Structural Impact Assessment of Cytochrome P450 2A13 Polymorphisms Using Molecular Dynamics Simulations.

One of the members of CYP, a monooxygenase, CYP2A13 is involved in the metabolism of nicotine, coumarin, and tobacco-specific nitrosamine. Genetic polymorphisms have been identified in CYP2A13, with reported loss or reduction in enzymatic activity in CYP2A13 allelic variants. This study aimed to unravel the mechanism underlying the diminished enzymatic activity of CYP2A13 variants by investigating their three-dimensional structures through molecular dynamics (MD) simulations. For each variant, MD simulations of 1000 ns were performed, and the obtained results were compared with those of the wild type. The findings indicated alterations in the interaction with heme in CYP2A13.4, .6, .8, and .9. In the case of CYP2A13.5, observable effects on the helix structure related to the interaction with the redox partner were identified. These conformational changes were sufficient to cause a decrease in enzyme activity in the variants. Our findings provide valuable insights into the molecular mechanisms associated with the diminished activity in the CYP2A13 polymorphisms.

Functional Characterization of 29 Cytochrome P450 4F2 Variants Identified in a Population of 8380 Japanese Subjects and Assessment of Arachidonic Acid ω-Hydroxylation.

Cytochrome P450 4F2 (CYP4F2) is an enzyme that is involved in the metabolism of arachidonic acid (AA), vitamin E and K, and xenobiotics including drugs. CYP4F2*3 polymorphism (rs2108622; c.1297G>A; p.Val433Met) has been associated with hypertension, ischemic stroke, and variation in the effectiveness of the anticoagulant drug warfarin. In this study, we characterized wild-type CYP4F2 and 28 CYP4F2 variants, including a Val433Met substitution, detected in 8380 Japanese subjects. The CYP4F2 variants were heterologously expressed in 293FT cells to measure the concentrations of CYP4F2 variant holoenzymes using carbon monoxide-reduced difference spectroscopy, where the wild type and 18 holoenzyme variants showed a peak at 450 nm. Kinetic parameters [Vmax , substrate concentration producing half of Vmax (S50 ), and intrinsic clearance (CL int ) as Vmax /S50 ] of AA ω-hydroxylation were determined for the wild type and 21 variants with enzyme activity. Compared with the wild type, two variants showed significantly decreased CL int values for AA ω-hydroxylation. The values for seven variants could not be determined because no enzymatic activity was detected at the highest substrate concentration used. Three-dimensional structural modeling was performed to determine the reason for reduced enzymatic activity of the CYP4F2 variants. Our findings contribute to a better understanding of CYP4F2 variant-associated diseases and possible future therapeutic strategies. SIGNIFICANCE STATEMENT: CYP4F2 is involved in the metabolism of arachidonic acid and vitamin K, and CYP4F2*3 polymorphisms have been associated with hypertension and variation in the effectiveness of the anticoagulant drug warfarin. This study presents a functional analysis of 28 CYP4F2 variants identified in Japanese subjects, demonstrating that seven gene polymorphisms cause loss of CYP4F2 function, and proposes structural changes that lead to altered function.

Identification of the Most Impactful Asparagine Residues for γS-Crystallin Aggregation by Deamidation.

Crystallin aggregation in the eye lens is involved in the pathogenesis of cataracts. The aggregation is considered to be promoted by non-enzymatic post-translational modifications, such as the deamidation and stereoinversion of amino acid residues. Although in a previous study, the deamidated asparagine residues were detected in γS-crystallin in vivo, it is unclear which deamidated residues have the most impact on the aggregation under physiological conditions. In this study, we investigated the deamidation impacts of all Asn residues in γS-crystallin for the structural and aggregation properties utilizing deamidation mimetic mutants (N14D, N37D, N53D, N76D, and N143D). The structural impacts were investigated using circular dichroism analysis and molecular dynamics simulations, and the aggregation properties were analyzed by gel filtration chromatography and spectrophotometric methods. No significant structural impacts of all mutations were detected. However, the N37D mutation decreased thermal stability and changed some intermolecular hydrogen-bond formations. Aggregation analysis indicated that the superiority of the aggregation rate in each mutant varied with temperature. Deamidation at any Asn residues promoted γS-crystallin aggregation, and the deamidation at Asn37, Asn53, and Asn76 were suggested to be the most impactful in the formation of insoluble aggregations.

Effect of the Addition of the Fifth Amino Acid to [GADV]-Protein on the Three-Dimensional Structure.

The [GADV]-protein, consisting only of glycine (G), alanine (A), aspartic acid (D), and valine (V), is frequently studied as a candidate for a primitive protein that existed at the beginning of life on Earth. The number of proteogenic amino acids increased during evolution, and glutamic acid may have been added as the fifth amino acid. In this study, we used molecular dynamics simulations to estimate the conformation of random peptides when glutamate is added to G, A, D, and V ([GADVE]), when leucine is added ([GADVL]), and when the frequency of alanine is doubled ([GADVA]). The results showed that the secondary structure contents of the [GADVE]-peptide and [GADVL]-peptide were higher than that of the [GADVA]-peptide. Although the [GADVL]-peptide had a higher secondary structure formation ability than the [GADVE]-peptide, it was less water soluble, suggesting that it may not be a primitive protein. The [GA(D/E)V]-peptide with G:A:D:V:E = 2:2:1:2:1 according to the occurrence ratio in the codon table also increased the secondary structure contents compared to the [GADV]-peptide, indicating that the addition of glutamic acid increased the structure formation ability of the primitive protein candidates.

Functional Characterization of 12 Dihydropyrimidinase Allelic Variants in Japanese Individuals for the Prediction of 5-Fluorouracil Treatment-Related Toxicity.

The drug 5-fluorouracil (5-FU) is the first-choice chemotherapeutic agent against advanced-stage cancers. However, 10% to 30% of treated patients experience grade 3 to 4 toxicity. The deficiency of dihydropyrimidinase (DHPase), which catalyzes the second step of the 5-FU degradation pathway, is correlated with the risk of developing toxicity. Thus, genetic polymorphisms within DPYS, the DHPase-encoding gene, could potentially serve as predictors of severe 5-FU-related toxicity. We identified 12 novel DPYS variants in 3554 Japanese individuals, but the effects of these mutations on function remain unknown. In the current study, we performed in vitro enzymatic analyses of the 12 newly identified DHPase variants. Dihydrouracil or dihydro-5-FU hydrolytic ring-opening kinetic parameters, Km and Vmax , and intrinsic clearance (CLint = Vmax /Km ) of the wild-type DHPase and eight variants were measured. Five of these variants (R118Q, H295R, T418I, Y448H, and T513A) showed significantly reduced CLint compared with that in the wild-type. The parameters for the remaining four variants (V59F, D81H, T136M, and R490H) could not be determined as dihydrouracil and dihydro-5-FU hydrolytic ring-opening activity was undetectable. We also determined DHPase variant protein stability using cycloheximide and bortezomib. The mechanism underlying the observed changes in the kinetic parameters was clarified using blue-native polyacrylamide gel electrophoresis and three-dimensional structural modeling. The results suggested that the decrease or loss of DHPase enzymatic activity was due to reduced stability and oligomerization of DHPase variant proteins. Our findings support the use of DPYS polymorphisms as novel pharmacogenomic markers for predicting severe 5-FU-related toxicity in the Japanese population. SIGNIFICANCE STATEMENT: DHPase contributes to the degradation of 5-fluorouracil, and genetic polymorphisms that cause decreased activity of DHPase can cause severe toxicity. In this study, we performed functional analysis of 12 DHPase variants in the Japanese population and identified 9 genetic polymorphisms that cause reduced DHPase function. In addition, we found that the ability to oligomerize and the conformation of the active site are important for the enzymatic activity of DHPase.

Structural investigation of pathogenic variants in dihydropyrimidinase using molecular dynamics simulations.

Dihydropyrimidinase (DHP) is an enzyme that catabolizes the degradation of pyrimidine and fluoropyrimidine drugs such as 5-fluorouracil. DHP deficiency triggers various clinical symptoms and increases the risk of fluoropyrimidine drug toxicity. Various pathogenic variants of DHP cause DHP deficiency, and their catalytic activities have been well studied. However, the three-dimensional structures of DHP variants have not been clarified. In this study, we investigated the effects of mutations on DHP structures using the molecular dynamics simulations. Simulations of the wild type and 10 variants were performed and compared. In the T68R, D81G, G278D, and L337P variants, the flexibilities of structures related to the interaction for oligomer formation increased in comparison with those of the wild type. W117R, T343A, and R412 M mutations affected the structures of stereochemistry gate loops or the substrate-binding pocket. The three-dimensional structures of W360R and G435R variants were suggested to collapse. On the other hand, only slight structural changes were observed in the R181W variant, whose experimentally observed activity was similar to that of the wild type. The computational results are expected to clarify the relationship between clinical mutations and structural effects of drug-metabolizing enzymes.

Predicting Reaction Mechanisms for the Threonine-Residue Stereoinversion Catalyzed by a Dihydrogen Phosphate Ion.

The stereoinversion of amino acid residues in proteins is considered to trigger various age-related diseases. Serine (Ser) residues are relatively prone to stereoinversion. It is assumed that threonine (Thr) residues also undergo stereoinversion, which results in the formation of the d-allo-Thr residue, by the same mechanisms as those for Ser-residue stereoinversion; however, d-allo-Thr residues have not been detected in vivo. To date, although Ser-residue stereoinversion has been suggested to progress via enolization, plausible reaction mechanisms for Thr-residue stereoinversion have not been proposed. In this study, we investigated the pathway of Thr-residue enolization and successfully identified the three types of plausible reaction pathways of Thr-residue stereoinversion catalyzed by a dihydrogen phosphate ion. The geometries of reactant complexes, transition states, and enolized product complexes were optimized using B3LYP density functional methods, and single-point calculations were performed for all optimized geometries using Møller-Plesset perturbation theory to obtain reliable energies. As a result, the calculated activation energies of Thr-residue stereoinversion were 105-106 kJ mol-1, which were comparable with those of Ser-residue stereoinversion reported previously. The infrequency of Thr-residue stereoinversion may be due to other factors, such as the hydrophobicity and/or the steric hindrance of the γ-methyl group, rather than the high activation energies.

Computational Analysis of the Mechanism of Nonenzymatic Peptide Bond Cleavage at the C-Terminal Side of an Asparagine Residue.

The nonenzymatic peptide bond cleavage at the C-terminal side of Asn residues is a protein post-translational modification that occurs under physiological conditions. This reaction proceeds much slower than the deamidation of the Asn side chain and causes denaturation and hypofunction of proteins. The peptide bond cleavage of Asn is detected primarily in crystallins and aquaporin 0 in the eye lens. Therefore, cleavage is thought to be involved in age-related cataracts. In this study, to clarify the mechanism underlying succinimide formation for the peptide bond cleavage of the Asn residue, we performed quantum chemical calculations on the model compound Ace-Asn-Gly-Nme (Ace = acetyl and Nme = methylamino). The density functional theory with the B3LYP/6-31+G(d,p) level of theory was used to obtain optimized geometries. The results suggested that the reaction proceeds through two steps, cyclization and C-terminal fragment release, and the required proton transfers can be mediated by H2PO4 - and HCO3 - ions. The conformational change of the main chain on the N-terminal side of Asn was needed for the C-terminal fragmentation step, and a separate conformational change at the C-terminal side was required for the cyclization step. Furthermore, the calculated activation barriers of the reactions catalyzed by the H2PO4 - ion (130 kJ mol-1) and the HCO3 - ion (123 kJ mol-1) were sufficiently low for the reactions to occur under normal physiological conditions.

Virtual Alanine Scan of the Main Protease Active Site in Severe Acute Respiratory Syndrome Coronavirus 2.

Recently, inhibitors of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mpro) have been proposed as potential therapeutic agents for COVID-19. Studying effects of amino acid mutations in the conformation of drug targets is necessary for anticipating drug resistance. In this study, with the structure of the SARS-CoV-2 Mpro complexed with a non-covalent inhibitor, we performed molecular dynamics (MD) simulations to determine the conformation of the complex when single amino acid residue in the active site is mutated. As a model of amino acid mutation, we constructed mutant proteins with one residue in the active site mutated to alanine. This method is called virtual alanine scan. The results of the MD simulations showed that the conformation and configuration of the ligand was changed for mutants H163A and E166A, although the structure of the whole protein and of the catalytic dyad did not change significantly, suggesting that mutations in His163 and Glu166 may be linked to drug resistance.

Deciphering Structural Alterations Associated with Activity Reductions of Genetic Polymorphisms in Cytochrome P450 2A6 Using Molecular Dynamics Simulations.

Cytochrome P450 (CYP) 2A6 is a monooxygenase involved in the metabolism of various endogenous and exogenous chemicals, such as nicotine and therapeutic drugs. The genetic polymorphisms in CYP2A6 are a cause of individual variation in smoking behavior and drug toxicities. The enzymatic activities of the allelic variants of CYP2A6 were analyzed in previous studies. However, the three-dimensional structures of the mutants were not investigated, and the mechanisms underlying activity reduction remain unknown. In this study, to investigate the structural changes involved in the reduction in enzymatic activities, we performed molecular dynamics simulations for ten allelic mutants of CYP2A6. For the calculated wild type structure, no significant structural changes were observed in comparison with the experimental structure. On the other hand, the mutations affected the interaction with heme, substrates, and the redox partner. In CYP2A6.44, a structural change in the substrate access channel was also observed. Those structural effects could explain the alteration of enzymatic activity caused by the mutations. The results of simulations provide useful information regarding the relationship between genotype and phenotype.

Functional Characterization of 21 Rare Allelic CYP1A2 Variants Identified in a Population of 4773 Japanese Individuals by Assessing Phenacetin O-Deethylation.

Cytochrome P450 1A2 (CYP1A2), which accounts for approximately 13% of the total hepatic cytochrome content, catalyzes the metabolic reactions of approximately 9% of frequently used drugs, including theophylline and olanzapine. Substantial inter-individual differences in enzymatic activity have been observed among patients, which could be caused by genetic polymorphisms. Therefore, we functionally characterized 21 novel CYP1A2 variants identified in 4773 Japanese individuals by determining the kinetic parameters of phenacetin O-deethylation. Our results showed that most of the evaluated variants exhibited decreased or no enzymatic activity, which may be attributed to potential structural alterations. Notably, the Leu98Gln, Gly233Arg, Ser380del Gly454Asp, and Arg457Trp variants did not exhibit quantifiable enzymatic activity. Additionally, three-dimensional (3D) docking analyses were performed to further understand the underlying mechanisms behind variant pharmacokinetics. Our data further suggest that despite mutations occurring on the protein surface, accumulating interactions could result in the impairment of protein function through the destabilization of binding regions and changes in protein folding. Therefore, our findings provide additional information regarding rare CYP1A2 genetic variants and how their underlying effects could clarify discrepancies noted in previous phenotypical studies. This would allow the improvement of personalized therapeutics and highlight the importance of identifying and characterizing rare variants.

Theoretical Studies on the Effect of Isomerized Aspartic Acid Residues on the Three-Dimensional Structures of Bovine Pancreatic Ribonucleases A.

Isomerized aspartic acid (Asp) residues have previously been identified in various aging tissues, and are suspected to contribute to age-related diseases. Asp-residue isomerization occurs nonenzymatically under physiological conditions, resulting in the formation of three types of isomerized Asp (i.e., L-isoAsp, D-Asp, and D-isoAsp) residues. Asp-residue isomerization often accelerates protein aggregation and insolubilization, making structural biology analyses difficult. Recently, Sakaue et al. reported the synthesis of a ribonuclease A (RNase A) in which Asp121 was artificially replaced with different isomerized Asp residues, and experimentally demonstrated that the enzymatic activities of these artificial mutants were completely lost. However, their structural features have not yet been elucidated. In the present study, the three-dimensional (3D) structures of these artificial-mutant RNases A were predicted using molecular dynamics (MD) simulations. The 3D structures of wild-type and artificial-mutant RNases A were converged by 3000-ns MD simulations. Our computational data show that the structures of the active site and the formation frequencies of the appropriate catalytic dyad structures in the artificial-mutant RNases A were quite different from wild-type RNase A. These computational findings may provide an explanation for the experimental data which show that artificial-mutant RNases A lack enzymatic activity. Herein, MD simulations have been used to evaluate the influences of isomerized Asp residues on the 3D structures of proteins.

Effects of substituent pattern on the intracellular target of antiproliferative benzo[b]thiophenyl chromone derivatives.

A new biological scaffold was produced by replacing the 6π-electron phenyl ring-B of a natural flavone skeleton with a 10π-electron benzothiophene (BT). Since aromatic rings are important for ligand protein interactions, this expansion of the π-electron system of ring-B might change the bioactivity profile. One of the resulting novel natural product-inspired compounds, 2-(benzo[b]thiophen-3-yl)-5-hydroxy-7-isopropoxy-6-methoxyflavone (6), effectively arrested the cell cycle at the G2/M phase and displayed significant antiproliferative effects with IC50 values of 0.05-0.08 µM against multiple human tumor cell lines, including a multidrug resistant line. A structure-activity relationship study revealed that a 10π-electron system with high aromaticity, juxtaposed 4-oxo and 5-hydroxy groups, and 7-alkoxy groups were important for potent antimitotic activity. Interestingly, two BT-flavonols (3-hydroxyflavone), 16 and 20, with 3-hydroxy and 5-alkoxy groups, induced distinct biological profiles affecting the cell cycle at the G1/S phase by inhibition of DNA replication through an interaction with topoisomerase I.

Modification of the pH Dependence of Assembly of Yeast Cargo Receptor Emp47p Coiled-Coil Domains: Computational Design and Experimental Mutagenesis.

The coiled-coil domains of the putative yeast cargo receptors Emp46p and Emp47p (Emp46pcc and Emp47pcc) assemble into heterocomplexes at neutral pH. Upon lowering the pH, the complex dissociates and reassembles into homo-oligomers. A glutamate residue (E303) located on the hydrophobic surface of Emp46pcc serves as the pH-sensing switch for assembly and segregation, and we have suggested that its side chains are protonated in the heterocomplex, even at neutral pH. To examine this hypothesis, we constructed two structural models in which the side chains of E303 were negatively charged or protonated and analyzed the effects of these charged states on the structure of the heterocomplex using molecular dynamics (MD) simulations. The calculated structures suggested the side chains of E303 to be protonated in the heterocomplex, even at neutral pH. Based on these computational results, the pH dependence of Emp47pcc homo-oligomer assembly was experimentally modified by a glutamate mutation on its hydrophobic surface. The Q306E mutant of Emp47pcc underwent a structural transition at physiological pH. Our results suggest a method for modifying pH-dependent protein-protein interactions.

Functional Assessment of 12 Rare Allelic CYP2C9 Variants Identified in a Population of 4773 Japanese Individuals.

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by CYP2C9 genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These CYP2C9 variants were heterologously expressed in 293FT cells, and the kinetic parameters (Km, kcat, Vmax, catalytic efficiency, and CLint) of (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype-phenotype associations based on the novel CYP2C9 rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.

Functional Characterization of 40 CYP3A4 Variants by Assessing Midazolam 1'-Hydroxylation and Testosterone 6ß-Hydroxylation.

CYP3A4 is among the most abundant liver and intestinal drug-metabolizing cytochrome P450 enzymes, contributing to the metabolism of more than 30% of clinically used drugs. Therefore, interindividual variability in CYP3A4 activity is a frequent cause of reduced drug efficacy and adverse effects. In this study, we characterized wild-type CYP3A4 and 40 CYP3A4 variants, including 11 new variants, detected among 4773 Japanese individuals by assessing CYP3A4 enzymatic activities for two representative substrates (midazolam and testosterone). The reduced carbon monoxide-difference spectra of wild-type CYP3A4 and 31 CYP3A4 variants produced with our established mammalian cell expression system were determined by measuring the increase in maximum absorption at 450 nm after carbon monoxide treatment. The kinetic parameters of midazolam and testosterone hydroxylation by wild-type CYP3A4 and 29 CYP3A4 variants (K m , k cat , and catalytic efficiency) were determined, and the causes of their kinetic differences were evaluated by three-dimensional structural modeling. Our findings offer insight into the mechanism underlying interindividual differences in CYP3A4-dependent drug metabolism. Moreover, our results provide guidance for improving drug administration protocols by considering the information on CYP3A4 genetic polymorphisms. SIGNIFICANCE STATEMENT: CYP3A4 metabolizes more than 30% of clinically used drugs. Interindividual differences in drug efficacy and adverse-effect rates have been linked to ethnicity-specific differences in CYP3A4 gene variants in Asian populations, including Japanese individuals, indicating the presence of CYP3A4 polymorphisms resulting in the increased expression of loss-of-function variants. This study detected alterations in CYP3A4 activity due to amino acid substitutions by assessing the enzymatic activities of coding variants for two representative CYP3A4 substrates.

Molecular Mechanisms of Succinimide Formation from Aspartic Acid Residues Catalyzed by Two Water Molecules in the Aqueous Phase.

Aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. The formation of isomerized Asp residues is considered to be associated with various age-related diseases, such as cataracts and Alzheimer's disease. In the present paper, we describe the reaction pathway of Suc residue formation from Asp residues catalyzed by two water molecules using the B3LYP/6-31+G(d,p) level of theory. Single-point energies were calculated using the MP2/6-311+G(d,p) level of theory. For these calculations, we used a model compound in which an Asp residue was capped with acetyl and methylamino groups on the N- and C-termini, respectively. In the aqueous phase, Suc residue formation from an Asp residue was roughly divided into three steps, namely, iminolization, cyclization, and dehydration, with the activation energy estimated to be 109 kJ mol-1. Some optimized geometries and reaction modes in the aqueous phase were observed that differed from those in the gas phase.

(S)-Erypoegin K, an isoflavone isolated from Erythrina poeppigiana, is a novel inhibitor of topoisomerase IIα: Induction of G2 phase arrest in human gastric cancer cells.

Erypoegin K, an isoflavone isolated from the stem bark of Erythrina poeppigiana, has a single chiral carbon in its structure and exists naturally as a racemic mixture. Our previous study showed (S)-erypoegin K selectively exhibits potent anti-proliferative and apoptosis-inducing activity against human leukemia HL-60 cells. To identify the target molecule of (S)-erypoegin K, we employed the human cancer cell panel analysis (termed JFCR39) coupled with a drug sensitivity database of pharmacologically well-characterized drugs for comparison using the COMPARE algorithm. (S)-erypoegin K exhibited a similar profile to that of etoposide, suggesting the molecular target for erypoegin K may be topoisomerase II (Topo II). Subsequent experiments using purified human Topo IIα established that the (S)-isomer selectively stabilizes the cleavage complex composed of double-stranded plasmid DNA and the enzyme. Moreover, (S)-erypoegin K inhibited decatenation of kinetoplast DNA. Molecular docking studies clearly indicated specific binding of the (S)-isomer to the active site of Topo IIα involving hydrogen bonds that help stabilize the cleavage complex. (S)-erypoegin K displayed potent cytotoxic activity against two human gastric cancer cells GCIY and MKN-1 with IC50 values of 0.270 and 0.327 µM, respectively, and induced enzyme activities of caspase 3 and 9. Cell cycle analysis showed marked cell cycle arrest at G2 phase in both cell lines. (S)-erypoegin K also displayed significant antitumor activity toward GCIY xenografted mice. The present study suggests (S)-erypoegin K acts as a Topo II inhibitor to block the G2/M transition of cancer cells.

Development of Force Field Parameters for p-Carborane to Investigate the Structural Influence of Carborane Derivatives on Drug Targets by Complex Formation.

Androgen receptor (AR) has a key role in the development and progression of prostate cancer, and AR antagonists are used for its remedy. Recently, carborane derivatives, which are carbon-containing boron clusters have attracted attention as new AR ligands. Here we determined the force field parameters of 10-vertex and 12-vertex p-carborane to facilitate in silico drug design of boron clusters. Then, molecular dynamics (MD) simulations of complexes of AR-carborane derivatives were performed to evaluate the parameters and investigate the influences of carborane derivatives on the three-dimensional structure of AR. Energy profiles were obtained using quantum chemical calculations, and the force-field parameters were determined by curve fitting of the energy profiles. The results of MD simulations indicated that binding of the antagonist-BA341 changed some hydrogen-bond formations involved in the structure and location of helix 12. Those results were consistent with previously reported data. The determined parameters are also useful for refining the structure of the carborane-receptor complex obtained by docking simulations and development of new ligands with carborane cages not only for AR but also for various receptors.

Mechanisms of Deamidation of Asparagine Residues and Effects of Main-Chain Conformation on Activation Energy.

Deamidation of asparagine (Asn) residues is a nonenzymatic post-translational modification of proteins. Asn deamidation is associated with pathogenesis of age-related diseases and hypofunction of monoclonal antibodies. Deamidation rate is known to be affected by the residue following Asn on the carboxyl side and by secondary structure. Information about main-chain conformation of Asn residues is necessary to accurately predict deamidation rate. In this study, the effect of main-chain conformation of Asn residues on deamidation rate was computationally investigated using molecular dynamics (MD) simulations and quantum chemical calculations. The results of MD simulations for γS-crystallin suggested that frequently deamidated Asn residues have common main-chain conformations on the N-terminal side. Based on the simulated structure, initial structures for the quantum chemical calculations were constructed and optimized geometries were obtained using the B3LYP density functional method. Structures that were frequently deamidated had a lower activation energy barrier than that of the little deamidated structure. We also showed that dihydrogen phosphate and bicarbonate ions are important catalysts for deamidation of Asn residues.