CryoEM structures of anion exchanger 1 capture multiple states of inward- and outward-facing conformations.

Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member.

Mechanistic insight into the impact of a bivalent ligand on the structure and dynamics of a GPCR oligomer.

Development of effective bivalent ligands has become the focus of intensive research toward modulation of G protein-coupled receptor (GPCR) oligomers, particularly in the field of GPCR pharmacology. Experimental studies have shown that they increased binding affinity and signaling potency compared to their monovalent counterparts, yet underlying molecular mechanism remains elusive. To address this, we performed accelerated molecular dynamics simulations on bivalent-ligand bound Adenosine 2A receptor (A2AR) dimer in the context of a modeled tetramer, which consists of A2AR and dopamine 2 receptor (D2R) homodimers and their cognate G proteins. Our results demonstrate that bivalent ligand impacted interactions between pharmacophore groups and ligand binding residues, thus modulating allosteric communication network and water channel formed within the receptor. Moreover, it also strengthens contacts between receptor and G protein, by modulating the volume of ligand binding pocket and intracellular domain of the receptor. Importantly, we showed that impact evoked by the bivalent ligand on A2AR dimer was also transmitted to apo D2R, which is part of the neighboring D2R dimer. To the best of our knowledge, this is the first study that provides a mechanistic insight into the impact of a bivalent ligand on dynamics of a GPCR oligomer. Consequently, this will pave the way for development of effective ligands for modulation of GPCR oligomers and hence treatment of crucial diseases such as Parkinson's disease and cancer.

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE.

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism.

Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO3-, CO32-, and Cl- necessary for pH regulation, transepithelial H+/base transport, and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl-/HCO3- exchange versus Na+-CO32- symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein-ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32- binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.

Drug repurposing studies of PARP inhibitors as a new therapy for inherited retinal degeneration.

The enzyme poly-ADP-ribose-polymerase (PARP) has important roles for many forms of DNA repair and it also participates in transcription, chromatin remodeling and cell death signaling. Currently, some PARP inhibitors are approved for cancer therapy, by means of canceling DNA repair processes and cell division. Drug repurposing is a new and attractive aspect of therapy development that could offer low-cost and accelerated establishment of new treatment options. Excessive PARP activity is also involved in neurodegenerative diseases including the currently untreatable and blinding retinitis pigmentosa group of inherited retinal photoreceptor degenerations. Hence, repurposing of known PARP inhibitors for patients with non-oncological diseases might provide a facilitated route for a novel retinitis pigmentosa therapy. Here, we demonstrate and compare the efficacy of two different PARP inhibitors, BMN-673 and 3-aminobenzamide, by using a well-established retinitis pigmentosa model, the rd1 mouse. Moreover, the mechanistic aspects of the PARP inhibitor-induced protection were also investigated in the present study. Our results showed that rd1 rod photoreceptor cell death was decreased by about 25-40% together with the application of these two PARP inhibitors. The wealth of human clinical data available for BMN-673 highlights a strong potential for a rapid clinical translation into novel retinitis pigmentosa treatments. Remarkably, we have found that the efficacy of 3 aminobenzamide was able to decrease PARylation at the nanomolar level. Our data also provide a link between PARP activity with the Wnt/ß-catenin pathway and the major intracellular antioxidant concentrations behind the PARP-dependent retinal degeneration. In addition, molecular modeling studies were integrated with experimental studies for better understanding of the role of PARP1 inhibitors in retinal degeneration.

Current status of multiscale simulations on GPCRs.

Membrane receptors couple signaling pathways using various mechanisms. G Protein-Coupled Receptors (GPCRs) represent the largest class of membrane proteins involved in signal transduction across the biological membranes. They are essential targets for cell signaling and are of great commercial interest to the pharmaceutical industry. Recent advances made in molecular biology and computational chemistry offer a range of simulation and multiscale modeling tools for the definition and analysis of protein-ligand, protein-protein, and protein-membrane interactions. The development of new techniques on statistical methods and free energy simulations help to predict novel optimal ligands, G protein specificity and oligomerization. The identification of the ligand-binding activation mechanisms and atomistic determinants as well as the interactions of intracellular binding partners that bind to GPCR targets in different coupling states will provide greater safety in human life. In this review, recent approaches and applications of multiscale simulations on GPCRs were highlighted.

Inhibition of acetylcholinesterase and butyrylcholinesterase with uracil derivatives: kinetic and computational studies.

Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) inhibitors are interesting compounds for different therapeutic applications, among which Alzheimer's disease. Here, we investigated the inhibition of these cholinesterases with uracil derivatives. The mechanism of inhibition of these enzymes was observed to be due to obstruction of the active site entrance by the inhibitors scaffold. Molecular docking and molecular dynamics (MD) simulations demonstrated the possible key interactions between the studied ligands and amino acid residues at different regions of the active sites of AChE and BuChE. Being diverse of the classical AChE and BuChE inhibitors, the investigated uracil derivatives may be used as lead molecules for designing new therapeutically effective enzyme inhibitors.

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.

Synthesis, anticholinesterase activity and molecular modeling study of novel carbamate-substituted thymol/carvacrol derivatives.

New thymol and carvacrol derivatives with the carbamate moiety were synthesized and their inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were evaluated. 5-isopropyl-2-methylphenyl(3-fluorophenyl)carbamate (29) was found to be the most potent AChE inhibitor with IC50 values of 2.22µM, and 5-isopropyl-2-methylphenyl (4-fluorophenyl)carbamate (30) exhibited the strongest inhibition against BuChE with IC50 value of 0.02µM. Additionally, the result of H4IIE hepatoma cell toxicity assay for compounds 18, 20, 29, 30 and 35 showed negligible cell death at 0.07-10µM. Moreover in order to better understand the inhibitory profiles of these molecules, molecular modeling studies were applied. Binding poses of studied compounds at the binding pockets of AChE and BuChE targets were determined. Predicted binding energies of these compounds as well as structural and dynamical profiles of molecules at the target sites were estimated using induced fit docking (IFD) algorithms and post-processing molecular dynamics (MD) simulations methods (i.e., Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) approaches).

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.

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.