Dynamics and Binding Modes of Free cdk2 and its Two Complexes with Inhibitors Studied by Computer Simulations

Abstract

This article presents a molecular dynamics (MD) study of the cdk2 enzyme and its two complexes with the inhibitors isopentenyladenine and roscovitine using the Cornell et al. force field from the AMBER software package. The results show that inserting an inhibitor into the enzyme active site does not considerably change enzyme structure but it seemingly changes the distribution of internal motions. The inhibitor causes differences in the domain motions in free cdk2 and in its complexes. It was found out that repulsion of roscovitine N9 substituent causes conformational change on Lys 33 side chain. Isopentenyladenine forms with Lys 33 side chain terminal amino group a hydrogen bond. It implies that the cavity, where N9 substituent of roscovitine is buried, can adopt larger substituent due to Lys 33 side chain flexibility. The composition of electrostatic and van der Waals interactions between the inhibitor and the enzyme were also calculated along both cdk2/inhibitor MD trajectories together with MM-PB/GBSA analysis. These results show that isopentenyladenine-like inhibitors could be more effective after modifications leading to an increase in their van der Waals contact with the enzyme. We suggest that a way leading to better inhibitors occupying isopentenyladenine binding mode could be: to keep N9 and N7 purine positions free, to keep 3,3-dimethylallylamino group at C6 position, and to add, e.g., benzylamino group at C2 position. The results support the idea that the isopentenyladenine binding mode can be used for cdk2 inhibitors design and that all possibilities to improve this binding mode were not uncovered yet.     

Analysis of CDK2 active-site hydration: a method to design new inhibitors

The interactions between the protein and the solvent were analyzed, and protein regions with a high density of water molecules, as well as structural water molecules, were determined by using molecular dynamics (MD) simulations. A number of water molecules that were in contact with the protein for the whole trajectory were determined. Their interaction energies and hydrogen bonds with protein residues were analyzed. Altogether, 39, 27, 49, and 32 water molecules bound to the protein were found for trajectories of the free CDK2, CDK2/ATP, CDK2/roscovitine, and CDK2/isopentenyladenine complexes, respectively. Positions of observed water molecules were compared with X-ray crystallography data. Special attention was paid to water molecules in the active site of the enzyme, and especially to the deep pocket, where the N9 roscovitine side-chain is buried. Exchange of active-site water molecules with bulk water through the tunnel from the pocket was observed. In the CDK2/isopentenyladenine complex simulation, two water molecules that arrange interaction between the inhibitor and the enzyme via an H-bond were observed. Two stable water molecules in the trajectory of the free CDK2 were found that occupy the same position as the nitrogens N3 and N9 of the isopentenyladenine or N1 and N6 nitrogens of the adenosine triphosphate (ATP). The positions of structural water molecules were compared with the positions of substrate polar groups and crystallographic water molecules found in the Brookhaven Protein Data Bank for various CDK2 complexes. It was concluded that tracing tightly bound water molecules may substantially help in designing new inhibitors.     

Structure-activity relations in the oxidation of phenethylamine analogues by recombinant human liver monoamine oxidase A

The interaction of recombinant human liver monoamine oxidase A (MAO A) with a series of phenethylamine substrate analogues has been investigated by steady-state and stopped-flow kinetic techniques. Substrate analogues with para substituents exhibit large deuterium kinetic isotope effect on k(cat), on k(cat)/K(m), and on the limiting rate of enzyme reduction in reductive half-reaction experiments. These kinetic isotope effect values range from 5 to 10 with the exception of tyramine, which exhibited smaller steady-state isotope effects (2.3-3.5) than that observed on the rate of flavin reduction (6.9). The stopped-flow data show that imine release from the reduced enzyme is slower than the rate of catalytic turnover. Phenethylamine oxidation by MAO A can be described as the C-H bond cleavage step being rate limiting in catalysis and with oxygen reacting with the reduced enzyme-imine complex. In the case of tyramine, the product release from the oxidized enzyme-imine complex contributes to the rate limitation in catalysis. The binding affinities of a series of para-substituted phenethylamine analogues to MAO A show an increase in affinity of the deprotonated amine with increasing van der Waals volume of the substituent. The limiting rate of enzyme reduction decreases with increasing van der Waals volume of the substituent in a linear manner with no observable electronic contribution as observed previously with benzylamine reduction of MAO A [Miller, J. R., and Edmondson, D. E. (1999) Biochemistry 38, 13670-13683]. Examination of side chain analogues of phenethylamine show 3-phenylpropylamine to be oxidized 2.5-fold more slowly and bound 75-fold more tightly than phenethylamine. 4-Phenylbutylamine is not a substrate for MAO A but is a good competitive inhibitor with a K(i) value of 31 +/- 5 microM. Analysis of the effect of alkyl side chain alterations on binding affinities of a series of arylalkylamine analogues taken from this study and from the literature show a linear correlation with the Taft steric value (E(s)) of the side chain. These results suggest that the binding site for the aryl ring is identical for phenethylamine and for benzylamine analogues and that steric interactions of the alkyl side chain with the enzyme strongly contribute to the binding affinities of a series of reversible inhibitors of MAO A.     

Induced fit docking,free energy calculation and molecular dynamics studies on Mycobacterium tuberculosis alanine racemase inhibitor

Alar, a Pyridoxal 5′-phosphate (PLP)-dependent bacterial enzyme is responsible for the racemisation of L-alanine into D-alanine which is essential for the peptidoglycan biosynthesis in both Gram-positive and Gram-negative bacteria. In the present study, we performed induced fit docking, binding free energy calculation and molecular dynamics simulation to elucidate the Alar inhibition potential of 1,2,4-thiadiazolidine-3,5-dione-based inhibitor 1. The inhibitor binds to the hydrophobic groove of Alar and the binding was found to be stable throughout 20-ns MD simulation. Induced fit docking result showed that Lys42, Tyr46, Tyr175 and Tyr364 residues are primarily responsible for the stabilisation of inhibitor–protein complex. Further, high negative van der Waals binding free energy value of –38.88 kcal/mol, indicated it as the main driving force for the inhibitor binding. Based on the information obtained from this study, we designed few molecules as potent Alar inhibitor. In order to gain structural insight and to validate the stability of complex, we performed 20-ns MD simulation of the designed molecule D1. Results obtained from this study can be used for the design of M. tuberculosis Alar potent inhibitors lacking affinity for the co-factor PLP.     

An explorative study on Staphylococcus aureus MurE inhibitor: induced fit docking,binding free energy calculation,and molecular dynamics

Staphylococcus aureus MurE enzyme catalyzes the addition of l-lysine as third residue of the peptidoglycan peptide moiety. Due to the high substrate specificity and its ubiquitous nature among bacteria, MurE enzyme is considered as one of the potential target for the development of new therapeutic agents. In the present work, induced fit docking (IFD), binding free energy calculation, and molecular dynamics (MD) simulation were carried out to elucidate the inhibition potential of 2-thioxothiazolidin-4-one based inhibitor 1 against S. aureus MurE enzyme. The inhibitor 1 formed majority of hydrogen bonds with the central domain residues Asn151, Thr152, Ser180, Arg187, and Lys219. Binding free-energy calculation by MM-GBSA approach showed that van der Waals (ΔG_vdW, ?57.30?kcal/mol) and electrostatic solvation (ΔG_solv, ?36.86?kcal/mol) energy terms are major contributors for the inhibitor binding. Further, 30-ns MD simulation was performed to validate the stability of ligand–protein complex and also to get structural insight into mode of binding. Based on the IFD and MD simulation results, we designed four new compounds D1–D4 with promising binding affinity for the S. aureus MurE enzyme. The designed compounds were subjected to the extra-precision docking and binding free energy was calculated for complexes. Further, a 30-ns MD simulation was performed for D1/4C13 complex.     

Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.     

Quantitative structure-activity relationship study on some 5-lipoxygenase inhibitors

A quantitative structure-activity relationship (QSAR) study has been made on some lipoxygenase inhibitors belonging to the series of omega-phenylalkyl hydroxamic acids, omega-naphthylalkyl hydroxamic acids, eicosatetraenoic acids, and 1H.benzimidazole-4-ols. It was found that the hydrophobic character of the molecules and the size of their substituents selectively govern their lipoxygenase inhibitory activity. The enzyme active site possesses a non-heme ferric ion, a hydrophobic domain, and a carboxylic acid binding site. It was found that while the functional group of inhibitors must interact with the ferric ion, the substituent on one side of it would be involved in hydrophobic interaction and that on the other side in van der Waals interaction with the enzyme so leading to an enhancement in the inhibitory activity of the inhibitors.     

Apoptosis of metastatic prostate cancer cells by a combination of cyclin-dependent kinase and AKT inhibitors

Effective treatments for advanced prostate cancer are much needed. Toward this goal, we show apoptosis and impaired long-term survival of androgen-independent prostate cancer cells (PC3 and PC3 derivatives) co-treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine and an AKT inhibitor (LY294002 or API-2). Apoptosis of PC3 cells by the drug combination required caspase-9 but not caspase-8 activity and thus is mitochondria-dependent. Roscovitine reduced amounts of the caspase inhibitor XIAP, and API-2 increased amounts of the BH3-only protein Bim. PC3 cells apoptosed when co-treated with API-2 and either cdk9 siRNA, dominant-negative cdk9, or the cdk9 inhibitor DRB; they did not apoptose when co-treated with API-2 and XIAP siRNA. Bax accumulated in mitochondria in response to API-2, whereas release of cytochrome c from mitochondria required both API-2 and roscovitine. We suggest that roscovitine elicits events that activate Bax once it translocates to mitochondria and that inactivation of cdk9 signals these events and the down-regulation of XIAP. Collectively, our data show apoptosis of prostate cancer cells by a drug combination and identify Bax activation as a basis of cooperation.     

Structural requirements for specific inhibition of microsomal aminopeptidase by mercaptoamines

L-Leucinthiol, a synthetic derivative of mercaptoethylamine with a hydrophobic side chain, was recently reported to be a potent inhibitor of microsomal aminopeptidase. The structural features necessary for interaction of mercaptoamines with this enzyme have now been explored more systematically. Optimal binding requires a primary amine linked to the mercapto group via two carbon atoms. Only a substituent with L-configuration at the 1 position increased the affinity toward the enzyme. The high degree of specificity and other evidence suggest that the mode of binding of these inhibitors is similar to that of substrates. Comparison of leucinthiol with other amino compounds suggest that the mercapto group makes a much greater contribution to the binding than the hydrophobic side chain. L-Leucinthiol is fairly specific for aminopeptidase although some inhibition of thermolysin and carboxypeptidase A is observed.     

Insights into drug resistance of mutations D30N and I50V to HIV-1 protease inhibitor TMC-114: Free energy calculation and molecular dynamic simulation

The single mutations D30N and I50V are considered as the key residue mutations of the HIV-1 protease drug resistance to inhibitors in clinical use. In this work, molecular dynamics (MD) simulations combined with the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method have been performed to investigate the drug-resistant mechanisms of D30N and I50V to an inhibitor TMC-114. The analyses of absolute binding free energies using the separate trajectory approach suggests that the decrease in the van der Waals energy and electrostatic energy in the gas phase results in the drug resistance of D30N to TMC-114, while for I50V, the decrease in the electrostatic energy mainly drive its drug resistance to TMC-114. Detailed binding free energies between TMC-114 and individual protein residues are computed by using a per-residue basis decomposition method, which provides insights into the inhibitor-protein binding mechanism and also explains the drug-resistant mechanisms of mutations D30N and I50V to TMC-114. The study shows that the loss of the hydrogen bond between TMC-114 and the side chain of Asn30′ is the main driving force of the resistance of D30N to TMC-114, and in the case of I50V, the increase in the polar solvation energies between TMC-114 and two residues Val50′ and Asp30′ definitively drives the resistance of I50V to TMC-114. We expect that this work can provide some helpful insights into the nature of mutational effect and aid the future design of better inhibitors.     

A comparative molecular dynamics study of methylation state specificity of JMJD2A

Histone modifications have great importance in epigenetic regulation. JMJD2A is a histone demethylase which is selective for di- and trimethyl forms of residues Lys9 and Lys36 of Histone 3 tail (H3K9 and H3K36). We present a molecular dynamics simulations of mono-, di- and trimethylated histone tails in complex with JMJD2A catalytic domain to gain insight into how JMJD2A discriminates between the methylation states of H3K9. The methyl groups are located at specific distances and orientations with respect to Fe(II) in methylammonium binding pocket. For the trimethyllysine the mechanism which provides the effectual orientation of methyl groups is the symmetry, whereas for the dimethyllysine case the determining factors are the interactions between methyllysine head and its environment and subsequently the restriction on angular motion. The occurrence frequency of methyl groups in a certain proximity of Fe(II) comes out as the explanation of the enzyme activity difference on di- and tri-methylated peptides. Energy analysis suggests that recognition is mostly driven by van der Waals and followed by Coulombic interactions in the enzyme-substrate interface. The number (mono, di or tri) and orientations of methyl groups and water molecules significantly affect the extent of van der Waals interaction strengths. Hydrogen bonding analysis suggests that the interaction between JMJD2A and its substrates mainly comes from main chain-side chain interactions. Binding free energy analysis points out Arg8 as an important residue forming an intra-substrate hydrogen bond with tri and dimethylated Lys9 of the H3 chain. Our study provides new insights into how JMJD2A discriminates between its substrates from both a structural and dynamical point of view.     

A computational analysis of pyrazole-based inhibitors binding to Hsp90 using molecular dynamics simulation and the MM-GBSA method

Owing to the key role of heat-shock protein 90 (Hsp90) in the evolution, development and disease pathogenesis of cancer, it has been an important target for anti-cancer chemotherapy over the years. A five-nanosecond molecular dynamics simulation combined with the calculation of the binding free energy was carried out to investigate the binding mechanisms of three Hsp90 inhibitors 4BH, 2E1 and 2D9 to Hsp90. The binding free energy of each complex was computed using the molecular mechanics–generalised Born surface area method. Detailed binding free energies between each inhibitor and residues of Hsp90 were calculated using a per-residue basis decomposition method. The detailed inhibitor–residue interaction provides insights into binding mechanisms and in-depth understanding of the structure–affinity relationship. This study suggests that van der Waals energy is primarily responsible for driving the binding of the inhibitors to Hsp90, and the three inhibitors bind to Hsp90 in a similar binding mode. However, a substituent in 2D9 leads to higher binding free energy than the other two inhibitors. These data may assist in designing new potent drugs to combat cancer.     

Evidence for alternative binding modes in the interaction of benzylamine analogues with bovine liver monoamine oxidase B

The interaction of purified bovine liver MAO B with the benzylamine analogues N,N-dimethylbenzylamine and alpha-methylbenzylamine has been investigated. Both classes of analogues are competitive inhibitors of benzylamine oxidase activity. The K(i) values were determined for nine different para-substituted N, N-dimethylbenzylamine analogues. Analysis of the binding affinities demonstrate the deprotonated forms of the tertiary amines are preferentially bound to MAO B and the affinity decreases with increasing van der Waals volume of the para-substituent. The correlation for this relation is:Log K(i)=-0.97+/-(0.28)sigma+(0. 75+/-0.11)(0.1xV(w))-4.24+/-(0.16)alpha-Methyl benzylamine analogues are also found to be competitive inhibitors of MAO B-catalyzed benzylamine oxidation. Similar K(i) values were determined using either the S or R stereoisomers. Analysis of the binding affinities of five para-substituted alpha-methylbenzylamine analogues to MAO B shows the deprotonated form also to be preferentially bound and the affinity is marginally increased with increasing van der Waals volume of the para-substituent:Log K(i)=-0.71sigma-(0.32)(0. 1xV(w))-3.50Comparison of these data with that previously published for para-substituted benzylamine binding to MAO B (Walker and Edmondson, Biochemistry 33 (1994) 7088-7098) demonstrates that these benzylamine analogues exhibit differing modes of binding to the active site of MAO B. The presence of an electronic substituent effect in the binding of these two classes of analogues compared with the lack of an observable electronic effect in the binding of benzylamine to MAO B is consistent with the proposal that orientation of the benzyl ring of the bound substrate is responsible for the absence of an electronic substituent effect on the rate of the reductive half reaction (Miller and Edmondson, Biochemistry 38 (1999) 13670-13683).     

The binding mechanism of a novel nicotinamide isostere inhibiting with TNKSs: a molecular dynamic simulation and binding free energy calculation

Tankyrases (TNKSs), a member of human poly (ADP-ribose) polymerase (PARP) protein superfamily, plays a key role in regulation of cell proliferation. Among the representative proteins of the PARPs family, it is found that the inhibitors have high selectivity for Tankyrase1 (TNKS1). The specific binding modes are investigated between the TNKS1 protein and nicotinamide isostere (ISX) which functions as an inhibitor of TNKS1. The stabilities of ISX-TNKS1 and AVA939-TNKS1 complexes are estimated by molecular dynamics (MD) simulations and free energy calculations; a good agreement with experimental results is reached. On the basis of the calculated results of MD simulations, we found that the inhibitors influence the conformational flexibility of TNKS1 and the XAV939 binding drive the peptide Ile1228-Gly1229-Gly1230 to form a helical structure while the ISX binding drive the peptide to form a turn structure. Moreover, the formed important hydrogen bonds of Tyr1203 residue with XVA939 and WAT1551 with ISX enhance stabilities of the complexes, and the electrostatic interactions in XAV939-TNKS1 and van der Waals interactions in ISX-TNKS1 system are main driving forces for affinity. According to the results of the decomposition of binding free energy, it is obvious that the residues Try1224 and Lys1220 make the most favorable contributions to the binding in, respectively, ISX and XAV939 complexes. Taken together, the obtained results are useful for studying the binding mechanisms of TNKSs and inhibitors and for designing potent inhibitors.     

Computational analysis of the binding affinities of the natural-product cyclopentapeptides argifin and argadin to chitinase B from Serratia marcescens

Molecular dynamics (MD) simulations and the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method were applied to study the interaction of the natural-product cyclopentapeptide chitinase inhibitors argifin and argadin with chitinase B (ChiB) from Serratia marcescens. Argadin inhibited ChiB with an inhibition constant (K(i)) value of 20 nM, which was three orders of magnitude greater than that of argifin (K(i)=33,000 nM). The MM-PBSA free-energy analysis provided absolute binding free energies of -6.98 and -11.16 kcal/mol for the argifin and argadin complexes, respectively. These estimates were in good agreement with the free energies derived from the experimental K(i) values (-6.36 and -10.92 kcal/mol for the argifin and argadin complexes, respectively). The energetic analysis revealed that the van der Waals and nonpolar solvation energies drove the binding of both argifin and argadin. We found that the binding of argadin gained approximately 12 kcal/mol more van der Waals energy than that of argifin, which was mainly responsible for the difference in binding free energy between argifin and argadin. In particular, W220 and W403 of ChiB were found to contribute to the more favorable van der Waals interaction with argadin. We also designed argifin derivatives with better binding affinity, in which a constituent amino-acid residue of argifin was mutated to one with a bulky side chain. The derivative in which D-Ala of argifin was replaced with D-Trp appeared to possess a binding affinity that was equally potent to that of argadin.     

Probing the electronic structures and properties of neutral and anionic ScSi n (0,−1) (n = 1–6) clusters using ccCA-TM and G4 theory

We apply molecular docking, molecular dynamics (MD) simulation, and binding free energy calculation to investigate and reveal the binding mechanism between five xanthine inhibitors and DPP-4. The electrostatic and van der Waals interactions of the five inhibitors with DPP-4 are analyzed and discussed. The computed binding free energies using MM-PBSA method are in qualitatively agreement with experimental inhibitory potency of five inhibitors. The hydrogen bonds of inhibitors with Ser630 and Asp663 can stabilize the inhibitors in binding sites. The van der Waals interactions, especially the key contacts with His740, Asn710, Trp629, and Tyr666 have larger contributions to the binding free energy and play important roles in distinguishing the variant bioactivity of five inhibitors.     

Inhibition of chymotrypsin by peptidyl trifluoromethyl ketones: determinants of slow-binding kinetics

A series of seven peptidyl trifluoromethyl ketone (TFK) inhibitors of chymotrypsin have been prepared which differ at the P1 and P2 subsites. Inhibition equilibria and kinetics of association and dissociation with chymotrypsin have been measured. The association rate of Ac-Phe-CF3 was measured at enzyme concentrations between 8 nM and 117 microM in order to examine the relation between the ketone/hydrate equilibrium of trifluoromethyl ketones and the "slow binding" by these inhibitors. The association rate decreases at high enzyme concentrations, indicating that TFK ketone is the reactive species and that conversion of TFK hydrate to ketone becomes rate limiting under these conditions. Inhibitors with hydrophobic side chains at P2 bind more tightly but more slowly to chymotrypsin, indicating that formation of van der Waals contacts between the P2 side chain and the His 57 and Ile 99 side chains of chymotrypsin is a relatively slow process. Inhibitor properties were compared to the Michaelis-Menten kinetic constants of a homologous series of peptide methyl ester and peptide amide substrates. Plots of log Ki vs log (kcat/Km) are linear with slopes of 0.65 +/- 0.2, indicating that these inhibitors are able to utilize 65% of the total binding energy between chymotrypsin and its hydrolytic transition state.     

Quantitative Structure-Activity Relationship Study on Some 5-Lipoxygenase Inhibitors

Abstract

A quantitative structure-activity relationship (QSAR) study has been made on some lipoxygenase inhibitors belonging to the series of ω-phenylalkyl hydroxamic acids, ω-naphthylalkyl hydroxamic acids, eicosatetraenoic acids, and 1H.benzimidazole-4-ols. It was found that the hydrophobic character of the molecules and the size of their substituents selectively govern their lipoxygenase inhibitory activity. The enzyme active site possesses a non-heme ferric ion, a hydrophobic domain, and a carboxylic acid binding site. It was found that while the functional group of inhibitors must interact with the ferric ion, the substituent on one side of it would be involved in hydrophobic interaction and that on the other side in van der Waals interaction with the enzyme so leading to an enhancement in the inhibitory activity of the inhibitors.