Analysis of the activating mutations within the activation loop of leukemia targets Flt-3 and c-Kit based on protein homology modeling

Molecular modeling provides a mechanistic hypothesis at the molecular level for the constitutive activation recently observed and reported for tyrosine protein kinases Flt-3 and c-Kit. Three-dimensional homology models for the active and inactive forms of these two kinases were made. Comparison of these models at the molecular level reveals that mutations of specific residues located in the activation loop (D835X and 836-deletion in Flt-3; D816V in c-Kit) as well as a 6-base pair (6-bp) insertion at residue 840 in Flt-3 operate in a similar way. Each mutation tends to weaken the forces that maintain the activation-loop folded inwards. None of the mutations are found to particularly stabilize the active state directly. The reason why the equilibrium is shifted towards the gate-open conformation of the protein is because, at least in these models, the mutations are found to critically destabilize the inactive conformational state of the kinase.     

Molecular insight into mutation-induced conformational change in metastasic bowel cancer BRAF kinase domain and its implications for selective inhibitor design

Oncogenic BRAF V600E mutation confers constitutive activation for the kinase and is closely related to the pathogenesis of metastasic bowel cancer (MBC). Here, the V600E-induced conformational change in MBC BRAF kinase domain is characterized systematically at structural, energetic and dynamic levels. The mutation is observed to cause a conformational conversion of the kinase’s activation loop from DFG-out to DFG-in, thus activating the kinase. Electrostatic force is primarily responsible for the conformational conversion and stabilization of DFG-in associated with the mutation. Molecular docking calculations are employed to analyze the binding mode difference of mutant-selective inhibitors between the DFG-out and DFG-in conformations of BRAF kinase. It is revealed that the mutation can reshape inhibitor selectivity profile by altering kinase loop conformation. Several compounds are determined to have a high or moderate selectivity for mutant over wild-type kinase. The selectivity is primarily originated from hydrogen bond interactions of inhibitor ligands with mutant rather than wild type due to the conformational difference in kinase domain.     

Molecular dynamics simulations of mutated Mycobacterium tuberculosis l-alanine dehydrogenase to illuminate the role of key residues

l-Alanine dehydrogenase from Mycobacterium tuberculosis (l-MtAlaDH) catalyzes the NADH-dependent interconversion of l-alanine and pyruvate, and it is considered to be a potential target for the treatment of tuberculosis. The experiment has verified that amino acid replacement of the conserved active-site residues which have strong stability and no great changes in biological evolutionary process, such as His96 and Asp270, could lead to inactive mutants [Ågren et al., J. Mol. Biol. 377 (2008) 1161–1173]. However, the role of these conserved residues in catalytic reaction still remains unclear. Based on the crystal structures, a series of mutant structures were constructed to investigate the role of the conserved residues in enzymatic reaction by using molecular dynamics simulations. The results show that whatever the conserved residues were mutated, the protein can still convert its conformation from open state to closed state as long as NADH is present in active site. Asp270 maintains the stability of nicotinamide ring and ribose of NADH through hydrogen bond interactions, and His96 is helpful to convert the protein conformation by interactions with Gln271, whereas, they would lead to the structural rearrangement in active site and lose the catalytic activity when they were mutated. Additionally, we deduce that Met301 plays a major role in catalytic reaction due to fixing the nicotinamide ring of NADH to prevent its rotation, and we propose that Met301 would be mutated to the hydrophobic residue with large steric hindrance in side chain to test the activity of the protein in future experiment.     

Computational analysis of Asp519 and Glu665 mutations of coagulation factor FVIIIa: Implications for enhanced binding affinity of A2-domain

The A2-domain of blood coagulation factor VIIIa is non-covalently bound to the A1 and A3 domains via weak intermolecular interactions. Functional instability due to rapid dissociation of A2-domain from the active FVIII in blood presents a major hurdle for the therapeutic applications of FVIIIa to treat Hemophilia-A. To identify the ideal hot-spot residues at the interface of A2 and A1/A3 domains that could enhance the structural stability of FVIIIa, we performed a comprehensive computational mutagenesis study of two A2-domain residues, Asp519 and Glu665, that interface the A1 and A3-domains. Each residue was mutated to 15 uncharged amino-acids and the mutant structures were refined by MD simulations. Based on the estimated relative binding affinities of mutant structures, we predict that the mutation of Asp519 to Leu, Gln, Thr, Val and the mutation of Glu665 to Val, Ile, Met, Asn and Trp enhance the A2-domain binding affinity by more than 20 kcal/mol, compared to the WT structure. We anticipate that these predictions will be valuable for enzymatic studies towards the rational design of FVIIIa synthetic constructs with improved A2-domain binding affinity.     

Flexibility of the murine prion protein and its Asp178Asn mutant investigated by molecular dynamics simulations.

Inherited forms of transmissible spongiform encephalopathy, e.g. familial Creutzfeldt-Jakob disease, Gerstmann-Str?ussler-Scheinker syndrome and fatal familial insomnia, segregate with specific point mutations of the prion protein. It has been proposed that the pathologically relevant Asp178Asn (D178N) mutation might destabilize the structure of the prion protein because of the loss of the Arg164-Asp178 salt bridge. Molecular dynamics simulations of the structured C-terminal domain of the murine prion protein and the D178N mutant were performed to investigate this hypothesis. The D178N mutant did not deviate from the NMR conformation more than the wild type on the nanosecond time scale of the simulations. In agreement with CD spectroscopy experiments, no major structural rearrangement could be observed for the D178N mutant, apart from the N-terminal elongation of helix 2. The region of structure around the disulfide bridge deviated the least from the NMR conformation and showed the smallest fluctuations in all simulations in agreement with hydrogen exchange data of the wild type prion protein. Large deviations and flexibility were observed in the segments which are ill-defined in the NMR conformation. Moreover, helix 1 showed an increased degree of mobility, especially at its N-terminal region. The dynamic behavior of the D178N mutant and its minor deviation from the folded conformation suggest that the salt bridge between Arg164 and Asp178 might not be crucial for the stability of the prion protein.     

Structural and dynamical properties of different protonated states of mutant HIV-1 protease complexed with the saquinavir inhibitor studied by molecular dynamics simulations

To understand the basis of drug resistance, particularly of the HIV-1 PR, three molecular dynamics (MD) simulations of HIV-1 PR mutant species, G48V, complexed with saquinavir (SQV) in explicit aqueous solution with three protonation states, diprotonation on Asp25 and Asp25' (Di-pro) and monoprotonation on each Asp residue (Mono-25 and Mono-25'). For all three states, H-bonds between saquinavir and HIV-1 PR were formed only in the two regions, flap and active site. It was found that conformation of P2 subsite of SQV in the Mono-25 state differs substantially from the other two states. The rotation about 177 degrees from the optimal structure of the wild type was observed, the hydrogen bond between P2 and the flap residue (Val48) was broken and indirect hydrogen bonds with the three residues (Asp29, Gly27, and Asp30) were found instead. In terms of complexation energies, interaction energy of -37.3 kcal/mol for the Mono-25 state is significantly lower than those of -30.7 and -10.7kcal/mol for the Mono-25' and Di-pro states, respectively. It was found also that protonation at the Asp25 leads to a better arrangement in the catalytic dyad, i.e., the Asp25-Asp25' interaction energy of -8.8 kcal/mol of the Mono-25 is significantly lower than that of -2.6kcal/mol for the Mono-25' state. The above data suggest us to conclude that interaction in the catalytic area should be used as criteria to enhance capability in drug designing and drug screening instead of using the total inhibitor/enzyme interaction.     

Solution conformation of phakellistatin 8 investigated by molecular dynamics simulations

Phakellistatin 8 is a cyclic decapeptide that inhibits cancer cell growth and has sequence and structure similar to antamanide. In molecular dynamics simulations of phakellistatin 8 in water, the decapeptide ring undergoes a conformational change from the saddle-like crystal structure to a more elongated conformation by a transition of the Tyr9 main chain from the alpha L to an extended structure. This is coupled to the loss of the NH9-O6 beta-turn hydrogen bond and the transient dissociation of the Pro7-Tyr9 side-chain packing. Furthermore, the water molecule acting as a transannular bridge forms an additional hydrogen bond with phakellistatin 8, namely with the NH group of Val5 besides those already present in the crystal structure, i.e., with the NH of Ile10 and the CO of Leu6. The alpha-turn hydrogen bond between the Phe4 amide hydrogen and the Ile10 carbonyl oxygen is always present. The solution conformations of the two cyclic decapeptides are similar, in particular in the region involving the NH4-O10 alpha turn of phakellistatin 8 and the NH5-O1 alpha turn of antamanide. The simulation results suggest that in aqueous solution the conformation of phakellistatin 8 is more extended than in the crystalline state, and on a nanosecond time scale phakellistatin 8 is more flexible than antamanide.     

Interaction between peroxisome proliferator-activated receptor γ and its agonists: docking study of oximes having 5-benzyl-2,4-thiazolidinedione

The molecular modelling of oximes having 5-benzyl-2,4-thiazolidinedione moieties, agonists of the peroxisome proliferator-activated receptor γ (PPARγ), was performed with respect to their structures complexed with the ligand binding domain of PPARγ. For each ligand molecule, the 5-benzyl-2,4-thiazolidinedione head group was used as an anchor and the conformation of the rest of the molecule was searched for the most energetically favorable interaction with the receptor by systematic conformation search and manual modelling. Although both tail-up and tail-down configurations, which have been observed in the crystal structure of eicosapentaenoic acid when complexed with PPARδ, appeared among the lowest energy structures for most of the compounds, potent agonists were found to adopt a configuration similar to that of rosiglitazone when bound to PPARγ, according to the crystal structure. The structure–activity relationships were analyzed based on the receptor–ligand interaction. The alkyl group and the aromatic ring of the tail group of the ligands had hydrophobic interactions with the receptor, and these interactions were found to be essential for the strong activity.     

Structural investigation of PAP derivatives by CoMFA and CoMSIA reveals novel insight towards inhibition of Bcr-Abl oncoprotein

Molecular modeling by 3D-QSAR comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were employed on a series of phenylaminopyrimidine-based (PAP) Bcr-Abl inhibitors. The chemical structures of 63 PAP analogues were aligned using a template extracted from the crystal structure of STI571 bound to Abl kinase. Subsequently, the structures built were divided into training and test sets that include 53 and 10 compounds, respectively. Statistical results showed that the 3D-QSAR models generated from CoMSIA were superior to CoMFA (CoMSIA; q2=0.66, r2=0.94, N=3, F=139.09, r2pred=0.64 while CoMFA; q2=0.53, r2=0.73, N=3, F=43.53, r2pred=0.61). Based on the contour interpretation, the attachment of hydrophobic and bulky groups to the phenyl and pyrrolidine (D- and E-ring of NS-187, respectively) along with highly electronegative groups around the D-ring are important structural features for the design of second-generation Bcr-Abl inhibitors. The generated models are predictive based on reproducible values of the predicted compared with experimental activities in the test set. Further, the complementary analysis of contour maps to the Bcr-Abl binding site suggested the anchor points for binding affinity.     

DFT,QTAIM, and NBO studies on the trimeric interactions in the protrusion domain of a piscine betanodavirus

Crystal structure of the protrusion domain (P-domain) of the grouper nervous necrosis virus (GNNV) shows the presence of three-fold trimeric protrusions with two asymmetrical calcium cations along the non-crystallographic three-fold axis. The trimeric interaction natures of the interacting residues and the calcium cations with the neighboring residues within the trimeric interface have been studied by the quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses in the framework of the density-functional theory (DFT) approach. The results revealed that residues Leu259, Val274, Trp280, and Gln322 of subunit A, Arg261, Asp275, Ala277, and Gln322 of subunit B, Leu259, Asp260, Arg261, Ala277, Val278, and Leu324 of subunit C are the main residues involved in the trimeric interactions. Charge-dipole, dipole-dipole, and hydrogen bonding interactions make the significant contributions to these trimeric interactions. Among different interacting residues within trimeric interface, residue pair Arg261 B-Leu259C forms the strongest hydrogen bond inside the interface between subunits B and C. It was also found that calcium cations interact with residues Asp273, Val274, and Asp275 of subunits A, B, and C through charge-charge and charge transfer interactions.     

Modeling the protonation states of β-secretase binding pocket by molecular dynamics simulations and docking studies

β-secretase (BACE1) is an aspartyl protease that processes the β-amyloid peptide in the human brain in patients with Alzheimer’s disease. There are two catalytic aspartates (ASP32 and ASP228) in the active domain of BACE1. Although it is believed that the net charge of the Asp dyad is −1, the exact protonation state still remains a matter of debate. We carried out molecular dynamic (MD) simulations for the four protonation states of BACE1 proteins. We applied Glide docking studies to 21 BACE1 inhibitors against the MD extracted conformations. The dynamic results infer that the protein/ligand complex remains stable during the entire simulation course for HD32D228 model. The results show that the hydrogen bonds between the inhibitor and the Asp dyad are maintained in the 10,000th ps snapshot of HD32D228 model. Our results also reveal the significant loop residues in maintaining the active binding conformation in the HD32D228 model. Molecular docking results show that the HD32D228 model provided the best enrichment factor score, suggesting that this model was able to recognize the most active compounds. Our observations provide an evidence for the preference of the anionic state (HD32D228) in BACE1 binding site and are in accord with reported computational data. The protonation state study would provide significant information to assign the correct protonation state for structure-based drug design and docking studies targeting the BACE1 proteins as a tactic to develop potential AD inhibitors.     

Construction and analysis of base-paired regions of the 16S rRNA in the 30S ribosomal subunit determined by constraint satisfaction molecular modelling

Structure models for each of the secondary structure regions from the Escherichia coli 16S rRNA (58 separate elements) were constructed using a constraint satisfaction modelling program to determine which helices deviated from classic A-form geometry. Constraints for each rRNA element included the comparative secondary structure, H-bonding conformations predicted from patterns of base-pair covariation, tertiary interactions predicted from covariation analysis, chemical probing data, rRNA–rRNA crosslinking information, and coordinates from solved structures. Models for each element were built using the MC-SYM modelling algorithm and subsequently were subjected to energy minimization to correct unfavorable geometry. Approximately two-thirds of the structures that result from the input data are very similar to A-form geometry. In the remaining instances, the presence of internal loops and bulges, some sequences (and sequence covariants) and accessory information require deviation from A-form geometry. The structures of regions containing more complex base-pairing arrangements including the central pseudoknot, the 530 region, and the pseudoknot involving base-pairing between G570-U571/A865-C866 and G861-C862/G867-C868 were predicted by this approach. These molecular models provide insight into the connection between patterns of H-bonding, the presence of unpaired nucleotides, and the overall geometry of each element.     

Conformational Study of the Structure of dibenzo-18-crown-6. Comparison with 18-crown-6

We report for the first time the conformational analysis of dibenzo-18-crown-6, db18c6. The conformational search was carried out using the CONFLEX conformational search method of cyclic molecules. Energies were calculated for the low-lying predicted conformations at different levels of theory up to the G3MP2 level. At the G3MP2 level, the predicted ground state (GS) conformation was more stable than the experimental conformation by only 1.60 kcal/mol. Strong similarity was found between the GS structure and experimental conformations of db18c6 and 18-crown-6, 18c6. The GS and experimental conformations of db18c6 are non-planar. This allows db18c6 to exist in optically active enantiomers. Similar to 18c6, it was concluded that the db18c6 structure is stabilized by intramolecular hydrogen bond. We also performed the computations for the water and chloroform solution phase, where the same conformation was predicted as the GS conformation.     

Homology modelling,docking, pharmacophore and site directed mutagenesis analysis to identify the critical amino acid residue of PknI from Mycobacterium tuberculosis

Tuberculosis is caused by Mycobacterium tuberculosis, an intracellular pathogen. PknI is one of the 11 functional Serine/Threonine Protein Kinases which is predicted to regulate the cell division of M. tuberculosis. In order to find newer drugs and vaccine we need to understand the pathogenesis of the disease. We have used the bioinformatics approach to identify the functionally active residues of PknI and to confirm the same with wet lab experiments. In the current study, we have created homology model for PknI and have done comparative structural analysis of PknI with other kinases. Molecular docking studies were done with a library of kinase inhibitors and T95 was found as the potent inhibitor for PknI. Based on structure based pharmacophore analysis of kinase substrate complexes, Lys 41 along with Asp90, Val92 and Asp96 were identified as functionally important residues. Further, we used site directed mutagenesis technique to mutate Lys 41 to Met resulting in defective cell division of Mycobacterium smegmatis mc². Overall, the proposed model together with its binding features gained from pharmacophore docking studies helped in identifying ligand inhibitor specific to PknI which was confirmed by laboratory experiments.     

Interaction between peroxisome proliferator-activated receptor gamma and its agonists: docking study of oximes having 5-benzyl-2,4-thiazolidinedione

The molecular modelling of oximes having 5-benzyl-2,4-thiazolidinedione moieties, agonists of the peroxisome proliferator-activated receptor gamma (PPAR gamma), was performed with respect to their structures complexed with the ligand binding domain of PPAR gamma. For each ligand molecule, the 5-benzyl-2,4-thiazolidinedione head group was used as an anchor and the conformation of the rest of the molecule was searched for the most energetically favorable interaction with the receptor by systematic conformation search and manual modelling. Although both tail-up and tail-down configurations, which have been observed in the crystal structure of eicosapentaenoic acid when complexed with PPAR delta, appeared among the lowest energy structures for most of the compounds, potent agonists were found to adopt a configuration similar to that of rosiglitazone when bound to PPAR gamma, according to the crystal structure. The structure-activity relationships were analyzed based on the receptor-ligand interaction. The alkyl group and the aromatic ring of the tail group of the ligands had hydrophobic interactions with the receptor, and these interactions were found to be essential for the strong activity.     

Effective fragment potentials and the enzyme active site

Optimization of the binding conformation of a substrate in an enzyme active site using ab initio quantum chemistry methods are intractable since the active site comprises several hundred atoms. However, the active site can be decomposed into an active and spectator region where the spectator residues are represented by effective fragment potentials and reducing the number of all-electron atoms involved in the chemistry to a reasonable level. The effective fragment potentials for electrostatics and polarization are implemented in GAMESS but the repulsive and charge transfer potentials are fit to interaction energies of water with models of the residues. These repulsive/charge transfer potentials are generated for the protein residues and the EFP are then used to optimize binding of a transition state analogue to chorismate mutase (B. subtilis) and small dianions to ribonuclease A. For chorismate mutase the calculated binding conformation compares well to the comparable X-ray structure. The binding of the inhibitor to the glutamate/glutamine mutant active site is then predicted with the optimization including the glutamine residue constrained only at the C alpha atom. The binding conformations suggest important roles for tyr108 and arg63, which have not been noted earlier. The electrostatic stabilization of the transition state by the active site charge distribution has to be augmented by a specific electronic activation by glu78. In ribonuclease A, the protons are found to move to provide a clustering of the charges to bind the small dianions, phosphate, thiophosphate, and sulfate.     

F1174V mutation alters the ALK active conformation in response to Crizotinib in NSCLC: Insight from molecular simulations

Crizotinib is an efficient antineoplastic drug for treatment of non-small cell lung carcinoma (NSCLC), which is identified as an anaplastic lymphoma kinase (ALK) inhibitor. F1174V is a recently identified acquired point mutation relating to the Crizotinib resistance in NSCLC patients. The mechanism of Crizotinib resistance relating to F1174V mutation as a non-active site mutation remains unclear. In this study, the molecular dynamic simulation was used to investigate the possible mechanisms by which F1174V mutation may affect the structure and activity of ALK kinase domain. The results suggested that F1174V mutation could cause two important secondary structure alterations, which led to the local conformational change in ALK kinase domain. This causes more positive free energy in the mutant complex in comparison with the wild-type one. In addition, our structural analyses illustrated that F1174V mutation could result in some important interactions, which represent the key characteristics of the ALK active conformation. This study provided a molecular mechanism for ALK Crizotinib resistance caused by F1174V mutation,which could facilitate designing more efficient drugs.     

Homology models of the mutated EGFR and their response towards quinazoline analogues

One of the most intensely studied tyrosine kinases is the epidermal growth factor receptor (EGFR). The tyrosine kinase receptors are known to be over expressed in some solid tumors and non-small cell lung cancers, causing differential susceptibility to the quinazoline inhibitors. In this study we have taken SYK tyrosine kinase coordinates from PDB database to model two new EGFR receptors with these mutations G695S and L834R and conducted all the docking studies of the inhibitors, also evaluated these two models for quality of structure using PROCHECK. Seven quinazoline analogues (gefitinib, erlotinib, CI-1033, and EKB-569 and other analogues) were selected for comparisons among the two new models. This study determined the receptor/inhibitor interactions, at that active domain binding sites consisting of 15 amino acids. We were able to calculate the energy data for each of the seven inhibitors. This data has been important in interpreting the affinity between the inhibitors evaluated against the three models of EGFR (wild-type and two mutated types). "Affinity"-based studies have indicated the order of response based on docking energy levels (Van der Waals and electrostatic interactions). The active ATP binding sites consisting of 15 amino acid residues were identified and the total energy (E(total)) which showed the affinity between the inhibitor molecules and the receptor (Van der Waals and electrostatic interactions). The selection of the quinazoline analogues was purely on their emergence as possible candidates in the drug discovery areas. This study describes the successful application of these models that we constructed for molecular docking studies to rationally design compounds predicted to bind favorably to the modeled EGFR catalytic sites.