Revealing binding selectivity of ligands toward murine double minute 2 and murine double minute X based on molecular dynamics simulations and binding free energy calculations

Abstract

It is well known that the interactions of p53 with murine double minute 2 and murine double minute X, namely MDM2 and MDMX, have been significant targets of efficient anti-cancer drug design. In this study, molecular dynamics (MD) simulations, principal component (PC) analysis and binding free energy calculations are combined to recognize binding selectivity of three ligands to MDM2 and MDMX. The binding free energies were estimated by using molecular mechanics generalized Born surface area (MM-GBSA) method and the obtained results display that the increase in the binding enthalpy of three ligands to MDM2 relative to MDMX mainly drives the binding selectivity of them toward MDM2 and MDMX. The information obtained from PC analysis shows that the associations of ligands exert important impacts on internal dynamics of MDM2 and MDMX. Meanwhile, the calculations of residue-based free energy decomposition not only identify the hot interaction spots of ligands with MDM2 and MDMX, but also show the residues (L54, M53), (Y67, Y66), (V93, V92), (H96, P95), (I99, I98) and (Y100, Y99) in (MDM2, MDMX) are responsible for most contributions to the binding selectivity of three ligands toward MDM2 and MDMX. It is believed that this work can provide useful information for design of highly selective and dual inhibitors targeting MDM2 and MDMX.

Communicated by Ramaswamy H. Sarma     

Structures of low molecular weight inhibitors bound to MDMX and MDM2 reveal new approaches for p53-MDMX/MDM2 antagonist drug discovery

Intensive anticancer drug discovery efforts have been made to develop small molecule inhibitors of the p53-MDM2 and p53-MDMX interactions. We present here the structures of the most potent inhibitors bound to MDM2 and MDMX that are based on the new imidazo-indole scaffold. In addition, the structure of the recently reported spiro-oxindole inhibitor bound to MDM2 is described. The structures indicate how the substituents of a small molecule that bind to the three subpockets of the MDM2/X-p53 interaction should be optimized for effective binding to MDM2 and/or MDMX. While the spiro-oxindole inhibitor triggers significant ligand-induced changes in MDM2, the imidazo-indoles share similar binding modes for MDMX and MDM2, but cause only minimal induced-fit changes in the structures of both proteins. Our study includes the first structure of the complex between MDMX and a small molecule and should aid in developing efficient scaffolds for binding to MDMX and/or MDM2.     

Casein Kinase 1α Regulates an MDMX Intramolecular Interaction To Stimulate p53 Binding

MDMX is an important regulator of p53 during embryonic development and malignant transformation. Previous studies showed that casein kinase 1α (CK1α) stably associates with MDMX, stimulates MDMX-p53 binding, and cooperates with MDMX to inactivate p53. However, the mechanism by which CK1α stimulates MDMX-p53 interaction remains unknown. Here, we present evidence that p53 binding by the MDMX N-terminal domain is inhibited by the central acidic region through an intramolecular interaction that competes for the p53 binding pocket. CK1α binding to the MDMX central domain and phosphorylation of S289 disrupts the intramolecular interaction, allowing the N terminus to bind p53 with increased affinity. After DNA damage, the MDMX-CK1α complex is disrupted by Chk2-mediated phosphorylation of MDMX at S367, leading to reduced MDMX-p53 binding. Therefore, CK1α is an important functional partner of MDMX. DNA damage activates p53 in part by disrupting CK1α-MDMX interaction and reducing MDMX-p53 binding affinity.     

Binding mode prediction of biologically active compounds from plant Salvia Miltiorrhiza as integrase inhibitor

Integrase (IN), an essential enzyme for HIV-1 replication, has been targeted in antiretroviral drug therapy. The emergence of HIV-1 variants clinically resistant to antiretroviral agents has lead to the development of alternative IN inhibitors. In the present work, binding modes of a high potent IN inhibitor, M₅22 and M₅32, within the catalytic binding site of wild type (WT) IN were determined using molecular docking calculation. Both M₅22 and M₅32 displayed similar modes of binding within the IN putative binding pocket and exhibited favorable interactions with the catalytic Mg²⁺ ions, the nearby amino acids and viral DNA through metal-ligand chelation, hydrogen bonding and π-π stacking interactions. Furthermore, the modes of action of these two compounds against the mutated Y212R, N224H and S217H PFV IN were also predicted. Although the replacement of amino acid could somehow disturb inhibitor binding mode, almost key interactions which detected in the WT complexes were fairly conserved. Detailed information could highlight the application of M₅22 and M₅32 as candidate IN inhibitors for drug development against drug resistant strains.     

Electrostatic interaction-mediated conformational changes of adipocyte fatty acid binding protein probed by molecular dynamics simulation

Abstract

Adipocyte fatty acid binding protein (A-FABP) is a potential drug target for treatment of diabetes, obesity and atherosclerosis. Molecular dynamics (MD) simulations, principal component (PC) analysis and binding free energy calculations were combined to probe effect of electrostatic interactions of residues R78, R106 and R126 with inhibitors ZGB, ZGC and IBP on structural stability of three inhibitor/A-FABP complexes. The results indicate that mutation R126A produces significant influence on polar interactions of three inhibitors with A-FABP and these interactions are main force for driving the conformational change of A-FABP. Analyses on hydrogen bond interactions show that the decrease in hydrogen bonding interactions of residues R126 and Y128 with three inhibitors and the increase in that of K58 with inhibitors ZGC and IBP in the R126A mutated systems mostly regulate the conformational changes of A-FABP. This work shows that R126A can generate a significant perturbation on structural stability of A-FABP, which implies that R126 is of significance in inhibitor bindings. We expect that this study can provide a theoretical guidance for design of potent inhibitors targeting A-FABP.

Communicated by Ramaswamy H. Sarma     

A computational insight into binding modes of inhibitors XD29, XD35, and XD28 to bromodomain-containing protein 4 based on molecular dynamics simulations

In the current work, conformational changes of bromodomain-containing protein 4 (1) (BRD4-1) induced by bindings of inhibitors XD29 (57G), XD35 (57F), and XD28 (L28) were investigated using molecular dynamics (MD) simulations and principal component analysis. The results demonstrate that inhibitor bindings produce significant effect on the motion of ZA loop in BRD4-1. Moreover, to further study binding modes of three inhibitors to BRD4-1, binding free energies of inhibitors to BRD4-1 were also calculated using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The results indicate that van der Waals interactions are main factors to modulate inhibitor bindings. Energy decomposition and hydrogen bond analysis demonstrate that residues Pro82, Leu92, Asn140, and Ile146 play important roles in binding processes of inhibitors to BRD4-1. This study is not only helpful for better understanding function and internal dynamics of BRD4-1, but also can provide a theoretical basis for rational designs of effective anticancer drugs targeting BRD4-1.     

Biophysical Studies of Bacterial Topoisomerases Substantiate Their Binding Modes to an Inhibitor

Bacterial DNA topoisomerases are essential for bacterial growth and are attractive, important targets for developing antibacterial drugs. Consequently, different potent inhibitors that target bacterial topoisomerases have been developed. However, the development of potent broad-spectrum inhibitors against both Gram-positive (G⁺) and Gram-negative (G⁻) bacteria has proven challenging. In this study, we carried out biophysical studies to better understand the molecular interactions between a potent bis-pyridylurea inhibitor and the active domains of the E-subunits of topoisomerase IV (ParE) from a G⁺ strain (Streptococcus pneumoniae (sParE)) and a G⁻ strain (Pseudomonas aeruginosa (pParE)). NMR results demonstrated that the inhibitor forms a tight complex with ParEs and the resulting complexes adopt structural conformations similar to those observed for free ParEs in solution. Further chemical-shift perturbation experiments and NOE analyses indicated that there are four regions in ParE that are important for inhibitor binding, namely, α2, the loop between β2 and α3, and the β2 and β6 strands. Surface plasmon resonance showed that this inhibitor binds to sParE with a higher K_D than pParE. Point mutations in α2 of ParE, such as A52S (sParE), affected its binding affinity with the inhibitor. Taken together, these results provide a better understanding of the development of broad-spectrum antibacterial agents.     

Structural and Regulatory Elements of HCV NS5B Polymerase – β-Loop and C-Terminal Tail – Are Required for Activity of Allosteric Thumb Site II Inhibitors

Elucidation of the mechanism of action of the HCV NS5B polymerase thumb site II inhibitors has presented a challenge. Current opinion holds that these allosteric inhibitors stabilize the closed, inactive enzyme conformation, but how this inhibition is accomplished mechanistically is not well understood. Here, using a panel of NS5B proteins with mutations in key regulatory motifs of NS5B – the C-terminal tail and β-loop – in conjunction with a diverse set of NS5B allosteric inhibitors, we show that thumb site II inhibitors possess a distinct mechanism of action. A combination of enzyme activity studies and direct binding assays reveals that these inhibitors require both regulatory elements to maintain the polymerase inhibitory activity. Removal of either element has little impact on the binding affinity of thumb site II inhibitors, but significantly reduces their potency. NS5B in complex with a thumb site II inhibitor displays a characteristic melting profile that suggests stabilization not only of the thumb domain but also the whole polymerase. Successive truncations of the C-terminal tail and/or removal of the β-loop lead to progressive destabilization of the protein. Furthermore, the thermal unfolding transitions characteristic for thumb site II inhibitor – NS5B complex are absent in the inhibitor – bound constructs in which interactions between C-terminal tail and β-loop are abolished, pointing to the pivotal role of both regulatory elements in communication between domains. Taken together, a comprehensive picture of inhibition by compounds binding to thumb site II emerges: inhibitor binding provides stabilization of the entire polymerase in an inactive, closed conformation, propagated via coupled interactions between the C-terminal tail and β-loop.     

Dendrimeric Template of Plasmodium falciparum Histidine Rich Protein II Repeat Motifs Bearing Asp→Asn Mutation Exhibits Heme Binding and β-Hematin Formation

Plasmodium falciparum (Pf) employs a crucial PfHRPII catalyzed reaction that converts toxic heme into hemozoin. Understanding heme polymerization mechanism is the first step for rational design of new drugs, targeting this pathway. Heme binding and hemozoin formation have been ascribed to PfHRPII aspartate carboxylate-heme metal ionic interactions. To investigate, if this ionic interaction is indeed pivotal, we examined the comparative heme binding and β-hematin forming abilities of a wild type dendrimeric peptide BNT1 {harboring the native sequence motif of PfHRPII (AHHAHHAADA)} versus a mutant dendrimeric peptide BNTM {in which ionic Aspartate residues have been replaced by the neutral Asparaginyl residues (AHHAHHAANA)}. UV and IR data reported here reveal that at pH 5, both BNT1 and BNTM exhibit comparable heme binding as well as β-hematin forming abilities, thus questioning the role of PfHRPII aspartate carboxylate-heme metal ionic interactions in heme binding and β-hematin formation. Based on our data and information in the literature we suggest the possible role of weak dispersive interactions like N-H···π and lone-pair···π in heme binding and hemozoin formation.     

Fluorinated Aromatic Amino Acids Distinguish Cation-π Interactions from Membrane Insertion

Cation-π interactions, where protein aromatic residues supply π systems while a positive-charged portion of phospholipid head groups are the cations, have been suggested as important binding modes for peripheral membrane proteins. However, aromatic amino acids can also insert into membranes and hydrophobically interact with lipid tails. Heretofore there has been no facile way to differentiate these two types of interactions. We show that specific incorporation of fluorinated amino acids into proteins can experimentally distinguish cation-π interactions from membrane insertion of the aromatic side chains. Fluorinated aromatic amino acids destabilize the cation-π interactions by altering electrostatics of the aromatic ring, whereas their increased hydrophobicity enhances membrane insertion. Incorporation of pentafluorophenylalanine or difluorotyrosine into a Staphylococcus aureus phosphatidylinositol-specific phospholipase C variant engineered to contain a specific PC-binding site demonstrates the effectiveness of this methodology. Applying this methodology to the plethora of tyrosine residues in Bacillus thuringiensis phosphatidylinositol-specific phospholipase C definitively identifies those involved in cation-π interactions with phosphatidylcholine. This powerful method can easily be used to determine the roles of aromatic residues in other peripheral membrane proteins and in integral membrane proteins.     

Insight into binding mechanisms of inhibitors MKP56, MKP73, MKP86, and MKP97 to HIV-1 protease by using molecular dynamics simulation

HIV-1 protease (PR) has been a significant target for design of potent inhibitors curing acquired immunodeficiency syndrome. Molecular dynamics simulations coupled with molecular mechanics Poisson–Boltzmann surface area method were performed to study interaction modes of four inhibitors MKP56, MKP73, MKP86, and MKP97 with PR. The results suggest that the main force controlling interactions of inhibitors with PR should be contributed by van der Waals interactions between inhibitors and PR. The cross-correlation analyses based on MD trajectories show that inhibitor binding produces significant effect on the flap dynamics of PR. Hydrogen bond analyses indicate that inhibitors can form stable hydrogen bonding interactions with the residues from the catalytic strands of PR. The contributions of separate residues to inhibitor bindings are evaluated by using residue-based free energy decomposition method and the results demonstrate that the CH–π and CH–CH interactions between the hydrophobic groups of inhibitors with residues drive the associations of inhibitors with PR. We expect that this study can provide a significant theoretical aid for design of potent inhibitors targeting PR.     

Anatomy of noncovalent interactions between the nucleobases or ribose and π-containing amino acids in RNA–protein complexes

A set of >300 nonredundant high-resolution RNA–protein complexes were rigorously searched for π-contacts between an amino acid side chain (W, H, F, Y, R, E and D) and an RNA nucleobase (denoted π–π interaction) or ribose moiety (denoted sugar–π). The resulting dataset of >1500 RNA–protein π-contacts were visually inspected and classified based on the interaction type, and amino acids and RNA components involved. More than 80% of structures searched contained at least one RNA–protein π-interaction, with π–π contacts making up 59% of the identified interactions. RNA–protein π–π and sugar–π contacts exhibit a range in the RNA and protein components involved, relative monomer orientations and quantum mechanically predicted binding energies. Interestingly, π–π and sugar–π interactions occur more frequently with RNA (4.8 contacts/structure) than DNA (2.6). Moreover, the maximum stability is greater for RNA–protein contacts than DNA–protein interactions. In addition to highlighting distinct differences between RNA and DNA–protein binding, this work has generated the largest dataset of RNA–protein π-interactions to date, thereby underscoring that RNA–protein π-contacts are ubiquitous in nature, and key to the stability and function of RNA–protein complexes.     

Affinity-based screening of MDM2/MDMX–p53 interaction inhibitors by chemical array: Identification of novel peptidic inhibitors

MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein–protein interactions (PPIs) of MDM2–p53 and MDMX–p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2–p53 and MDMX–p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2–p53 and MDMX–p53 interactions.     

Structure and Stability of Human Telomeric G-Quadruplex with Preclinical 9-Amino Acridines

G-quadruplexes are higher-order DNA structures formed from guanine-rich sequences, and have been identified as attractive anticancer drug targets. Elucidating the three-dimensional structure of G-quadruplex with 9-amino acridines and the specific interactions involved in binding selectivity are the key to understanding their mechanism of action. Fluorescence titration assays, competitive dialysis and NMR studies have been used to study the binding specificity of 9-amino acridines to DNA. Structural models of the complexes with the telomeric DNA G-quadruplex based on NMR measurements were developed and further examined by molecular dynamics simulations and free energy calculations. Selective binding of 9-amino acridines for G-quadruplex sequences were observed. These compounds bind between A and G-tetrads, involving significant π-π interactions and several strong hydrogen bonds. The specific interactions between different moieties of the 9-amino acridines to the DNA were examined and shown to play a significant role in governing the overall stabilities of DNA G-quadruplex complexes. Both 9-amino acridines, with similar binding affinities to the G-quadruplex, were shown to induce different level of structural stabilization through intercalation. This unique property of altering structural stability is likely a contributing factor for affecting telomerase function and, subsequently, the observed differences in the anticancer activities between the two 9-amino acridines.     

Computational revelation of binding mechanisms of inhibitors to endocellular protein tyrosine phosphatase 1B using molecular dynamics simulations

Endocellular protein tyrosine phosphatase 1B (PTP1B) is one of the most promising target for designing and developing drugs to cure type-II diabetes and obesity. Molecular dynamics (MD) simulations combined with molecular mechanics generalized Born surface area (MM-GBSA) and solvated interaction energy methods were applied to study binding differences of three inhibitors (ID: 901, 941, and 968) to PTP1B, the calculated results show that the inhibitor 901 has the strongest binding ability to PTP1B among the current inhibitors. Principal component (PC) analysis was also carried out to investigate the conformational change of PTP1B, and the results indicate that the associations of inhibitors with PTP1B generate a significant effect on the motion of the WPD-loop. Free energy decomposition method was applied to study the contributions of individual residues to inhibitor bindings, it is found that three inhibitors can generate hydrogen bonding interactions and hydrophobic interactions with different residues of PTP1B, which provide important forces for associations of inhibitors with PTP1B. This research is expected to give a meaningfully theoretical guidance to design and develop of effective drugs curing type-II diabetes and obesity.     

On the interaction mechanisms of a p53 peptide and nutlin with the MDM2 and MDMX proteins: A Brownian dynamics study

The interaction of p53 with its regulators MDM2 and MDMX plays a major role in regulating the cell cycle. Inhibition of this interaction has become an important therapeutic strategy in oncology. Although MDM2 and MDMX share a very high degree of sequence/structural similarity, the small-molecule inhibitor nutlin appears to be an efficient inhibitor only of the p53-MDM2 interaction. Here, we investigate the mechanism of interaction of nutlin with these two proteins and contrast it with that of p53 using Brownian dynamics simulations. In contrast to earlier attempts to examine the bound states of the partners, here we locate initial reaction events in these interactions by identifying the regions of space around MDM2/MDMX, where p53/nutlin experience associative encounters with prolonged residence times relative to that in bulk solution. We find that the initial interaction of p53 with MDM2 is long-lived relative to nutlin, but, unlike nutlin, it takes place at the N- and C termini of the MDM2 protein, away from the binding site, suggestive of an allosteric mechanism of action. In contrast, nutlin initially interacts with MDM2 directly at the clefts of the binding site. The interaction of nutlin with MDMX, however, is very short-lived compared with MDM2 and does not show such direct initial interactions with the binding site. Comparison of the topology of the electrostatic potentials of MDM2 and MDMX and the locations of the initial encounters with p53/nutlin in tandem with structure-based sequence alignment revealed that the origin of the diminished activity of nutlin toward MDMX relative to MDM2 may stem partly from the differing topologies of the electrostatic potentials of the two proteins. Glu25 and Lys51 residues underpin these topological differences and appear to collectively play a key role in channelling nutlin directly toward the binding site on the MDM2 surface and are absent in MDMX. The results, therefore, provide new insight into the mechanism of p53/nutlin interactions with MDM2 and MDMX and could potentially have a broader impact on anticancer drug optimization strategies.     

Aromatic-aromatic interactions: analysis of π-π interactions in interleukins and TNF proteins

Aromatic-aromatic hydrogen bonds are important in many areas of chemistry, biology and materials science. In this study we have analyzed the roles played by the π-π interactions in interleukins (ILs) and tumor necrosis factor (TNF) proteins. Majority of π-π interacting residues are conserved in ILs and TNF proteins. The accessible surface area calculations in these proteins reveal that these interactions might be important in stabilizing the inner core regions of these proteins. In addition to π-π interactions, the aromatic residues also form π-networks in ILs and TNF proteins. The results obtained in the present study indicate that π-π interactions and π-π networks play important roles in the structural stability of ILs and TNF proteins.     

Alpha-hydroxytropolones are noncompetitive inhibitors of human RNase H1 that bind to the active site and modulate substrate binding

The ribonucleases H (RNases H) of HIV and hepatitis B virus are type 1 RNases H that are promising drug targets because inhibiting their activity blocks viral replication. Eukaryotic ribonuclease H1 (RNase H1) is an essential protein and a probable off-target enzyme for viral RNase H inhibitors. α-hydroxytropolones (αHTs) are a class of anti-RNase H inhibitors that can inhibit the HIV, hepatitis B virus, and human RNases H1; however, it is unclear how these inhibitors could be developed to distinguish between these enzymes. To accelerate the development of selective RNase H inhibitors, we performed biochemical and kinetic studies on the human enzyme, which was recombinantly expressed in Escherichia coli. Size-exclusion chromatography showed that free RNase H1 is monomeric and forms a 2:1 complex with a substrate of 12 bp. FRET heteroduplex cleavage assays were used to test inhibition of RNase H1 in steady-state kinetics by two structurally diverse αHTs, 110 and 404. We determined that turnover rate was reduced, but inhibition was not competitive with substrate, despite inhibitor binding to the active site. Given the compounds’ reversible binding to the active site, we concluded that traditional noncompetitive and mixed inhibition mechanisms are unlikely. Instead, we propose a model in which, by binding to the active site, αHTs stabilize an inactive enzyme–substrate–inhibitor complex. This new model clarifies the mechanism of action of αHTs against RNase H1 and will aid the development of RNase H inhibitors selective for the viral enzymes.     

The Free Energy Profile of Tubulin Straight-Bent Conformational Changes,with Implications for Microtubule Assembly and Drug Discovery

αβ-tubulin dimers need to convert between a ‘bent’ conformation observed for free dimers in solution and a ‘straight’ conformation required for incorporation into the microtubule lattice. Here, we investigate the free energy landscape of αβ-tubulin using molecular dynamics simulations, emphasizing implications for models of assembly, and modulation of the conformational landscape by colchicine, a tubulin-binding drug that inhibits microtubule polymerization. Specifically, we performed molecular dynamics, potential-of-mean force simulations to obtain the free energy profile for unpolymerized GDP-bound tubulin as a function of the ∼12° intradimer rotation differentiating the straight and bent conformers. Our results predict that the unassembled GDP-tubulin heterodimer exists in a continuum of conformations ranging between straight and bent, but, in agreement with existing structural data, suggests that an intermediate bent state has a lower free energy (by ∼1 kcal/mol) and thus dominates in solution. In agreement with predictions of the lattice model of microtubule assembly, lateral binding of two αβ-tubulins strongly shifts the conformational equilibrium towards the straight state, which is then ∼1 kcal/mol lower in free energy than the bent state. Finally, calculations of colchicine binding to a single αβ-tubulin dimer strongly shifts the equilibrium toward the bent states, and disfavors the straight state to the extent that it is no longer thermodynamically populated.