How does multiple substrate binding lead to substrate inhibition of CYP2D6 metabolizing dextromethorphan? A theoretical study.

CYP2D6 is one of the most important metalloenzymes involved in the biodegradation of many drug molecules in the human body. It has been found that multiple substrate binding can lead to substrate inhibition of CYP2D6 metabolizing dextromethorphan (DM), but the corresponding theoretical mechanism is rarely reported. Therefore, we chose DM as the probe and performed molecular dynamics simulations and quantum mechanical calculations on CYP2D6-DM systems to investigate the mechanism of how the multiple substrate binding leads to the substrate inhibition of CYP2D6 metabolizing substrates. According to our results, three gate residues (Arg221, Val374, and Phe483) for the catalytic pocket are determined. We also found that the multiple substrate binding can lead to substrate inhibition by reducing the stability of CYP2D6 binding DM and increasing the reactive activation energy of the rate-determining step. Our findings would help to understand the substrate inhibition of CYP2D6 metabolizing the DM and enrich the knowledge of the drug-drug interactions for the cytochrome P450 superfamily.

The regioselectivity of the interaction between dextromethorphan and CYP2D6.

CYP2D6 is an important enzyme of the cytochrome P450 superfamily, and catalyzes nearly 25% of the drugs sold in the market. For decades, the interactions and metabolism between CYP2D6 and substrates have been a hot topic. However, the key factors of the catalytic regioselectivity for CYP2D6 still remain controversial. Here, we construct four systems to explore the interaction between dextromethorphan (DM) and CYP2D6. A new binding mode of CYP2D6 is defined, and two key residues (residue Asp301 and residue Glu216) are discovered working simultaneously to stabilize the DM at the reactive site by forming water bridge hydrogen bonds when CYP2D6 binds DM. Our results also indicate that the substrate concentration could mediate the binding mode between the substrate and CYP2D6 by decreasing the volume of the catalytic pocket, which is not conducive to the O-demethylation of DM but benefits the N-demethylation of DM. These results could shed light on the process of CYP2D6 binding to the substrate, and help to better understand the regioselectivity of CYP2D6 catalyzing the substrates.

Exploring the Distinct Binding and Activation Mechanisms for Different CagA Oncoproteins and SHP2 by Molecular Dynamics Simulations.

CagA is a major virulence factor of Helicobacter pylori. H. pylori CagA is geographically subclassified into East Asian CagA and Western CagA, which are characterized by the presence of a EPIYA-D or EPIYA-C segment. The East Asian CagA is more closely associated with gastric cancer than the Western CagA. In this study, molecular dynamic (MD) simulations were performed to investigate the binding details of SHP2 and EPIYA segments, and to explore the allosteric regulation mechanism of SHP2. Our results show that the EPIYA-D has a stronger binding affinity to the N-SH2 domain of SHP2 than EPIYA-C. In addition, a single EPIYA-D binding to N-SH2 domain of SHP2 can cause a deflection of the key helix B, and the deflected helix B could squeeze the N-SH2 and PTP domains to break the autoinhibition pocket of SHP2. However, a single EPIYA-C binding to the N-SH2 domain of SHP2 cannot break the autoinhibition of SHP2 because the secondary structure of the key helix B is destroyed. However, the tandem EPIYA-C not only increases its binding affinity to SHP2, but also does not significantly break the secondary structure of the key helix B. Our study can help us better understand the mechanism of gastric cancer caused by Helicobacter pylori infection.

In Silico Study of Membrane Lipid Composition Regulating Conformation and Hydration of Influenza Virus B M2 Channel.

The proton conduction of transmembrane influenza virus B M2 (BM2) proton channel is possibly mediated by the membrane environment, but the detailed molecular mechanism is challenging to determine. In this work, how membrane lipid composition regulates the conformation and hydration of BM2 channel is elucidated in silico. The appearance of several important hydrogen-bond networks has been discovered, as the addition of negatively charged lipid palmitoyloleoyl phosphatidylglycerol (POPG) and cholesterol reduces membrane fluidity and augments membrane rigidity. A more rigid membrane environment is beneficial to expand the channel, allow more water to enter the channel, promote channel hydration, and then even affect the proton conduction facilitated by the hydrated channel. Thus, membrane environment could be identified as an important influence factor of conformation and hydration of BM2. These findings can provide a unique perspective for understanding the mechanism of membrane lipid composition regulating conformation and hydration of BM2 and have important significance to the further study of anti-influenza virus B drugs.

Revealing the binding and drug resistance mechanism of amprenavir, indinavir, ritonavir, and nelfinavir complexed with HIV-1 protease due to double mutations G48T/L89M by molecular dynamics simulations and free energy analyses.

Infection by human immunodeficiency virus type 1 (HIV-1) not only destroys the immune system bringing about acquired immune deficiency syndrome (AIDS), but also induces serious neurological diseases including behavioral abnormalities, motor dysfunction, toxoplasmosis, and HIV-1 associated dementia. The emergence of HIV-1 multidrug-resistant mutants has become a major problem in the therapy of patients with HIV-1 infection. Focusing on the wild type (WT) and G48T/L89M mutated forms of HIV-1 protease (HIV-1 PR) in complex with amprenavir (APV), indinavir (IDV), ritonavir (RTV), and nelfinavir (NFV), we have investigated the conformational dynamics and the resistance mechanism due to the G48T/L89M mutations by conducting a series of molecular dynamics (MD) simulations and free energy (MM-PBSA and solvated interaction energy (SIE)) analyses. The simulation results indicate that alterations in the side-chains of G48T/L89M mutated residues cause the inner active site to increase in volume and induce more curling of the flap tips, which provide the main contributions to weaker binding of inhibitors to the HIV-1 PR. The results of energy analysis reveal that the decrease in van der Waals interactions of inhibitors with the mutated PR relative to the wild-type (WT) PR mostly drives the drug resistance of mutations toward these four inhibitors. The energy decomposition analysis further indicates that the drug resistance of mutations can be mainly attributed to the change in van der Waals and electrostatic energy of some key residues (around Ala28/Ala28' and Ile50/Ile50'). Our work can give significant guidance to design a new generation of anti-AIDS inhibitors targeting PR in the therapy of patients with HIV-1 infection.

Insight on mutation-induced resistance to anaplastic lymphoma kinase inhibitor ceritinib from molecular dynamics simulations.

Ceritinib, an advanced anaplastic lymphoma kinase (ALK) next-generation inhibitor, has been proved excellent antitumor activity in the treatment of ALK-associated cancers. However, the accumulation of acquired resistance mutations compromise the therapeutic efficacy of ceritinib. Despite abundant mutagenesis data, the structural determinants for reduced ceritinib binding in mutants remains elusive. Focusing on the G1123S and F1174C mutations, we applied molecular dynamics (MD) simulations to study possible reasons for drug resistance caused by these mutations. The MD simulations predict that the studied mutations allosterically impact the configurations of the ATP-binding pocket. An important hydrophobic cluster is identified that connects P-loop and the αC-helix, which has effects on stabilizing the conformation of ATP-binding pocket. It is suggested, in this study, that the G1123S and F1174C mutations can induce the conformational change of P-loop thereby causing the reduced ceritinib affinity and causing drug resistance.

Molecular dynamics simulations study of influence of Tyr422Ala mutation on transcriptional enhancer activation domain 4 (TEAD4) and transcription co-activators complexes.

Transcriptional enhancer activation domain (TEAD) proteins are the downstream transcriptional factor of the Hippo pathway. The transcription co-activators Yes-associated protein (YAP) and its paralog transcription co-activators with PDZ-binding motif (TAZ), binding to TEAD to promote transcription of genes in cell proliferation and anti-apoptosis, are key effectors of the Hippo pathway. TEAD4, one member of TEAD proteins, is specifically required in embryo implantation. The recently reported crystal structure of TEAD4-TAZ complex (PDB Code 5GN0) in mouse reveals that the interactions between the two helices of YAP/TAZ and TEAD4 are highly conserved. Point mutation of the residue Tyr422 of TEAD4 protein would disrupt the relevant hydrogen bond and even abolish the interaction. However, detailed information affected by the mutation at the atom level are still unrevealed. Molecular dynamics (MD) simulations and the molecular mechanics/Generalized-Born surface area (MM/GBSA) free energy calculations were used to explore the effects of mutation Tyr422Ala on the structural flexibility and conformational dynamics. The non-polar interactions play an indispensable role in the binding process of TEAD4 and YAP/TAZ. The helices α1 and α2 of YAP/TAZ provide a primary function to anchor YAP/TAZ well bound to TEAD4. The mutation Tyr422Ala disrupts the hydrogen-bonding network but do not obviously influence the secondary structure stability of TEAD4. The binding conformation of YAP/TAZ distorted by decreased non-polar interaction and the lost hydrogen bonds would lead to reduced interaction activity. The present study would provide important insights into the structure-function relationships of TEAD protein and give a new explanation for the affinity of YAP/TAZ with TEAD.

MD Simulation Investigation on the Binding Process of Smoke-Derived Germination Stimulants to Its Receptor.

Karrikins (KARs) are a class of smoke-derived seed germination stimulants with great significance in both agriculture and plant biology. By means of direct binding to the receptor protein KAI2, the compounds can initiate the KAR signal transduction pathway, hence triggering germination of the dormant seeds in the soil. In the research, several molecular dynamics (MD) simulation techniques were properly integrated to investigate the binding process of KAR1 to KAI2 and reveal the details of the whole binding event. The calculated binding free energy, -7.00 kcal/mol, is in good agreement with the experimental measurement, -6.83 kcal/mol. The obtained PMF profile indicates the existence of three intermediate states in the binding process. The analysis of the simulation trajectories demonstrates that, in the intermediate structures, KAR1 is stabilized by some hydrophobic residues (Phe26, Phe134, Leu142, Trp153, Phe157, Leu160, Phe194), along with several bridging water molecules, and meanwhile, the significant shifting occurs in the local conformation of the protein as the ligand's binding. A series of the residues (Gln141-Phe157) on the so-called "cap domain" are proposed to be responsible for capturing the ligand at the initial stage of the binding. Besides, the changes of the ligand's poses are also quantitatively characterized by the proper choice of the coordinate system. Our work will contribute to the more penetrating understanding of the ligand binding process and the receptor affinity difference between several members in the KAR family and help design new, more effective germination stimulants.

What are the effects of the serine triad on proton conduction of an influenza B M2 channel? An investigation by molecular dynamics simulations.

The tetrameric influenza B M2 channel (BM2), an acid activated proton channel, is important in the influenza virus B lifecycle. A conserved HxxxW motif is responsible for proton conduction and channel gating. In this study, to explore the effects of the serine triad (S9, S12 and S16) on proton conduction, we performed classical molecular dynamics (CMD) simulations and adaptive steered molecular dynamics (ASMD) simulations at different protonation states of the H19 tetrad. The results of the pore radius and the C-terminal tilt angle show that the electrostatic repulsion induced by protonated H19 is the key driving force for opening the BM2 channel. The open states could be stabilized by the hydrogen bonds between S16 and protonated H19. The solvent accessible surface area and water density indicate that the polar hydrophilic environment provided by the serine triad facilitates the formation of a water wire, and then exhibits favourable effects on proton conduction. The mutant research verifies and supports these views. Our work clarifies the effects of the serine triad on proton conduction in the BM2 channel, which would help us deeply understand the proton conduction mechanism in BM2 and provides a new perspective for antiviral drug design against BM2.

Humanization of fibroblast growth factor 1 single-chain antibody and validation for its antitumorigenic efficacy in breast cancer and glioma cells.

Single-chain variable fragment (scFv) antibodies are the smallest immunoglobulins with high antigen-binding affinity. We have previously reported that fibroblast growth factor 1 played pivotal roles in cancer development and generated a mouse scFv (mscFv1C9) could effectively prohibit cancer cell proliferation in vitro and in vivo. Here, we further humanized this scFv (hscFv1C9) using a structure-guided complementarity determining region grafting strategy. The purified hscFv1C9 maintained similar antigen-binding affinity and specificity as mscFv1C9, and it was capable of inhibiting growth of different tumours in vitro and in vivo. These data strongly suggested that hscFv1C9 has antitumour potentials.

Molecular dynamics investigation of stereoselective inhibition mechanism of HIF-2α/ARNT heterodimer.

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors related with the onset and progression of solid tumors. Studies demonstrated a class of tetrazole containing chiral inhibitors could stereoselectively disrupt the HIF-2 dimerization and reduce the target gene expression. However, the dynamical features and structural motifs of the HIF-2 heterodimer caused by the binding of enantiomers have not been rationalized at the atomistic level. In this work, molecular dynamics (MD) simulations combined with adaptive steered MD (ASMD) simulations were used to investigate stereoselective interrupting mechanism of HIF-2. Our results decipher that the binding of ligand A (S, R)-24 begets the significant conformation changes of ß-sheets and interrupts the HIF-2α/ARNT heterodimerization, which may be attributed to the disruption of the hydrogen bond and salt bridge interactions formed by the 4 foremost residues (Asp240, Arg247, Glu362, and Arg366) and the destruction of hydrophobic interactions on the binding interface. By contrast, the binding of ligand B (R, S)-24 does not disrupt protein dimerization and causes the motion of Fα helix in HIF-2α PAS-B domain to further change the major tunnel for ligand ingress and engress. The present work provides important molecular-level insight into the effect of the binding enantiomers on HIF-2 heterodimerization and bridges the gap between theory and the experimental results, which may conduce to develop highly potent antagonists for intervening the HIF-2-driven tumors.

Exploring the inhibition mechanism on HIF-2 by inhibitor PT2399 and 0X3 using molecular dynamics simulations.

Targeting transcription factors HIF-2 is currently considered to be the most direct way for the therapy of clear cell renal cell carcinoma. The preclinical inhibitor PT2399 and artificial inhibitor 0X3 have been identified as promising on-target inhibitors to inhibit the heterodimerization of HIF-2. However, the inhibition mechanism of PT2399 and 0X3 on HIF-2 remains unclear. To this end, molecular dynamics (MD) simulations and molecular docking were applied to investigate the effects of 2 inhibitors on structural motifs and heterodimerization of HIF-2. Our simulation results reveal that the binding of inhibitors disrupts the crucial hydrogen bond and hydrophobic interactions of interdomain of HIF-2 heterodimer due to the local conformational changes of binding interface, confirming the hypothesis that the perturbation of few residues is sufficient to disrupt the heterodimerization of HIF-2. In addition, it can be found that PT2399 with dominant substituents (cyano, fluorine, sulfuryl, and hydroxyl) is more preferred than 0X3 as HIF-2 inhibitor and these substituents play a crucial role in involving more hydrogen bond interactions with residues of interface and then cause the larger structural change of protein. This study may provide a deeper atomic-level insight into the effect of on-target inhibitors on HIF-2 heterodimer, which is expected to contribute to further rational design of effective clear cell renal cell carcinoma drugs.

Exploring the influence of hyperthermophilic protein Ssh10b on the stability and conformation of RNA by molecular dynamics simulation.

The hyperthermophilic Ssh10b from Sulfolobus shibatae is a member of the Sac10b family, which binds RNA in vivo as a physiological substrate, and it has been postulated to play a key role in chromosomal organization in Archaea. Even though the crystal structure of Ssh10b-RNA was resolved successively by X-ray diffraction (Protein Data Bank [PDB] code: 3WBM), the detailed dynamic characteristics of Ssh10b-RNA are still unclear. In this study, molecular dynamics (MDs) simulations at 6 temperatures (300, 350, 375, 400, 450, and 500 K) and molecular mechanics Generalized-Born surface area (MM-GB/SA) free energy calculations were performed to investigate the mechanism of how Ssh10b protects and stabilizes RNA. The simulation results indicate that RNA is stabilized by Ssh10b when the temperature rises up to 375 K. RNA is found to undergo conformational transition between A-RNA and A'-RNA when Ssh10b binds to RNA at 3 different temperatures (300, 350, and 375 K). Salt bridges, hydrogen bonds and hydrophobic interactions are observed, and some residues have significant impact on the structural stability of the complex. This study increases our understanding of the dynamics and interaction mechanism of hyperthermophilic proteins and RNA at the atomic level, and offers a model for studying the structural biology of hyperthermophilic proteins and RNA.

Exploring the structure characteristics and major channels of cytochrome P450 2A6, 2A13, and 2E1 with pilocarpine.

The majority of cytochromes P450 play a critical role in metabolism of endogenous and exogenous substrates, some of its products are carcinogens. Therefore, inhibition of P450 enzymes activity can promote the detoxification and elimination of chemical carcinogens. In this study, molecular dynamics (MD) simulations and adaptive steered molecular dynamics (ASMD) simulations were performed to explore the structure features and channel dynamics of three P450 isoforms 2A6, 2A13, and 2E1 bound with the common inhibitor pilocarpine. The binding free energy results combined with the PMF calculations give a reasonable ranking of binding affinity, which are consistent with the experimental data. Our results uncover how a sequence divergence of different CYP2 enzymes causes individual variations in major channel selections. On the basis of channel bottleneck and energy decomposition analysis, we propose a gating mechanism of their respective major channels in three enzymes, which may be attributed to a reversal of Phe209 in CYP2A6/2A13, as well as the rotation of Phe116 and Phe298 in CYP2E1. The hydrophobic residues not only make strong hydrophobic interactions with inhibitor, but also act as gatekeeper to regulate the opening of channel. The present study provides important insights into the structure-function relationships of three cytochrome P450s and the molecular basis for development of potent inhibitors.

Exploring the interactions of EGFR with phosphorylated Mig6 by molecular dynamics simulations and MM-PBSA calculations.

Mig6, a negative regulator, directly binds to epidermal growth factor receptor (EGFR), including Mig6-segment1 and Mig6-segment2. Mig6 requires phosphorylation of Y394 on Mig6-segment2 in order to inhibit EGFR. Two phosphorylation pathways for Y394 have been previously reported and the first way may phosphorylate Y394 primed by Y395 phosphorylation. Besides, the binding mechanism of phosphorylated Mig6-segment2 with EGFR has not been elucidated clearly. Focused on EGFR complex with phosphorylated Mig6-segment2, molecular dynamics (MD) simulations were performed to explore the interactions of Mig6-segment2 with EGFR. Our results indicate a probable phosphorylation pathway on Y394 and some key residues of EGFR play important roles in binding to phosphorylated Mig6-segment2. In addition, a special L-shaped structure was found to be possibly associated with irreversible inhibition of EGFR by Mig6. Our work can give meaningful information to better understand the phosphorylation pathways for Y394 and the interactions of EGFR binding to phosphorylated Mig6-segment2.

Theoretical research in structure characteristics of different inhibitors and differences of binding modes with CBP bromodomain.

The CBP (CREB (cAMP responsive element binding protein) binding protein) bromodomain (BRD) could recognize and bind with acetyl K382 of human tumor suppressor protein p53 which the mutation of encoding gene might cause human cancers. CBP-BRD serves as a promising drug target for several disease pathways and a series of effective drug have been discovered. In this study, molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GB/SA) approaches were performed to investigate the different binding modes between five inhibitors with CBP-BRD. Based on the energy and conformation analyses, a potent core fragment is chosen to act as the starting point for new inhibitor design by means of LUDI and rational drug design approaches. Then, T.E.S.T and molinspirition were applied to evaluate oral bioavailability and drug promiscuity of the new molecules. These results shed light on the idea for further inhibitor design.

Structural features and dynamic investigations of the membrane-bound cytochrome P450 17A1.

Cytochrome P450 (CYP) 17A1 is a dual-function monooxygenase with a critical role in the synthesis of many human steroid hormones. The enzyme is an important target for treatment of breast and prostate cancers that proliferate in response to estrogens and androgens. Despite the crystallographic structures available for CYP17A1, no membrane-bound structural features of this enzyme at atomic level are available. Accumulating evidence has indicated that the interactions between bounded CYPs and membrane could contribute to the recruitment of lipophilic substrates. To this end, we have investigated the effects on structural characteristics in the presence of the membrane for CYP17A1. The MD simulation results demonstrate a spontaneous insertion process of the enzyme to the lipid. Two predominant modes of CYP17A1 in the membrane are captured, characterized by the depths of insertion and orientations of the enzyme to the membrane surface. The measured heme tilt angles show good consistence with experimental data, thereby verifying the validity of the structural models. Moreover, conformational changes induced by the membrane might have impact on the accessibility of the active site to lipophilic substrates. The dynamics of internal aromatic gate formed by Trp220 and Phe224 are suggested to regulate tunnel opening motions. The knowledge of the membrane binding characteristics could guide future experimental and computational works on membrane-bound CYPs so that various investigations of CYPs in their natural, lipid environment rather than in artificially solubilized forms may be achieved.

Exploring the mechanism how AF9 recognizes and binds H3K9ac by molecular dynamics simulations and free energy calculations.

Histone acetylation is a very important regulatory mechanism in gene expression in the chromatin context. A new protein family-YEATS domains have been found as a novel histone acetylation reader, which could specific recognize the histone lysine acetylation. AF9 is an important one in the YEATS family. Focused on the AF9-H3K9ac (K9 acetylation) complex (ALY) (PDB code: 4TMP) and a serials of mutants, MUT (the acetyllsine of H3K9ac was mutated to lysine), F59A, G77A, and D103A, we applied molecular dynamics simulation and molecular mechanics Poisson-Boltzmann (MM-PBSA) free energy calculations to examine the role of AF9 protein in recognition interaction. The simulation results and analysis indicate that some residues of the protein have significant influence on recognition and binding to H3K9ac peptides and hydrophobic surface show the hydrophobic interactions play an important role in the binding. Our work can give important information to understand how the protein AF9 recognizes the peptides H3K9ac. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 779-786, 2016.

Effect of External Electric Field on Substrate Transport of a Secondary Active Transporter.

Substrate transport across a membrane accomplished by a secondary active transporter (SAT) is essential to the normal physiological function of living cells. In the present research, a series of all-atom molecular dynamics (MD) simulations under different electric field (EF) strengths was performed to investigate the effect of an external EF on the substrate transport of an SAT. The results show that EF both affects the interaction between substrate and related protein's residues by changing their conformations and tunes the timeline of the transport event, which collectively reduces the height of energy barrier for substrate transport and results in the appearance of two intermediate conformations under the existence of an external EF. Our work spotlights the crucial influence of external EFs on the substrate transport of SATs and could provide a more penetrating understanding of the substrate transport mechanism of SATs.

Bio-activation of 4-alkyl analogs of 1,4-dihydropyridine mediated by cytochrome P450 enzymes.

4-Alkyl-substituted 1,4-dihydropyridines (DHP) exhibit inhibitory activity toward certain cytochrome P450 enzymes (P450) during their biotransformation by these enzymes, which is called mechanism-based inactivation. Though much experimental evidence had proved the essentiality of alkyl radical for P450 inactivation, the underlying mechanism of such radical formation remains elusive. In the present study, density functional calculations were employed to investigate the dealkylation mechanism of 4-alkyl-substituted DHPs mediated by P450. Interestingly, our results indicate that the initial N-H activation proceeds via a proton-coupled electron transfer process, not via the long presumed hydrogen atom transfer mechanism or the stepwise electron transfer/proton transfer one, to form the amino radical and Cpd II complex. Subsequently, homolytic C-C bond cleavage at the 4-position occurs to afford the product complex involving an alkyl radical, an aromatic pyridine derivative. This C-C cleavage step is rate determining for the whole metabolic reaction, with an energy barrier of 7.9/7.9 kcal/mol on the quartet/doublet state, to which aromatization contributes as an essential intrinsic driving force. The 4-substituent groups induce differences in activation energy barriers and in the transition state structures of hydrogen abstraction process. The substrate reactivity correlates well with the stability of the generated alkyl radical as well as the C-C bond dissociation energy. Understanding the metabolic mechanism of DHP analogs is indeed essential for the related design of safer and more efficient drugs. Furthermore, our findings also enrich the mechanistic picture of amine oxidation catalyzed by P450.