Computational Survey of Recent Experimental Developments in the Hydroxylation Mechanism of Kynurenine 3-Monooxygenase.

Recently, two new mechanistic proposals for the kynurenine 3-monooxygenase (KMO) catalyzed hydroxylation reaction of l-Kynurenine (l-Kyn) have been proposed. According to the first proposal, instead of the distal oxygen, the proximal oxygen of the hydroperoxide intermediate of flavin adenine dinucleotide (FAD) is transferred to the substrate ring. The second study proposes that l-Kyn participates in its base form in the reaction. To address these proposals, the reaction was reconsidered with a 386 atom quantum cluster model that is based on a recent X-ray structure (PDB id: 6FOX). The computations were carried out at the UB3LYP/6-311+G(2d,2p)//UB3LYP/6-31G(d,p) level with solvation (polarizable continuum model) and dispersion (DFT-D3(BJ)) corrections. To supplement the results of the density functional theory (DFT) calculations, molecular dynamics (MD) simulations of the protein-substrate complex were employed. The comparison of a proximal oxygen transfer mechanism to the distal oxygen transfer mechanism revealed that the former requires too high of a barrier energy while the latter validated our previous results. According to the MD simulations, the hydroperoxy moiety does not favor an alignment that might promote the proximal oxygen transfer mechanism. In the second part of the study, hydroxylation reaction with the base form of l-Kyn was sought. Although DFT calculations confirmed a much more facile reaction with the base form of l-Kyn, a mechanism which would allow the deprotonation of the l-Kyn before the oxygen transfer could not be determined with the X-ray-based positions. A concerted mechanism with both the oxygen transfer and the deprotonation required a high barrier energy. A stepwise mechanism involving the deprotonation of l-Kyn was found, starting from an MD frame. The overall barrier of the oxygen transfer step of this model was found to be in the range of that of with neutral l-Kyn. MD simulations supported the idea of ineffectiveness of the nearby shell surrounding the utilized active site core on the deprotonation of l-Kyn.

In silico methods predict new blood-brain barrier permeable structure for the inhibition of kynurenine 3-monooxygenase.

Kynurenine 3-monooxygenase (KMO) regulates the levels of bioactive substances in the kynurenine pathway of tryptophan catabolism and its activity is tied to so many diseases that finding an appropriate inhibitor for KMO has become an urgent task. This especially proved to be difficult for the central nervous system related diseases due to the requirement that the supposed inhibitor should be both blood brain barrier permeable and should not cause hydrogen peroxide as a harmful side product. In this in silico study, we present our step-wise approach, whose starting point is based on the important experimental observations. To tackle the problem, a library of 7561938 structures was obtained from Zinc15 database utilizing the tranche browser. From this library, a subset of 501777 structures was determined with the considerations of their functional groups that constrain their applicability. Then, the binding affinity ranking of this set of structures was determined via virtual screening. Starting from the structures whose affinities are the highest among this subset, the ADMET properties were checked through in silico methods and the binding properties of the selected inhibitor candidates were further investigated via molecular dynamics simulations and MM/GBSA calculations. According to the computational results of this study, ZINC_71915355 has passed all the evaluations and is a potentially BBB permeable structure that can inhibit KMO. Additionally, ZINC_19827377 was identified as a new potential KMO inhibitor which may be more suitable for peripheral administration. From the in silico study presented herein, ZINC_71915355 and ZINC_19827377 structures, which showed high binding affinity without harmful H2O2 production, along with the tailored properties can now serve as powerful candidates for KMO inhibition and these hits are worth of further experimental validation.

Mechanism of Kynurenine 3-Monooxygenase-Catalyzed Hydroxylation Reaction: A Quantum Cluster Approach.

The mechanism of the hydroxylation reaction between l-Kyn and model flavin adenine dinucleotide (FAD)-hydroperoxide was investigated via density functional theory (DFT) calculations in the absence and in the presence of the kynurenine 3-monooxygenase (KMO) enzyme by considering possible pathways that can lead to the product 3-hydroxykynurenine (3-HK). Crystal structure (pdb code: 5NAK )-based calculations involved a quantum cluster model in which the active site of the enzyme with the substrate l-Kyn was represented with 348 atoms. According to the deduced mechanism, KMO-catalyzed hydroxylation reaction takes place with four transformations. In the initial transition state, FAD delivers its peroxy hydroxyl to the l-Kyn ring, creating an sp3-hybridized carbon center. Then, the hydrogen on the hydroxyl moiety is immediately transferred back to the proximal oxygen that remained on FAD. These consequent transformations are in line with the somersault rearrangement previously described for similar enzymatic systems. The second step corresponds to a hydride shift from the sp3-hybridized carbon of the substrate ring to its adjacent carbon, producing the keto form of 3-HK. Then, keto-3-HK is transformed into its enol form (3-HK) with a water-assisted tautomerization. Lastly, FAD is oxidized with a water-assisted dehydration, which also involves 3-HK as a catalyst. In the proposed pathway, Asn54, Pro318, and a crystal water molecule were seen to play significant roles in the proton relays. The energies obtained via the cluster approach were calculated at the B3LYP/6-311+G(2d,2p)//B3LYP/6-31G(d,p) level with solvation (polarizable continuum model) and dispersion (DFT-D3(BJ)) corrections.

Computational investigation of the control of the thermodynamics and microkinetics of the reductive amination reaction by solvent coordination and a co-catalyst.

Amines are among the most important and frequently used chemical compounds due to their biological activity and a wide range of applications in industry. Reductive amination reactions are an efficient and facile route to synthesize long chain amines from sustainable sources by using a different available aldehydes and ketones, and a large variety of amines including primary, secondary and tertiary forms. The pathway of the reaction process is critically dependent on reaction parameters such as the pH of the reaction medium, choice of solvent (explicitly coordinating solvent) and process conditions. These parameters are affecting the reaction performance and the selectivity but are still not fully rationalized. Here, we investigate the microkinetics and thermodynamics of the individual steps of the reductive amination reaction by exploring the systems' parameters. Explicit water coordination to the aldehyde leads to a stepwise rather than concerted nucleophilic addition with a lower activation barrier by 6-10 kcal mol-1. At low pH, the pathway is changed by a direct protonation of the amine substrate. This protonation does not strongly affect the kinetics of the reaction, but the thermodynamic equilibria. The presence of an acid as a co-catalyst leads to the formation of an iminium intermediate and this drives the reaction forward. Thus, the presence of an acid as a co-catalyst clearly renders this pathway the thermodynamically preferred one. Consequently, altering the reaction parameters does not only influence the reaction kinetics, but also the thermodynamic profile of the pathways in all cases. Further understanding of the reaction dynamics is essential to develop a microkinetic model of the reaction to then control and engineer the process in order to rationally design routes to tailor-made products.

Structural investigation of vesnarinone at the pore domains of open and open-inactivated states of hERG1 K+ channel.

In this study, the dynamics of vesnarinone bounded hERG1 K+ channels are investigated using in silico approaches such as molecular docking, molecular dynamics (MD) simulations, MM/PBSA (Molecular Mechanics/Poisson Boltzmann Surface Area) calculations and Principal Component Analysis (PCA). Vesnarinone (a cardiotonic agent) falls into a category of drugs that inhibit phosphodiesterase 3-type (PDE3) enzymes. PDE3 enzymes have specific roles in the dehydyrolysis of intracellular second messengers 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Thus, PDE3 inhibitors elevate the intracellular concentrations of these substrates. However, it is also known that vesnarinone inhibits the human ether-à-go-go-related gene (hERG) channels. Since inhibition of hERG channels may cause life-threatening arrhythmias, leading to Torsades de pointes (TdP) and long QT syndrome (LQTS), it is important to understand the particular residue-drug interactions and hERG channel dynamics. Applying the computational approaches in this study, have helped to elucidate the possible binding patterns and time evaluation dynamics of this drug at hERG1 channel models (both in its open and open-inactivated states) together with the crucial amino acid residues that mostly contribute in binding processes via interaction binding energy decomposition analysis.

Investigation of PDE5/PDE6 and PDE5/PDE11 selective potent tadalafil-like PDE5 inhibitors using combination of molecular modeling approaches, molecular fingerprint-based virtual screening protocols and structure-based pharmacophore development.

The essential biological function of phosphodiesterase (PDE) type enzymes is to regulate the cytoplasmic levels of intracellular second messengers, 3',5'-cyclic guanosine monophosphate (cGMP) and/or 3',5'-cyclic adenosine monophosphate (cAMP). PDE targets have 11 isoenzymes. Of these enzymes, PDE5 has attracted a special attention over the years after its recognition as being the target enzyme in treating erectile dysfunction. Due to the amino acid sequence and the secondary structural similarity of PDE6 and PDE11 with the catalytic domain of PDE5, first-generation PDE5 inhibitors (i.e. sildenafil and vardenafil) are also competitive inhibitors of PDE6 and PDE11. Since the major challenge of designing novel PDE5 inhibitors is to decrease their cross-reactivity with PDE6 and PDE11, in this study, we attempt to identify potent tadalafil-like PDE5 inhibitors that have PDE5/PDE6 and PDE5/PDE11 selectivity. For this aim, the similarity-based virtual screening protocol is applied for the "clean drug-like subset of ZINC database" that contains more than 20 million small compounds. Moreover, molecular dynamics (MD) simulations of selected hits complexed with PDE5 and off-targets were performed in order to get insights for structural and dynamical behaviors of the selected molecules as selective PDE5 inhibitors. Since tadalafil blocks hERG1 K channels in concentration dependent manner, the cardiotoxicity prediction of the hit molecules was also tested. Results of this study can be useful for designing of novel, safe and selective PDE5 inhibitors.

In silico design of novel hERG-neutral sildenafil-like PDE5 inhibitors.

Cyclic nucleotide phosphodiesterase enzymes (PDEs) have functions in regulating the levels of intracellular second messengers, 3', 5'-cyclic adenosine monophosphate (cAMP) and 3', 5'-cyclic guanosine monophosphate (cGMP), via hydrolysis and decomposing mechanisms in cells. They take essential roles in modulating various cellular activities such as memory and smooth muscle functions. PDE type 5 (PDE5) inhibitors enhance the vasodilatory effects of cGMP in the corpus cavernosum and they are used to treat erectile dysfunction. Patch clamp experiments showed that the IC50 values of the human ether-à-go-go-related gene (hERG1) potassium (K) ion channel blocking affinity of PDE5 inhibitors sildenafil, vardenafil, and tadalafil as 33, 12, and 100 µM, respectively. hERG1 channel is responsible for the regulation of the action potential of human ventricular myocyte by contributing the rapid component of delayed rectifier K+ current (IKr) component of the cardiac action potential. In this work, interaction patterns and binding affinity predictions of selected PDE5 inhibitors against the hERG1 channel are studied. It is attempted to develop PDE5 inhibitor analogs with lower binding affinity to hERG1 ion channel while keeping their pharmacological activity against their principal target PDE5 using in silico methods. Based on detailed analyses of docking poses and predicted interaction energies, novel analogs of PDE5 inhibitors with lower predicted binding affinity to hERG1 channels without loosing their principal target activity were proposed. Moreover, molecular dynamics (MD) simulations and post-processing MD analyses (i.e. Molecular Mechanics/Generalized Born Surface Area calculations) were performed. Detailed analysis of molecular simulations helped us to better understand the PDE5 inhibitor-target binding interactions in the atomic level. Results of this study can be useful for designing of novel and safe PDE5 inhibitors with enhanced activity and other tailored properties.

Ag-catalyzed azide alkyne cycloaddition: a DFT approach.

In this study, the mechanism of AgAAC reaction has been studied by quantum mechanical calculations to gain insights into this promising reaction and the first successful application of a Ag catalyst alone in AAC. Elucidating the reaction mechanism will enable more control over the synthesis and help to obtain tailor made products in good yields without copper. The feasibility of the experimentally proposed reaction mechanism was investigated by modelling the profound intermediates and the transition state structures connecting them. The DFT calculations with the wB97XD functional with MWB28 effective core potential and 6-31+G* basis set combination herein show that once the silver acetylide structure forms, triazole synthesis via the experimentally proposed cycloaddition is a facile reaction in terms of energetics. The number of metal atoms involved in a click reaction is one of the main questions considered in mechanistic studies. In AgAAC reaction, comparison of mononuclear and binuclear paths shows that the barrier for binuclear cases is lower than that of mononuclear cases.

Stereoselective propagation in free radical polymerization of acrylamides: a DFT study.

In this study stereospecific free radical polymerization of N,N-alkylamides [N,N-dimethylacrylamide (DMAAm), N-methyl-N-phenylacrylamide (MphAAm) and N,N-diphenylacrylamide (DPAAm)] is investigated with density functional theory (DFT) calculations. Model propagation reactions at dimeric stage are used to elucidate the effect of substituent bulkiness, temperature and solvent polarity on stereospecific addition modes. In calculations all the monomers favor gauche conformation in their pro-meso and pro-racemo additions in general. The DFT calculations have reproduced the stereospecificity seen in these monomers. The implicit solvent calculations performed with IEFPCM have further refined the quantitative agreement. The calculations of DMAAm in solvents of different polarity (toluene, THF, chloroform and 2-propanol) have successfully reproduced the experimental trend both qualitatively and quantitatively. Tartrate molecules as stereospecifity inducer in DMAAm are considered and the experimentally observed change in stereospecificity from iso to syn in their presence have been elucidated by modeling the possible orientations of transition states in the propagation step. The favorable stereospecific addition modes are explained via interplay between the steric effects and the hydrogen bonding interactions.

The mechanism of copper-catalyzed azide-alkyne cycloaddition reaction: a quantum mechanical investigation.

In this study, the mechanism of CuAAC reaction and the structure of copper acetylides have been investigated with quantum mechanical methods, namely B3LYP/6-311+G(d,p). A series of possible copper-acetylide species which contain up to four copper atoms and solvent molecules as ligand has been evaluated and a four-copper containing copper-acetylide, M1A, was proposed more likely to form based on its thermodynamic stability. The reaction has been modeled with a representative simple alkyne and a simple azide to concentrate solely on the electronic effects of the mechanism. Later, the devised mechanism has been applied to a real system, namely to the reaction of 2-azido-1,1,1-trifluoroethane and ethynylbenzene in the presence of copper. The copper catalyst transforms the concerted uncatalyzed reaction to a stepwise process and lowers the activation barrier. The pre-reactive complexation of the negatively charged secondary nitrogen of azide and the positively charged copper of copper-acetylide brings the azide and the alkyne to a suitable geometry for cycloaddition to take place. The calculated activation barrier difference between the catalyzed and the uncatalyzed reactions is consistent with faster and the regioselective synthesis of triazole product.

Modeling the effect of substitution on the Pb(OAc)4 mediated oxidative cleavage of steroidal 1,2-diols.

[reaction: see text] We comment on the effects of angular substitution on the outcome of a Pb(OAc)4 (LTA) mediated heterodomino reaction, with selected bicyclic unsaturated 1,2-diols, which is considered to proceed through a series of transformations in a single vessel. The first two, oxidative and pericyclic, are followed by the key step, an electrophilic addition of LTA to the olefin, responsible for the course of the domino process. In this study, the electrophilic addition of LTA to the double bond has been modeled with B3LYP, where the 6-31G* basis set is used for C, O, and H atoms and the LANL2DZ method is used for Pb. The modeling in the gaseous phase and in solution has revealed the concerted nature of the addition of LTA to the double bond of the intermediate. The fact that LTA adds from the same side as the substituent R, for R=H and from the opposite side when R=CH3 has been attributed to steric hindrance, which causes deformation of the olefinic intermediate.