Effects of synergistic and non-synergistic anions on the iron binding site from serum transferrin: A molecular dynamic simulation analysis

The role of the anions in transferrin chemistry highlights the importance of the anion binding site in transferrin family. A synergistic anion as carbonate is an anion that is required for iron binding by transferrin while non-synergistic anions do not act as the synergistic anions to promote iron binding, but affect the iron binding and release. Some questions remain unclear about the difference between synergistic and non-synergistic anion functions. In the present work, molecular dynamic simulation techniques were employed in order to gain access into a molecular level understanding of the iron binding site of the human serum transferrin during the synergistic and non-synergistic anion binding. For this purpose, a comparative analysis was performed to illustrate the observed changes. In addition to the comparison between the synergistic and non-synergistic anions, structural differences between two synergistic anions, Carbonate and Oxalate were studied. Meanwhile,the simulation of the open (Apo), partially closed (Carbonate) and fully closed (Carbonate-Fe) forms of the transferrin structure allows a direct comparison between the iron binding site of these three states.On the basis of results, synergistic anions form high affinity binding site, while non-synergistic anions act like Apo state of the transferrin structure and change the proper conformation of the binding site. In order to act as a synergistic anion and form high affinity binding site, anion stereochemistry and interactions must be able to achieve a Carbonate-like configuration. Carbonate complex showed the highest binding affinity and electrostatic energy is the major favorable contributor to synergistic anion-transferrin interaction. Carbonate and Oxalatecomplexes as synergistic anions have many features in common, without a significant change in the transferrin structure. Only the residues in the vicinity of the binding site showed a little different conformation depending on whether the synergistic anion is Carbonate orOxalate.Finally, the results show thatASP63, GLY65 and HIS249 residues have the maximum displacement during the Carbonate and iron binding. ASP63 and HIS249 are the residues, which are coordinated to the iron and GLY65 is in the second shell residuesof the transferrin structure.     

Molecular recognition pattern of cytotoxic alkaloid vinblastine with multiple targets

Vinblastine (VLB), a cytotoxic alkaloid is used extensively against various cancer types and the crystal structure of its tubulin complex is already known. Multitarget affinity of vinblastine has been investigated and the nature of binding with biological receptors namely, duplex DNA and Human serum albumin (HSA) has been compared to the binding characteristics of its known complex with natural high affinity receptor tubulin using molecular docking and QM–MM calculations. VLB is found to interact with DNA as well as HSA protein, though, with weaker affinity as compared to tubulin. Analysis of various docked complexes revealed that the H-bonds and cation–pi bonds do not have significant contribution to the binding interactions and despite its large size, VLB remains in relaxed conformation and fits in the hydrophobic regions on the receptors.     

A theory of mode of action of azolylalkylquinolines as DNA binding agents using automated flexible ligand docking

Azolylalkylquinolines (AAQs) are a family of quinolines with varying degrees of cytotoxic activity (comparable or moderately superior to adriamycin in some cases) developed in the past decade in our group where their exact mode of action is still unclear. In this study the most probable DNA binding mode of AAQs was investigated employing a novel flexible ligand docking approach by using AutoDock 3.0. Forty-nine AAQs with known experimental inhibitory activity were docked onto d(CGCAAATTTGCG)(2), d(CGATCG)(2) and d(CGCG)(2) oligonucleotides retrieved from the Protein Data Bank (PDB IDs: 102D, 1D12 and 1D32, respectively) as the representatives of the three plausible models of interactions between chemotherapeutic agents and DNA (groove binding, groove binding plus intercalation and bisintercalation, respectively). Good correlation (r(2)=0.64) between calculated binding energies and experimental inhibitory activities was obtained using groove binding plus intercalation model for phenyl-azolylalkylquinoline (PAAQ) series. Our findings show that the most probable mode of action of PAAQs as DNA binding agents is via intercalation of quinolinic moiety between CG base pairs with linker chain and azole moiety binding to the minor groove.     

Binding of the tautomeric forms of isoniazid-NAD adducts to the active site of the Mycobacterium tuberculosis enoyl-ACP reductase (InhA): a theoretical approach

The front-line antituberculosis drug isoniazid (INH) inhibits InhA, the NADH-dependent fatty acid biosynthesis enoyl ACP-reductase from Mycobacterium tuberculosis, via formation of covalent adducts with NAD (INH-NAD adducts). While ring tautomers were found the main species formed in solution, only the 4S chain INH-NAD tautomer was evidenced in the crystallized InhA:INH-NAD complex. In this study we attempted to explore the modes of interaction and energy binding of the different isomers placed in the active site of InhA with the help of various molecular modelling techniques. Ligand and enzyme models were generated with the help of the Vega ZZ program package. Resulting ligands were then docked into the InhA active site individually using computational automated docking package AUTODOCK 3.0.5. The more relevant docked conformations were then used to compute the interaction energy between the ligands and the InhA cavity. The AM1 Hamiltonian and the QM/MM ONIOM methodologies were used and the results compared. The various tautomers were found docked in almost the same place where INH-NAD was present as predicted by earlier X-ray crystallographic studies. However, some changes of ligand conformation and of the interactions ligand-protein were evidenced. The lower binding energy was observed for the 4S chain adduct that probably represents the effective active form of the INH-NAD adducts, as compared to the 4R epimer. The two 4S,7R and 4R,7S ring tautomers show intermediate and similar binding energies contrasting with their different experimental inhibitory potency on InhA. As a possible explanation based on calculated conformations, we formulated the hypothesis of an initial binding of the two ring tautomers to InhA followed by opening of only the ring hemiamidal 4S,7R tautomer (possibly catalyzed by Tyr158 phenolate basic group) to give the 4S chain INH-NAD tight-binding inhibitor. The predictions of ligand-protein interactions at the molecular level can be of primary importance in elucidating the mechanisms of action of isoniazid and InhA-related resistances, in identifying the effective mycobactericidal entities and, in further step, in the design of a new generation of antitubercular drugs.     

Prediction of the binding mode of biarylpropylsulfonamide allosteric AMPA receptor modulators based on docking, GRID molecular interaction fields and 3D-QSAR analysis

A novel approach of combining flexible molecular docking, GRID molecular interaction fields, analysis of ligand-protein hydrogen bond interactions, conformational energy penalties and 3D-QSAR analysis was used to propose a binding mode in the dimer interface of the iGluR2 receptor for the biarylpropylsulfonamide class of positive allosteric AMPA modulators. Possible binding poses were generated by flexible molecular docking. GRID molecular interaction fields of the binding site, ligand-protein hydrogen bonding interactions and conformational energy penalties were used to select the most likely binding mode. The selected binding poses were subjected to a 3D-QSAR analysis using previously published activity data. The resulting model (2 LVs, R2=0.89, q2=0.61) predicted the activities of the compounds in the test set with a standard deviation on error of prediction of 0.17. The proposed binding mode was validated by interpretation of the PLS-coefficient regions from the 3D-QSAR analysis in terms of interactions between the receptor and the modulators.     

Modeling binding modes of angiotensin II and pseudopeptide analogues to the AT2 receptor

The 3D model of the AT2 receptor has been built employing homology to the transmembrane domain of rhodopsin and a novel build-up procedure for restoring the extracellular loops. By docking a model peptide of angiotensin II in the AT2 receptor model two plausible binding modes were identified. These binding modes were in agreement with most of the suggested ligand-receptor contact points reported in the literature. Eight active and one inactive pseudopeptide angiotensin II analogue were also docked in the receptor and four of the active pseudopeptides were found to mimic the binding mode of angiotensin II. An alternative binding mode for the other four active pseudopeptides was found.     

Interactions of acetylcholine binding site residues contributing to nicotinic acetylcholine receptor gating: Role of residues Y93, Y190, K145 and D200

The nicotinic acetylcholine receptor exhibits multiple conformational states, resting (channel closed), active (channel open) and desensitized (channel closed). The resting state may be distinguished from the active and desensitized states by the orientation of loop C in the extracellular ligand binding domain (LBD). Homology modeling was used to generate structures of the Torpedo californica α₂βδγ nAChR that initially represent the resting state (loop C open) and the desensitized state (loop C closed). Molecular dynamics (MD) simulations were performed on the extracellular LBD on each nAChR conformational state, with and without the agonist anabaseine present in each binding site (the αγ and the αδ sites). Three MD simulations of 10 ns each were performed for each of the four conditions. Comparison of dynamics revealed that in the presence of agonist, loop C was drawn inward and attains a more stable conformation. Examination of side-chain interactions revealed that residue αY190 exhibited hydrogen-bonding interactions either with residue αY93 in the ligand binding site or with residue αK145 proximal to the binding site. αK145 also exhibited side chain (salt bridge) interactions with αD200 and main chain interactions with αY93. Residues αW149, αY198, γY116/δT119, γL118/δL121 and γL108/δL111 appear to play the role of stabilizing ligand in the binding site. In MD simulations for the desensitized state, the effect of ligand upon the interactions among αK145, αY190, and αY93 as well as ligand-hydrogen-bonding to αW149 were more pronounced at the αγ interface than at the αδ interface. Differences in affinity for the desensitized state were determined experimentally to be 10-fold. The changes in side chain interactions observed for the two conformations and induced by ligand support a model wherein hydrogen bond interactions between αD200 and αY93 are broken and rearrange to form a salt-bridge between αK145 and αD200 and hydrogen bond interactions between αY93 and αY190 and between αK145 and αY190.     

Docking,molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin

CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the β-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable “tense” state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin.     

In silico identification and computational analysis of the nucleotide binding site in the C-terminal domain of Hsp90

Hsp90 contains two distinct Nucleotide Binding Sites (NBS), in its N-terminal domain (NTD) and C-terminal domain (CTD), respectively. The NTD site belongs to the GHKL super-family of ATPases and has been the subject of extensive characterization. However, a structure of the nucleotide-bound form of CTD is still unavailable. In this study molecular modeling was employed to incorporate experimental data using partial constructs of the CTD, from work published by many research groups, onto existing structural models of its apo- form. Our attempts to locate potential nucleotide ligand-binding sites or cavities yielded one major candidate—a structurally unconventional site—exhibiting the requisite shape and volume for accommodation of tri-phosphate nucleotides. Its structure was refined by molecular dynamics (MD)-based techniques. We reproducibly docked the Mg²⁺ complexed form of ATP, GTP, CTP, TTP and UTP to this putative NBS. These docking simulations and calculated ligand-binding scores are in general agreement with published data about experimentally measured binding to the CTD. The overall pattern of interactions between residues lining the site and docked nucleotides is conserved and broadly similar to that of other nucleotide-binding sites. Our docking simulations suggest that nucleotide binding stabilizes the only structurally labile region, thereby providing a rationale for the increased resistance to thermal denaturation and proteolysis. The docked nucleotides do not intrude onto the surface of residues involved in dimerization or chaperoning. Our molecular modeling permitted recognition of larger structural changes in the nucleotide-bound CTD dimer, including stabilization of helix-2 in both chains and intra- and inter- chain interactions between three residues (I613, Q617, R620).     

A structural insight into the inhibitory mechanism of an orally active PI3K/mTOR dual inhibitor,PKI-179 using computational approaches

The PI3K/AKT/mTOR signaling pathway has been identified as an important target for cancer therapy. Attempts are increasingly made to design the inhibitors against the key proteins of this pathway for anti-cancer therapy. The PI3K/mTOR dual inhibitors have proved more effective than the inhibitors against only single protein targets. Recently discovered PKI-179, an orally effective compound, is one such dual inhibitor targeting both PI3K and mTOR. This anti-cancer compound is efficacious both in vitro and in vivo. However, the binding mechanisms and the molecular interactions of PKI-179 with PI3K and mTOR are not yet available. The current study investigated the exact binding mode and the molecular interactions of PKI-179 with PI3Kγ and mTOR using molecular docking and (un)binding simulation analyses. The study identified PKI-179 interacting residues of both the proteins and their importance in binding was ranked by the loss in accessible surface area, number of molecular interactions of the residue, and consistent appearance of the residue in (un)binding simulation analysis. The key residues involved in binding of PKI-179 were Ala-805 in PI3Kγ and Ile-2163 in mTOR as they have lost maximum accessible surface area due to binding. In addition, the residues which played a role in binding of the drug but were away from the catalytic site were also identified using (un)binding simulation analyses. Finally, comparison of the interacting residues in the respective catalytic sites was done for the difference in the binding of the drug to the two proteins. Thus, the pairs of the residues falling at the similar location with respect to the docked drug were identified. The striking similarity in the interacting residues of the catalytic site explains the concomitant inhibition of both proteins by a number of inhibitors. In conclusion, the docking and (un)binding simulation analyses of dual inhibitor PKI-179 with PI3K and mTOR will provide a suitable multi-target model for studying drug–protein interactions and thus help in designing the novel drugs with higher potency.     

Combining docking, scoring and molecular field analyses to probe influenza neuraminidase-ligand interactions

In this project, several docking conditions, scoring functions and corresponding protein-aligned molecular field analysis (CoMFA) models were evaluated for a diverse set of neuraminidase (NA) inhibitors. To this end, a group of inhibitors were docked into the active site of NA. The docked structures were utilized to construct a corresponding protein-aligned CoMFA models by employing probe-based (H+, OH, CH3) energy grids and genetic partial least squares (G/PLS) statistical analysis. A total of 16 different docking configurations were evaluated, of which some succeeded in producing self-consistent and predictive CoMFA models. However, the best model coincided with docking the ionized ligands into the hydrated form of the binding site via PLP1 scoring function (r2LOO=0.735, r2PRESS against 24 test compounds=0.828). The highest-ranking CoMFA models were employed to probe NA-ligand interactions. Further validation by comparison with a co-crystallized ligand-NA crystallographic structure was performed. This combination of docking/scoring/CoMFA modeling provided interesting insights into the binding of different NA inhibitors.     

Comparison of templates for homology model of ρ1 GABAC receptors: More insights to the orthosteric binding site’s structure and functionality

Five sets of ρ1 GABA_C homology models were generated based on X-ray crystal structures of the acetylcholine binding protein (AChBP), the ion channel from Caenorhabditis elegans (GLIC), the ion channel from Erwinia chrysanthemi (ELIC), the homomeric GABA_A β₃ ion channel, and the homomeric α-subunit of glutamate-gated homopentameric chloride channel (GluCl). The GluCl based model was found to the represent the structure of ρ1 GABA_C receptors. The GABA pose docked in the selected best model was confirmed by QM-polarized ligand docking and induced fit docking protocol, and used to study molecular interactions in the ρ1 GABA binding site. The potential interactions of identified residues are discussed. This study identified several residues with potential ligand interactions located on loops F and G with their side chain oriented toward the binding site such as Ser215 and Gln83. The partial agonists muscimol and imidazole-4-acetic acid (I4AA) were docked into the binding site of the most reliable ‘GABA bound’ homology model. The potency and efficacy of these partial agonists in activating recombinant ρ1 receptors were correlated with their docking results. The model predicts that muscimol resembles GABA in the docking pose with similar interactions. However, I4AA has a very different docking pose to GABA and was predicted by the model to form π–π stacking with aromatic residues in the orthosteric binding site. A set of TPMPA bound ρ1 homology models based on the GluClα ‘apo state’ template was built in order to study a competitive antagonist in the ρ1 orthosteric binding site. The results demonstrated the ability of our model to explain most experimental findings and predict potential roles of residues within the orthosteric binding site.     

Identification of a putative binding site for 5-alkyl-benzothiadiazides in the AMPA receptor dimer interface

Crystal structures of three different allosteric modulators co-crystallized with the iGluR2 ligand-binding domain are currently available. The modulators, cyclothiazide, aniracetam and CX614, bind at overlapping binding sites in the dimer interface between two iGluR2 subunits. However, pharmacological data indicate that there are one or more additional binding sites for this class of compounds. Based on differences in structure-activity relationship data we show that 5-alkyl-benzothiadiazide (5ABTD) modulators and a series of close analogs of cyclothiazide, despite having a common core structure, do not have the same binding site. In the present work, a new potential binding site for allosteric modulators has been identified in the dimer interface of the iGluR2 ligand-binding domain. By comparing different iGluR2 crystal structures including different co-crystallized agonists, this cavity is shown to be a structurally conserved part of the dimer interface. The cavity is characterized with respect to shape and potential favorable interactions with ligands and docking is used to find a reasonable binding mode for the core structure of the 5ABTDs. The extensive structure-activity data available for this series of compounds are in agreement with the proposed binding mode, supporting the conclusion that the identified cavity most likely is the binding site for the 5ABTDs.     

A piezoelectric sensor with propidium as a recognition element for cholinesterases

A piezoelectric biosensor has been developed on the basis of the reversible acetylcholinesterase (AChE) inhibitor propidium. The propidium cation was bound to a 11-mercaptoundecanoic acid monolayer on gold-coated quartz crystals. The immobilization was done via activation of carboxyl groups by 1,3-dicyclohexylcarbodiimide (DCC). Different types of cholinesterases (acetyl- and butyryl-ChE) from different origins were tested for their binding ability towards the immobilized propidium. Binding studies were performed in a flow system. Furthermore, catalytically active and organophosphate-inhibited enzyme were compared regarding their binding capability. The binding constants were derived by using an one to one binding model and a refined model also including rebinding effects. It was shown that organophosphorylation of the active site hardly influences the affinity of AChE towards propidium. Furthermore the propidium-based biosensor provides equal sensitivity as compared with piezolelectric sensors with immobilized paraoxon—an active site ligands of AChE.     

Conformational analyses and docking studies of a series of 5-nitrofuran- and 5-nitrothiophen-semicarbazone derivatives in three possible binding sites of trypanothione and glutathione reductases

To explore three possible binding sites of trypanothione and glutathione reductase, namely, the active, the dimer interface and the coenzyme NADPH binding site, a series of eight compounds, nitrofurans and nitrothiophenes derivatives, were docked, using their crystallographic and modeled conformations. Docking results showed that, for both families and both enzymes, compounds are more likely to bind in the interface site, even though there is some probability of binding in the active site. These studies are in agreement with experimental data, which suggest that these class of compounds can act either as uncompetitive or mixed type inhibitors, and also with the finding that there is an alpha-helix which connects the active with the interface site, thus allowing charge transference between them.     

Structural determinants of the alpha2 adrenoceptor subtype selectivity

Alpha2-adrenergic receptor (α2-AR) subtypes, acting mainly on the central nervous and cardiovascular systems, represent important targets for drug design, confirmed by the high number of studies published so far. Presently, only a few α2-AR subtype selective compounds are known. Using homology modeling and ligand docking, the present study analyzes the similarities and differences between binding sites, and between extracellular loops of the three subtypes of α2-ARs. Several α2-AR subtype selective ligands were docked into the active sites of the three α2-AR subtypes, key interactions between ligands and receptors were mapped, and the predicted results were compared with the available experimental data. Binding site analysis reveals a strong identity between important amino acid residues in each receptor, the very few differences being the key toward modulating selectivity of α2-AR ligands. The observed differences between binding site residues provide an excellent starting point for virtual screening of chemical databases, in order to identify potentially selective ligands for α2-ARs.     

Three-dimensional models of histamine H3 receptor antagonist complexes and their pharmacophore

Molecular modeling was used to analyze the binding mode and activities of histamine H₃ receptor antagonists. A model of the H₃ receptor was constructed through homology modeling methods based on the crystal structure of bovine rhodopsin. Known H₃ antagonists were interactively docked into the putative antagonist binding pocket and the resultant model was subjected to molecular mechanics energy minimization and molecular dynamics simulations which included a continuum model of the lipid bilayer and intra- and extracellular aqueous environments surrounding the transmembrane helices. The transmemebrane helices stayed well embedded in the dielectric slab representing the lipid bilayer and the intra- and extracellular loops remain situated in the aqueous solvent region of the model during molecular dynamics simulations of up to 200 ps in duration. A pharmacophore model was calculated by mapping the features common to three active compounds three-dimensionally in space. The 3D pharmacophore model complements our atomistic receptor/ligand modeling. The H₃ antagonist pharmacophore consists of two protonation sites (i.e. basic centers) connected by a central aromatic ring or hydrophobic region. These two basic sites can simultaneously interact with Asp 114 (3.32) in helix III and a Glu 206 (5.46) in helix V which are believed to be the key residues that histamine interacts with to stabilize the receptor in the active state. The interaction with Glu 206 is consistent with the enhanced activity resulting from the additional basic site. In addition to these two salt bridging interactions, the central region of these antagonists contains a lipophilic group, usually an aromatic ring, that is found to interact with several nearby hydrophobic side chains. The picture of antagonist binding provided by these models is consistent with earlier pharmacophore models for H₃ antagonists with some exceptions.     

Investigating detailed interactions between novel PAR1 antagonist F16357 and the receptor using docking and molecular dynamic simulations

Currently, Vorapaxar is the only recently FDA-approved antiplatelet drug targeting Protease-activated receptor 1 (PAR1). However, a novel antagonist, F16357, has been shown to prevent painful bladder syndrome, also known as interstitial cystitis (IC). Unfortunately, there is no high resolution structure of the F16357-receptor complex, hindering its optimization as a therapeutic agent. In this study, we used docking and molecular dynamic (MD) simulations to investigate the detailed interactions between F16357 and PAR1 at a molecular level. The recently solved crystal structure of human PAR1 complexed with Vorapaxar was used in our docking of F16357 into the binding pocket of the receptor. To enhance binding pose selection, F16357 was docked first without constraints and then with a positional constraint to invert its orientation to become similar to that of Vorapaxar. The three systems, with crystal Vorapaxar, F16357 and an inverted F16357, were subjected to 3.0 μs MD simulations. The MM-GBSA binding energy analysis showed that F16357 binds more strongly in a pose obtained from an unrestrained docking than in the inverted pose from a restrained docking; and Vorapaxar binds more strongly than F17357. This ordering is consistent with the experimental pIC₅₀ values. Our structural data showed subtle changes in the binding pose between Vorapaxar and F16357. Transmembrane helices 1, 2, 5, and 7 were most significantly affected; most notably a large kink at F279^5.47 in TM helix 5 of the Vorapaxar complex was completely absent in the F16357 complex. The results of this study facilitate the future development of other therapeutic PAR1 antagonists.     

Effect of NaeI-L43K mutation on protein dynamics and DNA conformation: Insights from molecular dynamics simulations

Protein-DNA interactions are an important class of biomolecular interactions inside the cell. Delineating the mechanisms of protein-DNA interactions and more specifically, how proteins search and bind to their specific cognate sequences has been the quest of many in the scientific community. Restriction enzymes have served as useful model systems to this end. In this work, we have investigated using molecular dynamics simulations the effect of L43K mutation on NaeI, a type IIE restriction enzyme. NaeI has two domains, the Topo and the Endo domains, each binding to identical strands of DNA sequences (GCCGGC)₂. The binding of the DNA to the Topo domain is thought to enhance the binding and cleavage of DNA at the Endo domain. Interestingly, it has been found that the mutation of an amino acid that is distantly-located from the DNA cleavage site (L43K) converts the restriction endonuclease to a topoisomerase. Our investigations reveal that the L43K mutation not only induces local structural changes (as evidenced by changes in hydrogen bond propensities and differences in the percentage of secondary structure assignments of the residues in the ligase-like domain) but also alters the overall protein dynamics and DNA conformation which probably leads to the loss of specific cleavage of the recognition site. In a larger context, our study underscores the importance of considering the role of distantly-located amino acids in understanding protein-DNA interactions.