑PGJ2 in competition assays. Of note, b‑15d‑PGJ2 displayed an improved PPARγ agonist activity, as measured by transactivation experiments. Molecular docking analyses revealed a similar insertion of b‑15d‑PGJ2 and 15d‑PGJ2 into the ligand binding domain of PPARγ via a covalent bond with Cys285. Finally, PPARγ silencing markedly decreased the cleavage of the apoptotic markers, poly(ADP-ribose) polymerase 1 (PARP‑1) and caspase‑7, that usually occurs following b‑15d‑PGJ2 treatment. Taken together, our data indicate that biotinylation enhances the anti-proliferative and pro-apoptotic activity of 15d‑PGJ2, and that this effect is partly mediated via a PPARγ-dependent pathway. These results may aid in the development of novel therapeutic strategies for breast cancer treatment.

Semiempirical quantum methods from the Neglect of Differential Diatomic Overlap (NDDO) family such as MNDO, AM1, or PM3 are fast albeit approximate quantum methods. By combining them with linear scaling methods like the Divide & Conquer (D&C) method, it is possible to quickly evaluate the energy of systems containing hundreds to thousands of atoms. We here present our implementation in the Amber biomolecular package of a SEBOMD module that provides a way to run SemiEmpirical Born-Oppenheimer Molecular Dynamics. At each step of a SEBOMD molecular dynamics, a fully converged SCF calculation is performed to obtain the semiempirical quantum potential energy of a molecular system encaged or not in periodic boundary conditions. We describe the implementation and the features of our SEBOMD implementation. We show the equirements to conserve the total energy in NVE simulations, and how to accelerate SCF convergence through density matrix extrapolation. Specific ways of handling periodic boundary conditions using mechanical embedding or electrostatic embedding through a tailored quantum Ewald summation is developed. The parallel performance of SEBOMD simulations using the D&C scheme are presented for liquid water systems of various sizes, and a comparison between the traditional full diagonalization scheme and the D&C approach for the reproduction of the structure of liquid water illustrates the potentiality of SEBOMD to simulate molecular systems containing several hundreds of atoms for hundreds of picoseconds with a quantum mechanical potential in a reasonable amount of CPU time.

Despite the growing use of poly-l-lysine dendrigrafts in biomedical applications, a deeper understanding of the molecular level properties of these macromolecules is missing. Herein, we report a simple methodology for the construction of three-dimensional structures of poly-l-lysine dendrigrafts and the subsequent investigation of their structural features using microsecond molecular dynamics simulations. This methodology relies on the encoding of the polymers' experimental characterizations (i.e., composition, degrees of polymerization, branching ratios, charges) into alphanumeric strings that are readable by the Amber simulation package. Such an original approach opens avenues toward the in silico exploration of dendrigrafts and hyperbranched polymers.

Aminoacyl-tRNA synthetases (aaRSs) catalyze the activation of the corresponding amino acids and attachment to their cognate tRNAs with extremely high fidelity due to pre and post-transfer editing processes. We have computationally elucidated a pretransfer editing mechanism in yeast mitochondrial threonyl-tRNA synthetase (MSTI) via the combined application of classical molecular dynamics and QM/MM-MD free energy calculations. It is concluded that the pretransfer editing mechanism against seryl-AMP occurs in two steps via an oxyanion intermediate. Importantly, its formation is central to, within the aminoacylation active site, the differential rates at which threonyl-tRNA synthetase (ThrRS) is able to hydrolyze its cognate threonyl-AMP and noncognate seryl-AMP substrates. More specifically, in contrast to that observed when threonyl-AMP is bound in the ThrRS active site, the binding of serylAMP enables a greater number of waters to permeate into the active site. This results in greater stabilization of the seryl-AMP derived oxyanion intermediate via hydrogen bonding such that it is thermodynamically more stable by 7-10 kcal mol(-1) than is the case for threonyl-AMP. Moreover, the barrier associated with the first step of the hydrolytic pretransfer mechanism is less for seryl-AMP than for threonyl-AMP by 4-10 kcal mol(-1). These results help to explain the preferential degradation of noncognate seryl-AMP over threonyl-AMP by ThrRS.

The mechanistic cysteinyl of GlmS can activate its thiol using its own α-amine without the need for a bridging water.

In a first step in the discovery of novel potent inhibitor structures for the PDE4B family with limited side effects, we present a protocol to rank newly designed molecules through the estimation of their IC\(_{50}\) values. Our protocol is based on reproducing the linear relationship between the logarithm of experimental IC\(_{50}\) values [\(\log\)(IC\(_{50}\))] and their calculated binding free energies (\(\Delta G_\mathrm{binding}\)). From 13 known PDE4B inhibitors, we show here that (1) binding free energies obtained after a docking process by AutoDock are not accurate enough to reproduce this linear relationship; (2) MM-GB/SA post-processing of molecular dynamics (MD) trajectories of the top ranked AutoDock pose improves the linear relationship; (3) by taking into account all representative structures obtained by AutoDock and by averaging MM-GB/SA computations on a series of 40 independent MD trajectories, a linear relationship between \(\log\)(IC\(_{50}\)) and the lowest \(\Delta G_\mathrm{binding}\) is achieved with \(R^2=0.944\).

The pyridoxal 5-phosphate (PLP) cofactor is a significant organic molecule in medicinal chemistry. It is often found covalently bound to lysine residues in proteins to form PLP dependent enzymes. An example of this family of PLP dependent enzymes is γ-aminobutyric acid aminotransferase (GABA-AT) which is responsible for the degradation of the neurotransmitter GABA. Its inhibition or inactivation can be used to prevent the reduction of GABA concentration in brain which is the source of several neurological disorders. As a test case for PLP dependent enzymes, we have performed molecular dynamics simulations of GABA-AT to reveal the roles of the protein residues and its cofactor. Three different states have been considered: the apoenzyme, the holoenzyme, and the inactive state obtained after the suicide inhibition by vigabatrin. Different protonation states have also been considered for PLP and two key active site residues: Asp298 and His190. Together, 24 independent molecular dynamics trajectories have been simulated for a cumulative total of 2.88 µs. Our results indicate that, unlike in aqueous solution, the PLP pyridine moiety is protonated in GABA-AT. This is a consequence of a pKa shift triggered by a strong charge-charge interaction with an ionic "diad" formed by Asp298 and His190 that would help the activation of the first half-reaction of the catalytic mechanism in GABA-AT: the conversion of PLP to free pyridoxamine phosphate (PMP). In addition, our MD simulations exhibit additional strong hydrogen bond networks between the protein and PLP: the phosphate group is held in place by the donation of at least three hydrogen bonds while the carbonyl oxygen of the pyridine ring interacts with Gln301; Phe181 forms a π-π stacking interaction with the pyridine ring and works as a gate keeper with the assistance of Val300. All these interactions are hypothesized to help maintain free PMP in place inside the protein active site to facilitate the second half-reaction in GABA-AT: the regeneration of PLP-bound GABA-AT (i.e., the holoenzyme). Proteins 2016; 84:875-891. © 2016 Wiley Periodicals, Inc.

Modeling biomolecular systems by explicitly taking into account the quantum mechanical behavior of the electrons represents one of the greatest challenges for theoretical chemistry studies. This is often achieved by coupling Molecular Dynamics (MD) with quantum chemistry methods. However, demanding computational efforts are usually necessary. We recently developed a new scheme allowing us to perform reasonably long Born-Oppenheimer MD simulations of large systems using a quantum electronic Hamiltonian described at the SemiEmpirical (SE) level (SEBOMD [1]). This technique has already been successfully applied to simulate liquid water [1] and N-methylacetamide [2] in aqueous solution.

Deamidation is the uncatalyzed process by which asparagine or glutamine can be transformed into aspartic acid or glutamic acid, respectively. In its active homodimeric form, mammalian triosephosphate isomerase (TPI) contains two deamidation sites per monomer. Experimental evidence shows that the primary deamidation site (Asn71-Gly72) deamidates faster than the secondary deamidation site (Asn15-Gly16). To evaluate the factors controlling the rates of these two deamidation sites in TPI, we have performed graphics processing unit-enabled microsecond long molecular dynamics simulations of rabbit TPI. The kinetics of asparagine dipeptide and two deamidation sites in mammalian TPI are also investigated using quantum mechanical/molecular mechanical tools with the umbrella sampling technique. Analysis of the simulations has been performed using independent global and local descriptors that can influence the deamidation rates: desolvation effects, backbone acidity, and side chain conformations. Our findings show that all the descriptors add up to favor the primary deamidation site over the secondary one in mammalian TPI: Asn71 deamidates faster because it is more solvent accessible, the adjacent glycine NH backbone acidity is enhanced, and the Asn side chain has a preferential near attack conformation. The crucial impact of the backbone amide acidity of the adjacent glycine on the deamidation rate is shown by kinetic analysis. Our findings also shed light on the effect of high-order structure on deamidation: the deamidation in a small peptide is favored first because of the higher reactivity of the asparagine residue and then because of the stronger stability of the tetrahedral intermediate.