Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein‐coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example

Homology modeling has been applied to fill in the gap in experimental G protein‐coupled receptors structure determination. However, achievement of G protein‐coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein‐coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein‐coupled receptors with ligand selectivity. To validate this integrated strategy, the A_2A adenosine receptor (A_2AAR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active‐state A_2AAR structure based on the inactive‐state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand–receptor interactions and conformational changes of key structural elements related to the activation of A_2AAR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein‐coupled receptor modeling, which should aid in the discovery of more effective and selective G protein‐coupled receptor ligands.     

Homology and Molecular Dynamics Models of Toll‐Like Receptor 7 Protein and Its Dimerization

Toll‐like receptor protein 7 is a transmembrane protein playing a crucial role in the signaling pathways involved in innate immunity. Its crystal structure is not yet available, but there are several proteins possessing domains of sufficiently high homology, which enabled us to build a model of the toll‐like receptor protein 7 monomer and gain insights into dimer formation. To obtain a reliable structure prediction, we subjected this model to equilibration using molecular dynamics simulations. Furthermore, the equilibrated monomer structure was used to construct models of dimerization and to predict binding sites for small ligands. Docking studies were performed for some of the known toll‐like receptor protein 7 ligands. We determined that a new homology model generated by the LOOPP server provides a good alternative to a previously reported model. Our docking results indicate that the addition of either imiquimod or 1V209 to a toll‐like receptor protein 7 dimer changes an unfavorable interaction into a favorable one. We found that eight small molecules docked to two pockets in toll‐like receptor protein 7 bind to both pockets at pH 7 and at pH 5.5. This work provides a realistic model that could be used for drug discovery aimed at finding toll‐like receptor protein 7 dimerization activators, with potential clinical applications to a host of diseases, including cancer.     

Molecular Modeling of 5HT2A Receptor – Arylpiperazine Ligands Interactions

In this paper, we report the molecular modeling of the 5HT_2A receptor and the molecular docking of arylpiperazine‐like ligands. The focus of the research was on explaining the effects the ligand structure has on the binding properties of the 5HT_2A receptor and on the key interactions between the ligands and the receptor‐binding site. To see what the receptor–ligand interactions were, various substituents were introduced in one part of the ligand, keeping the rest unchanged. In this way, using a docking analysis on the proposed 5HT_2A receptor model, we identified key receptor–ligand interactions and determined their properties. Those properties were correlated with experimentally determined binding affinities in order to determine the structure to activity relationship of the examined compounds.     

Prediction of the Human EP1 Receptor Binding Site by Homology Modeling and Molecular Dynamics Simulation

The prostanoid receptor EP1 is a G-protein-coupled receptor (GPCR) known to be involved in a variety of pathological disorders such as pain, fever and inflammation. These receptors are important drug targets, but design of subtype specific agonists and antagonists has been partially hampered by the absence of three-dimensional structures for these receptors. To understand the molecular interactions of the PGE2, an endogen ligand, with the EP1 receptor, a homology model of the human EP1 receptor (hEP1R) with all connecting loops was constructed from the 2.6 Å resolution crystal structure (PDB code: 1L9H) of bovine rhodopsin. The initial model generated by MODELLER was subjected to molecular dynamics simulation to assess quality of the model. Also, a step by step ligand-supported model refinement was performed, including initial docking of PGE2 and iloprost in the putative binding site, followed by several rounds of energy minimizations and molecular dynamics simulations. Docking studies were performed for PGE2 and some other related compounds in the active site of the final hEP1 receptor model. The docking enabled us to identify key molecular interactions supported by the mutagenesis data. Also, the correlation of r²=0.81 was observed between the Ki values and the docking scores of 15 prostanoid compounds. The results obtained in this study may provide new insights toward understanding the active site conformation of the hEP1 receptor and can be used for the structure-based design of novel specific ligands.     

Molecular Modeling on Berberine Derivatives toward BuChE: An Integrated Study with Quantitative Structure–Activity Relationships Models,Molecular Docking,and Molecular Dynamics Simulations

A dataset of 67 berberine derivatives for the inhibition of butyrylcholinesterase (BuChE) was studied based on the combination of quantitative structure–activity relationships models, molecular docking, and molecular dynamics methods. First, a series of berberine derivatives were reported, and their inhibitory activities toward butyrylcholinesterase (BuChE) were evaluated. By 2D‐ quantitative structure–activity relationships studies, the best model built by partial least‐square had a conventional correlation coefficient of the training set (R²) of 0.883, a cross‐validation correlation coefficient () of 0.777, and a conventional correlation coefficient of the test set () of 0.775. The model was also confirmed by Y‐randomization examination. In addition, the molecular docking and molecular dynamics simulation were performed to better elucidate the inhibitory mechanism of three typical berberine derivatives (berberine, C2, and C55) toward BuChE. The predicted binding free energy results were consistent with the experimental data and showed that the van der Waals energy term (ΔE_vdw) difference played the most important role in differentiating the activity among the three inhibitors (berberine, C2, and C55). The developed quantitative structure–activity relationships models provide details on the fine relationship linking structure and activity and offer clues for structural modifications, and the molecular simulation helps to understand the inhibitory mechanism of the three typical inhibitors. In conclusion, the results of this study provide useful clues for new drug design and discovery of BuChE inhibitors from berberine derivatives.     

Discovery of BMS-753426: A Potent Orally Bioavailable Antagonist of CC Chemokine Receptor 2

To improve the metabolic stability profile of BMS-741672 (1a), we undertook a structure–activity relationship study in our trisubstituted cyclohexylamine series. This ultimately led to the identification of 2d (BMS-753426) as a potent and orally bioavailable antagonist of CCR2. Compared to previous clinical candidate 1a, the tert-butyl amine 2d showed significant improvements in pharmacokinetic properties, with lower clearance and higher oral bioavailability. Furthermore, compound 2d exhibited improved affinity for CCR5 and good activity in models of both monocyte migration and multiple sclerosis in the hCCR2 knock-in mouse. The synthesis of 2d was facilitated by the development of a simplified approach to key intermediate (4R)-9b that deployed a stereoselective reductive amination which may prove to be of general interest.     

Unraveling the Role of Arg4 and Arg6 in the Auto‐Inhibition Mechanism of GSK3β From Molecular Dynamics Simulation

Glycogen synthase kinase 3β (GSK3β) is a multifunctional serine/threonine protein kinase that is involved in several biological processes including insulin and Wnt signaling pathways. GSK3β can be phosphorylated by the protein kinase B (PKB). The mutations of Arg4 and Arg6 to alanine at N‐terminal GSK3β have been reported to impair its ability to autophosphorylate at Ser9. Despite the extensive experimental observations, the detailed mechanism for the auto‐inhibition of GSK3β has not been rationalized at the molecular level. In this study, we have demonstrated the structural consequences of GSK3β R4A and R6A mutations and the atomic changes that influenced the loss of PKB‐binding affinity. Molecular dynamics simulation results suggested significant loss in atomic contacts in the R4A and R6A mutant systems compared to the wild‐type system. Furthermore, we observed many notable changes (such as conformation, residues motions, hydrogen bonds, and binding free energy) in the mutated GSK3β–PKB complexes. Loss of binding affinity in the mutated systems rendered the decrease in GSK3β phosphorylation, which, in turn, impaired the auto‐inhibition of GSK3β. The significant outcomes obtained from this study can explain the auto‐inhibition of GSK3β and maybe facilitate type 2 diabetes mellitus researches and in developing the potent drug therapies.     

Distribution and Effect of Water Content on Molecular Mobility in Poly(vinylpyrrolidone) Glasses: A Molecular Dynamics Simulation

Purpose This work explores the distribution of water and its effects on molecular mobilities in poly(vinylpyrrolidone) (PVP) glasses using molecular dynamics (MD) simulation technology.Methods PVP glasses containing 0.5% and 10% w/w water and a small amount of ammonia and Phe-Asn-Gly were generated. Physical aging processes and associated structural and dynamic properties were monitored vs. time for periods up to 0.1 μs by MD simulation.Results Increasing water content from 0.5% to 10% w/w was found to reduce the T_g by about 90 K and increase the rates of volume and enthalpy relaxation. At 0.5% w/w, water molecules are mostly isolated and uniformly distributed while at 10% w/w, water distribution is markedly heterogeneous, with strands of water molecules occupying channels between the polymer chains. At 10% w/w, each water molecule has an average of 2.0 neighboring water molecules. The plasticization effects of water were revealed in diffusion coefficient increases of 3.7-, 7.3-, and 7.6-fold for water, ammonia, and the individual polyvinylpyrrolidone segments, respectively, and in shorter relaxation times (37- to 47-fold) for rotation of polymer segments with an elevation in water content from 0.5% to 10% w/w. Water diffusivity was found to linearly correlate with the number of neighboring water molecules. Rotation of the PVP segments is comprised of a fast wobble motion within a highly restrained cavity and a slow rotation over a wider angular space. Only the slow rotation was shown to be significantly affected by water content.Conclusions Water distribution in the PVP glass is highly heterogeneous at 10% w/w water, reflecting the formation of water strands or small clusters rather than complete phase separation. Local enhancement of mobility with increasing water content has been demonstrated using MD simulations.     

Probing the Conformational Dynamics of the Bioactive Peptide TLQP‐21 in Solution: A Molecular Dynamics Study

VGF‐derived peptide, TLQP‐21, is a physiologically active neuropeptide exhibiting important roles in energy expenditure and balance, gastric contractility, reproduction, pain modulation, and stress. Although the physiological functions of the peptide constitute a research area of considerable interest, structural information is clearly lacking. Here, using extensive 550 nanoseconds molecular dynamics simulation in explicit water model, we have explored the folding energy landscape of the peptide. Principal component analysis and cluster analysis have been used to identify highly populated conformational states of the peptide in solution. The most populated structure of the peptide adopts a highly compact globular form stabilized by several hydrogen‐bonding interactions and π‐cationic interactions. Strong surface complementarity of hydrophobic residues allows tighter spatial fit of the residues within the core region of the peptide. Our simulation also predicts that the peptide is highly flexible in solution and that the region A₇‐R₉ and three C‐terminal residues, P₁₉‐R₂₁, possess strong helical propensity.     

Structural Behaviour of 2-Hydroxypropyl-β-Cyclodextrin in Water: Molecular Dynamics Simulation Studies

Purpose To investigate the effect of 2-hydroxypropyl side group substitutions on the structure of β-cyclodextrin (CD) in water. Methods Molecular dynamics simulations were carried out on four HPBCDs that broadly represent a range of degree of substitutions in order to investigate the effect of substitution of β-cyclodextrin with 2-hydroxypropyl groups at various O2 and O6 positions of the glucose units. Results The 2-hydroxypropyl side groups located at the O2 positions widen the cavity entrance at the secondary OH position of the CD molecule. These groups are spatially more spread out but dynamically more restricted, due to the formation of a hydrogen bond network between the hydroxyl groups of the side chains and the glucose units. On the other hand, the 2-hydroxypropyl groups at the O6 positions are dynamically more flexible. Conclusions The extent and the location of the substitution can affect the cavity structure of the CD molecule, and thus possibly the molecular encapsulation capabilities.     

A new approach for 11C–C bond formation: Synthesis of 17α‐(3′‐[11C]prop‐1‐yn‐1‐yl)‐3‐methoxy‐3,17β‐estradiol

A new approach for ¹¹C–C bond formation via a Sonogashira‐like cross‐coupling reaction of terminal alkynes with [¹¹C]methyl iodide was exemplified by the synthesis of 17α‐(3′‐[¹¹C]prop‐1‐yn‐1‐yl)‐3‐methoxy‐3,17β‐estradiol. The LC‐purified title compound was obtained in decay‐corrected radiochemical yields of 27–47% (n=8) based on [¹¹C]methyl iodide within 21–27 min after EOB. In a typical synthesis starting from 9.6 GBq [¹¹C]methyl iodide, 1.87 GBq of 17α‐(3′‐[¹¹C]prop‐1‐yn‐1‐yl)‐3‐methoxy‐3,17β‐estradiol was synthesized in radiochemical purity >99%. The specific radioactivity ranged between 10 and 19 GBq/µmol, and the labeling position was verified by ¹³C‐NMR analysis of the corresponding ¹³C‐labeled compound. Copyright © 2003 John Wiley & Sons, Ltd.     

The Stereoselectivity of CYP2C19 on R‐ and S‐isomers of Proton Pump Inhibitors

PPIs are mainly metabolized by CYP2C19. It has a stereoselectivity effect on R‐ and S‐isomers of PPIs according to previous studies. However, no study has been reported to elucidate the binding mechanism at the atomic level based on the CYP2C19 crystal structure. Recently, the advent of the first crystal structure of CYP2C19 allowed us to take in silico approaches including MD simulation, MM/GBSA calculation, energy decomposition, and alanine scanning to explore the stereoselectivity of CYP2C19 on R‐ and S‐isomers of PPIs. The key residues responsible for the selective binding for R‐ and S‐isomers of omeprazole, lansoprazole, and pantoprazole are disclosed by free energy and alanine scanning analysis. Structural analysis showed that chiral isomers of PPIs alter their conformations to have strong binding affinities with CYP2C19. Furthermore, a theoretical pharmacophore model of PPIs was obtained with the importance of pharmacophore feature being weighted, basing on our results. Our results are valuable for designing and synthesizing new generation of PPIs in the future.     

A Molecular Dynamics Investigation of Mycobacterium tuberculosis Prenyl Synthases: Conformational Flexibility and Implications for Computer‐aided Drug Discovery

With the rise in antibiotic resistance, there is interest in discovering new drugs active against new targets. Here, we investigate the dynamic structures of three isoprenoid synthases from Mycobacterium tuberculosis using molecular dynamics (MD) methods with a view to discovering new drug leads. Two of the enzymes, cis‐farnesyl diphosphate synthase (cis‐FPPS) and cis‐decaprenyl diphosphate synthase (cis‐DPPS), are involved in bacterial cell wall biosynthesis, while the third, tuberculosinyl adenosine synthase (Rv3378c), is involved in virulence factor formation. The MD results for these three enzymes were then compared with previous results on undecaprenyl diphosphate synthase (UPPS) by means of active site volume fluctuation and principal component analyses. In addition, an analysis of the binding of prenyl diphosphates to cis‐FPPS, cis‐DPPS, and UPPS utilizing the new MD results is reported. We also screened libraries of inhibitors against cis‐DPPS, finding ~1 μm inhibitors, and used the receiver operating characteristic–area under the curve (ROC‐AUC) method to test the predictive power of X‐ray and MD‐derived cis‐DPPS receptors. We found that one compound with potent M. tuberculosis cell growth inhibition activity was an IC₅₀ ~0.5‐ to 20‐μm inhibitor (depending on substrate) of cis‐DPPS, a ~660‐nm inhibitor of Rv3378c as well as a 4.8‐μm inhibitor of cis‐FPPS, opening up the possibility of multitarget inhibition involving both cell wall biosynthesis and virulence factor formation.     

The molecular mechanism of pH‐regulating C3d‐CR2 interactions: Insights from molecular dynamics simulation

The interactions of complement receptor 2 (CR2) and the degradation fragment C3d of complement component C3 mediate the innate and adaptive immune systems. Due to the importance of C3d‐CR2 interaction in the design of vaccines, many studies have indicated the interactions are pH‐dependent. Moreover, C3d‐CR2 interactions at pH 5.0 are unknown. To investigate the molecular mechanism of pH‐regulating C3d‐CR2 interaction, molecular dynamics simulations for C3d‐CR2 complex in different pH are performed. Our results revealed that the protonation of His9 in C3d at pH 6.0 slightly weakens C3d‐CR2 association as reducing pH from 7.4 to 6.0, initiated from a key hydrogen bond formed between Gly270 and His9 in C3d at pH 6.0. When reducing pH from 6.0 to 5.0, the protonation of His33 in C3d weakens C3d‐SCR1 association by changing the hydrogen‐bond network of Asp36, Glu37, and Glu39 in C3d with Arg13 in CR2. In addition, the protonation of His90 significantly enhances C3d‐SCR2 association. This is because the enhanced hydrogen‐bond interactions of His90 with Glu63 and Ser69 of the linker change the conformations of the linker, Cys112‐Asn116 and Pro87‐Gly91 regions. This study uncovers the molecular mechanism of the mediation of pH on C3d‐CR2 interaction, which is valuable for vaccine design.     

Insights into the Interaction Mechanism of Ligands with Aβ42 Based on Molecular Dynamics Simulations and Mechanics: Implications of Role of Common Binding Site in Drug Design for Alzheimer's Disease

Aggregation of β‐amyloid (Aβ) into oligomers and further into fibrils is hypothesized to be a key factor in pathology of Alzheimer's disease (AD). In this study, mapping and docking were used to study the binding of ligands to protofibrils. It was followed by molecular simulations to understand the differences in interactions of known therapeutic agents such as curcumin, fluorescence‐based amyloid staining agents such as thioflavin T, and diagnostic agents such as florbetapir (AV45), with Aβ protofibrils. We show that therapeutic agents bind to and distort the protofibrils, thus causing destabilization or prevention of oligomerization, in contrast to diagnostic agents which bind to but do not distort such structures. This has implications in the rational design of ligands, both for diagnostics and therapeutics of AD.     

Clinical assessment of the lag‐time and tmax of pellets with controlled release of glucose: in vitro/in vivo comparison using 13C–breath test

Maintaining a stable glycaemia in diabetes mellitus type 1 requires flexible insulin administration and carbohydrate intake to affected individuals. In real life, there might be some situations limiting the insulin–sugar balance control, e.g. night sleep or prolonged sporting activities. Glucose pellets with a pre‐determined time lag between the pellet administration and glucose release were developed to mimic a ‘snack eaten in advance’. In this article, a ¹³C–glucose breath test was introduced to translate laboratory dissolution testing to clinical confirmation of the glucose release pattern using 5% δ abundance to differentiate the appearance of in ¹³C exhaled breath. An independent two‐sample t‐test (p  = 0.20) confirmed an average clinical lag time of 300 min and an in vitro time of 338 min to be identical at a level of significance of α = 0.05. Moreover, using the same statistical method, the clinical t_max (564 min) and the in vitro t₅₀ (594 min) were also considered identical (p  = 0.34). It was concluded that dissolution testing is a relevant method to determine the time lags of dosage forms with controlled release of glucose and that the ¹³C–glucose breath test is a suitable clinical tool for lag time verification in clinical studies.     

Structure–function relationship of Chikungunya nsP2 protease: A comparative study with papain

Chikungunya virus is a growing human pathogen transmitted by mosquito bite. It causes fever, chills, nausea, vomiting, joint pain, headache, and swelling in the joints. Its replication and propagation depend on the protease activity of the Chikungunya virus‐nsP2 protein, which cleaves the nsP1234 polyprotein replication complex into individual functional units. The N‐terminal segment of papain is structurally identical with the Chikungunya virus‐nsP2 protease. Hence, molecular dynamics simulations were performed to compare molecular mechanism of these proteases. The Chikungunya virus‐snP2 protease shows more conformational changes and adopts an alternate conformation. However, N‐terminal segment of these two proteases has identical active site scaffold with the conserved catalytic diad. Hence, some of the non‐peptide inhibitors of papain were used for induced fit docking at the active site of the nsP2 to assess the binding mode. In addition, the peptides that connect different domains/protein in Chikungunya virus poly‐protein were also subjected for docking. The overall results suggest that the active site scaffold is the same in both the proteases and a possibility exists to experimentally assess the efficacy of some of the papain inhibitors to inhibit the Chikungunya virus‐nsP2.     

Structure–activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A

A series of analogues of neurokinin A(4–10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser⁵ were assessed at rat fundus NK2 receptors. In membrane binding studies with [¹²⁵I]-[Lys⁵,Tyr(I₂)⁷,MeLeu⁹,Nle¹⁰]-NKA(4–10), all compounds except [Asp⁵]NKA(4–10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4–10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe⁵]NKA(4–10) is in fact neurokinin B(4–10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn⁵]NKA(4–10) was equally as weak in contractile studies as the acidic [Asp⁵]NKA(4–10). However, [Glu⁵]NKA(4–10) and [Gln⁵]NKA(4–10) were no different from NKA(4–10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.     

Aporphinoid Antagonists of 5‐HT2A Receptors: Further Evaluation of Ring A Substituents and the Size of Ring C

A series of ring A‐modified analogs of nantenine as well as structural variants in ring C were synthesized and evaluated for antagonist activity at 5‐HT_2A and α_1A receptors. Halogenation improves 5‐HT_2A antagonist potency in molecules containing a C1 methoxyl/C2 methoxyl or C1 methoxyl/C2 hydroxyl moiety. Bromination or iodination (but not chlorination) with the latter moiety also significantly increased α_1A antagonist potency. Homologation or contraction of ring C adversely affected antagonist activity at both receptors, implying that a six‐membered ring C motif is beneficial for high antagonist potency at both receptors. Molecular docking studies suggest that the improved antagonist activity (by virtue of improved affinity) of C3‐halogenated aporphines in this study is attributable to favorable interactions with the C3 halogen and F339 and/or F340.