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Results for “molecular dynamic simulation assay”

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PubMed2002

Rational design of a polymer specific for microcystin-LR using a computational approach.

A computational approach for the design of a molecularly imprinted polymer (MIP) specific for Cyanobacterial toxin microcystin-LR is presented. By using molecular modeling software, a virtual library of functional monomers was designed and screened against the target toxin, employed as a template. The monomers giving the highest binding energy were selected and used in a simulated annealing (molecular dynamics) process to investigate their interaction with the template. The stoichiometric ratio observed from the simulated annealing study was used in MIP preparation for microcystin-LR. The monomers were copolymerized with a cross-linker in the presence of the template. A control (blank) polymer was prepared under the same conditions but in the absence of template. A competitive assay with microcystin-horseradish peroxidase conjugate was optimized and used to evaluate the affinity and cross-reactivity of the polymer. The performance of the artificial receptor was compared to the performance of monoclonal and polyclonal antibodies raised against the toxin. The results indicate that imprinted polymer has affinity and sensitivity comparable to those of polyclonal antibodies (the detection limit for microcystin-LR using the MIP-based assay was found to be 0.1 microg L-1), while superior chemical and thermal stabilities were obtained. Moreover, cross-reactivity to other toxin analogues was very low for the imprinted polymer, in contrast to the results achieved for antibodies. It is anticipated that the polymer designed could be used in assays, sensors, and solid-phase extraction.

Computer Simulation↗

PubMed2005

Synthesis, biological evaluation, and molecular modeling of ribose-modified adenosine analogues as adenosine receptor agonists.

A number of 3'-C-methyl analogues of selective adenosine receptor agonists such as CPA, CHA, CCPA, 2'-Me-CCPA, NECA, and IB-MECA was synthesized to further investigate the subdomain of the receptor that binds the ribose moiety of the ligands. Affinity data at A(1), A(2A), and A(3) receptors in bovine brain membranes showed that the 3'-C-modification in adenosine resulted in a decrease of the affinity at all three receptor subtypes. When this modification was combined with N(6)-substitution with groups that induce high potency and selectivity at A(1) receptor, the affinity and selectivity were increased. However, all 3'-C-methyl derivatives proved to be very less active than the corresponding 2'-C-methyl analogues. The most active compound was found to be 3'-Me-CPA which displayed a K(i) value of 0.35 microM at A(1) receptor and a selectivity for A(1) vs A(2A) and A(3) receptors higher than 28-fold. 2'-Me-CCPA was confirmed to be the most selective, high affinity agonist so far known also at human A(1) receptor with a K(i) value of 3.3 nM and 2903- and 341-fold selective vs human A(2A) and A(3) receptors, respectively. In functional assay, 3'-Me-CPA, 3'-Me-CCPA, and 2-Cl-3'-Me-IB-MECA inhibited forskolin-stimulated adenylyl cyclase activity with IC(50) values ranging from 0.3 to 4.9 microM, acting as full agonists. A rhodopsin-based model of the bovine A(1)AR was built to rationalize the higher affinity and selectivity of 2'-C-methyl derivatives of N(6)-substituted-adenosine compared to that of 3'-C-methyl analogues. In the docking exploration, it was found that 2'-Me-CCPA was able to form a number of interactions with several polar residues in the transmembrane helices TM-3, TM-6, and TM-7 of bA(1)AR which were not preserved in the molecular dynamics simulation of 3'-Me-CCPA/bA(1)AR complex.

Adenosine↗

PubMed2005

The very C-terminus of protein kinase Cepsilon is critical for the full catalytic competence but its hydrophobic motif is dispensable for the interaction with 3-phosphoinositide-dependent kinase-1.

In this article, we explore the role of the C-terminus (V5 domain) of PKCepsilon plays in the catalytic competence of the kinase using serial truncations followed by immune-complex kinase assays. Surprisingly, removal of the last seven amino acid residues at the C-terminus of PKCepsilon resulted in a PKCepsilon-Delta731 mutant with greatly reduced intrinsic catalytic activity while truncation of eight amino acid residues at the C-terminus resulted in a catalytically inactive PKCepsilon mutant. Computer modeling and molecular dynamics simulations showed that the last seven and/or eight amino acid residues of PKCepsilon were involved in interactions with residues in the catalytic core. Further truncation analyses revealed that the hydrophobic phosphorylation motif was dispensable for the physical interaction between PKCepsilon and 3-phosphoinositide-dependent kinase-1 (PDK-1) as the PKCepsilon mutant lacking both the turn and the hydrophobic motifs could still be co-immunoprecipitated with PDK-1. These results provide fresh insights into the biochemical and structural basis underlying the isozyme-specific regulation of PKC and suggest that the very C-termini of PKCs constitute a promising new target for the development of novel isozyme-specific inhibitors of PKC.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2026

FusionTarget: Computational framework for drug repurposing against modeled fusion protein structures from genomic breakpoints.

Many fusion genes have been recognized as biomarkers and therapeutic targets. However, the lack of knowledge on protein structures and targeting approaches made it challenging to develop effective targeting therapeutics. To fill this, we developed a computational pipeline, FusionTarget, which annotates the genomic DNA breakage to RNA and protein sequences, predicts the 3D structures of fusion proteins, and performs comparative virtual screening, comparative molecular dynamics simulation, and quantitative analyses to identify the fusion protein-selective small molecules by selecting drugs with consistent high-fold binding affinity between fusion and wild-type proteins in multiple isoforms. We applied our pipeline to EWSR1::FLI1 in Ewing sarcoma and KMT2A::AFF1 in infant acute lymphoblastic leukemia. Further cell assay experiments confirmed that cells expressing individual fusion genes were more sensitive to the suggested drugs, and the key downstream genes were affected by our drugs. FusionTarget provides a unique foundation for developing therapeutics targeting fusion proteins.

applied computing in medical science↗

PubMed1999

Localization and mobility of coenzyme Q in lipid bilayers and membranes.

We have studied the mobility of coenzyme Q (CoQ) in lipid bilayers and mitochondrial membranes in relation to the control of electron transfer activities. A molecular dynamics computer simulation in the vacuum yielded a folded structure for CoQ10, with a length of only 21 A. Using this information we were able to calculate diffusion coefficients in the range of 10(-6) cm2/s in good agreement with those found experimentally by fluorescence quenching of pyrene derivatives. To investigate if CoQ diffusion may represent the rate-limiting step of electron transfer, we reconstituted complexes I and III and assayed the resulting NADH-cytochrome c reductase activity in presence of different CoQ10 levels and at different distances between complexes; the experimental turnovers were higher than the collision frequencies calculated using diffusion coefficients of 10(-9) cm2/s but compatible with values found by us by fluorescence quenching. Since the experimental turnovers are independent of the distance between complexes, we conclude that CoQ diffusion is not rate-limiting for electron transfer.

Animals↗

PubMed2003

Cyclorocaglamide, the first bridged cyclopentatetrahydrobenzofuran, and a related "open chain" rocaglamide derivative from Aglaia oligophylla.

Two rocaglamide-related natural products, the previously known compound 6-demethoxy-10-hydroxy-11-methoxy-6,7-methylendioxyrocaglamide (3), and cyclorocaglamide (4), its 8b,10-anhydro analogue, have been isolated from the tropical plant Aglaia oligophylla. Compound 4 is the first bridged cyclopentatetrahydrobenzofuran natural product, and it exhibited a CD spectrum virtually opposite that of all the other rocaglamide natural products known so far, but it still has the same absolute configuration at all stereogenic centers of the basic molecular framework. This was shown unequivocally by quantum chemical CD calculations (here based on molecular dynamics-weighted force field structures) and was finally confirmed experimentally, by a "biomimetic-type" cyclization of 3 to give 4, with the expected "inversion" of the CD spectrum. The opposite chiroptical properties of 3 and 4, despite their homochiral character, underline the necessity of handling chiroptical data with the greatest care, e.g., by simulating them by quantum chemical CD calculations. Compound 3 exhibited an LC(50) of 2.5 ppm when evaluated against neonate larvae of Spodoptera littoralis, while 4 was inactive in this assay up to 100 ppm.

Animals↗

PubMed2005

Protein C Sapporo (protein C Glu 25 --> Lys): a heterozygous missense mutation in the Gla domain provides new insight into the interaction between protein C and endothelial protein C receptor.

Interaction of the gamma-carboxyglutamic acid (Gla) domain of protein C with endothelial protein C receptor (EPCR) is a critical step for efficient activation of protein C, though interactions by mutants in the Gla domain of protein C with EPCR have been rarely evaluated. We identified a 44-year-old Japanese woman with a history of recurrent thromboembolism as an inherited missense mutation, the first such case reported in Japan, which involved a protein C Gla 25 mutation. Total protein C antigen and Gla protein C antigen levels in the proband were normal. Protein C activity measured with an anticoagulant assay was reduced, whereas that measured with an amidolytic assay was normal. She was therefore phenotypically diagnosed as type IIb protein C deficiency. Direct sequencing of the PCR fragments revealed a heterozygous G to A transition at nucleotide position 1462 in exon 3, which predicted an amino acid substitution of Glu 25 by Lys. Her mother and one son were also heterozygous for this mutation. A molecular dynamics simulation of Gla 25-->Lys/EPCR complex in water suggested that the affinity between the molecules was decreased compared to the wild type Gla domain/EPCR complex. Since Gla 25 has been shown to play an important role in protein C function, not only in membrane phospholipid binding but also in binding to EPCR, our findings provide new insight into the mechanism by which the Glu 25-->Lys mutation induces type IIb protein C deficiency in individuals.

Adult↗

PubMed2000

Molecular correlates of the action of bis(ethyl)polyamines in breast cancer cell growth inhibition and apoptosis.

Polyamines are known to be involved in cell growth regulation in breast cancer. To evaluate the efficacy of bis(ethyl)polyamine analogs for breast cancer therapy and to understand their mechanism of action we measured the effects of a series of polyamine analogs on cell growth, activities of enzymes involved in polyamine metabolism, intracellular polyamine levels, and the uptake of putrescine and spermidine using MCF-7 breast cancer cells. The IC50 values for cell growth inhibition of three of the compounds, N1,N12-bis(ethyl)spermine, N1,N11-bis(ethyl)norspermine, and N1,N14-bis(ethyl)homospermine, were in the range of 1-2 microM. Another group of three compounds showed antiproliferative activity at about 5 microM level. These compounds are also capable of suppressing colony formation in soft agar assay and inducing apoptosis of MCF-7 cells. The highly effective growth inhibitory agents altered the activity of polyamine biosynthetic and catabolic enzymes and down-regulated the transport of natural polyamines, although each compound produced a unique pattern of alterations in these parameters. HPLC analysis showed that cellular uptake of bis(ethyl)polyamines was highest for bis(ethyl)spermine. We also analyzed polyamine analog conformations and their binding to DNA minor or major grooves by molecular modelling and molecular dynamics simulations. Results of these analyses indicate that tetramine analogs fit well in the minor groove of DNA whereas, larger compounds extend out of the minor groove. Although major groove binding was also possible for the short tetramine analogs, this interaction led to a predominantly bent conformation. Our studies show growth inhibitory activities of several potentially important analogs on breast cancer cells and indicate that multiple sites are involved in the mechanism of action of these analogs. While the activity of an analog may depend on the sum of these different effects, molecular modelling studies indicate a correlation between antiproliferative activity and stable interactions of the analogs with major or minor grooves of DNA.

Acetyltransferases↗

PubMed2026

Stable simulations do not guarantee functional engagement: a case study of off-target prediction for Seladelpar and Zanamivir.

Identifying off-target interactions of approved drugs is important to anticipate side effects and uncover repurposing opportunities. Computational pipelines combining structural homology, structure prediction, and molecular dynamics (MD) simulations offer a promising strategy, but it remains unclear whether stable, control-like MD trajectories reliably indicate functional engagement. We examined this in a case study of two approved drugs. Using the Evolutionary Classification of Protein Domains (ECOD) framework to select candidate off-targets, we modeled each drug-protein complex as two independent AlphaFold3 models and simulated both by MD, for Seladelpar (a PPARδ agonist) and Zanamivir, an influenza neuraminidase inhibitor that also inhibits human Sialidase-2 (NEU2). Candidates were ranked by the similarity of global MD descriptors to the on-target control. For Seladelpar, the three top-ranked candidates (FXR, RARγ, ERRγ) were tested experimentally; the Zanamivir set was analyzed computationally only. None showed measurable activity in reporter or thermal shift assays, despite stable simulations and descriptor values comparable to the control. Including PPARα and PPARγ as weak-positive comparators, these descriptors did not rank genuine interactions closer to the control than inactive candidates. Residue-level comparison with experimental structures showed the predicted poses reproduced only part of the canonical contacts. Where experimental drug-bound structures existed, AlphaFold3 reproduced the pose for PPARα but not PPARγ, and its per-model confidence did not track pose accuracy. Within this case study, the specific global descriptors examined reflect complex stability rather than functional engagement, which does not mean MD-based approaches cannot make this distinction.

Zanamivir↗

PubMed2003

Synthesis, activity, and molecular modeling studies of novel human aldose reductase inhibitors based on a marine natural product.

Aldose reductase (ALR2) has been implicated in the etiology of diabetic complications, including blindness. Because of the limited number of currently available drugs for the prevention of these long-term complications, the discovery of new ALR2 inhibitors appears highly desirable. In this study, a polybrominated diphenyl ether (1) naturally occurring in a marine sponge was found to inhibit recombinant human ALR2 with an IC(50) of 6.4 microM. A series of polyhalogenated analogues that were synthesized and tested in vitro to explore the structure-activity relationships displayed various degrees of inhibitory activity. The most active compounds were also capable of preventing sorbitol accumulation inside human retinal cells. In this cell-based assay, the most potent synthesized analogue (16) showed a 17-fold increase in inhibitory activity compared to that of sorbinil (IC(50) = 0.24 vs 4 microM). A molecular representation of human ALR2 in complex with the natural product was built using homology modeling, automated docking, and energy refinement methods. AMBER parameters for the halogen atoms were derived and calibrated using condensed phase molecular dynamics simulations of fluorobenzene, chlorobenzene, and bromobenzene. Inhibitor binding is proposed to cause a conformational change similar to that recently reported for zenarestat. A free energy perturbation thermodynamic cycle allowed us to assess the importance of a crucial bromine atom that distinguishes the active lead compound from a much less active close natural analogue. Remarkably, the spatial location of this bromine atom is equivalent to that occupied by the only bromine atom present in zenarestat.

Aldehyde Reductase↗

PubMed2006

Phosphorylated and nonphosphorylated epitopes of the La/SSB autoantigen: comparison of their antigenic and conformational characteristics.

La/SSB phosphoprotein is the target antigen of autoantibodies in sera of patients with Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Among other structural and function motifs, four phosphorylation sites are encompassed in the primary sequence of La/SSB. Two of them (Thr-362 and Ser-366) are located within GSGKGKVQFQGKKTKFASDD (346-368) and one (Thr-302) within VTWEVLEGEVEKEALKKI (301-318), which are main B-cell epitopes of La/SSB. With the aim to investigate how phosphorylation, one of the most common posttranslational protein modifications, affects the antigenic and conformational characteristics of the La/SSB epitopes, we synthesized and studied the phosphorylated epitopes La/SSB(346-368)-P, La/SSB(359-368)-P, and La/SSB(301-318)-P with respect to their nonphosphorylated counterparts. Anti-La/SSB positive sera from SS and SLE patients are better recognized by the phosphorylated epitopes compared to their nonphosphorylated counterparts. Conformational analysis by (1)H nuclear magnetic resonance spectroscopy and molecular dynamics showed that the phosphorylated epitopes adopt different structural characteristics from those of the corresponding nonphosphorylated epitopes. It is concluded that phosphorylation can create neoepitopes with altered functions, compared to the nonphosphorylated epitopes, which might be seen from the immune system as "foreign."

Amino Acid Sequence↗

PubMed2006

A 40.7 kDa Rpp30/Rpp1 homologue is a protein subunit of Dictyostelium discoideum RNase P holoenzyme.

RNase P is an essential and ubiquitous endonuclease that mediates the maturation of the 5' ends of all precursor tRNA molecules. The holoenzyme from Dictyostelium discoideum possesses RNA and protein subunits essential for activity, but the exact composition of the ribonucleoprotein complex is still under investigation. Bioinformatic analysis of D. discoideum genome identified seven open reading frames encoding candidate RNase P protein subunits. The gene named drpp30 encodes a protein with a predicted molecular mass of 40.7 kDa that clusters with Rpp1 and Rpp30 RNase P protein subunits from Saccharomyces cerevisiae and human respectively, which have significantly lower molecular masses. Cloning and heterologous expression of DRpp30 followed by immunochemical analysis of RNase P active fractions demonstrates its association with RNase P holoenzyme. Furthermore, we show that DRpp30 can bind D. discoideum RNase P RNA and tRNA transcripts in vitro, giving a first insight of its possible role in D. discoideum RNase P function. Homology modeling using as a template the archaeal Ph1887p, and molecular dynamics simulations of the modeled structure suggest that DRpp30 adopts a TIM-barrel fold.

Amino Acid Sequence↗

PubMedDate not supplied

High-affinity rat anti-fluorescein monoclonal antibody with unique fine specificity properties including differential recognition of dynamic ligand analogues.

The ability of antibodies to specifically select and stabilize through binding one or more isomers of highly dynamic ligands remains a relatively unexplored immunochemical problem. The experimental strategy employed in this study was to elicit homogeneous antibodies to polyaromatic fluorescein which exists in one isomeric form. The binding properties of a monoclonal rat antifluorescein antibody specific to a given isomer were quantitatively studied to determine the capacity to bind dynamic analogues of fluorescein which exists in multiple isomers. To generate monoclonal anti-fluorescein antibodies that reacted with specific dynamic analogues of fluorescein possessing unconjugated aromatic ring systems, immune spleenocytes from Lou/M rats immunized with FITC(I)-KLH were fused with Balb/c SP2/0-Ag14 murine myeloma cells forming rat-mouse hybridomas. Cell line P2A12-1-C8 was selected for further characterization from the original 23 stable rat hybrids, since it produced a monoclonal antibody with a binding affinity 2.0 x 10(10)/M for fluorescein based on dissociation rate measurements. P2A12-1-C8 exhibited significant reactivity with HPF and phenol red, which are dynamic structural analogues of the homologous fluorescein ligand. No reactivity was demonstrated with phenolphthalein, which based on relative chemical structures was expected to be more reactive than phenol red. Computer-based molecular modeling and energy minimization studies of fluorescein, HPF, phenol red, and phenolphthalein showed that in terms of the most energetically favorable orientation of the three aromatic rings, phenol red more closely simulated fluorescein than phenolphthalein. The results were analyzed in terms of the mechanisms of dynamic ligand stabilization and binding involving accommodation of specific ligand isomers by energetically permissible conformational states exhibited by an antibody active site. Thus, antibody reactivity of an anti-fluorescein antibody with phenol red and phenolphthalein was dictated more by ligand dynamics and aromatic orientation than by chemical structure similarities.

Absorption↗

PubMed2006

Inspection of the binding sites of proteinase3 for the design of a highly specific substrate.

Proteinase3 (PR3) and human neutrophil elastase (HNE) are homologous proteases from the polymorphonuclear neutrophils and have been thought for a long time to have close enzymatic specificity. We have used molecular dynamics simulations to investigate and compare the interactions between different peptides and the two enzymes. The important role played especially by the C-terminal part of the peptides is confirmed. We provide a map of the subsites of PR3 and a description of the interaction scheme for six ligands. The main difference between HNE and PR3 concerns S2, S1', S2', and S3'. The recognition subsites in PR3 are interconnected; in particular, Lys99 participates to a hydrophobic (S4) and a polar (S2) pocket. On the basis of the simulations, we suggest that VADVKDR is a highly specific sequence for PR3; enzymatic assays confirm that it is cleaved by PR3 with a high specificity constant (k(cat)/K(m) = 3,400,000 M(-1) s(-1)) and not by HNE.

Amino Acid Sequence↗

PubMed2003

Docking and electron transfer studies between rubredoxin and rubredoxin:oxygen oxidoreductase.

The interaction and electron transfer (ET) between rubredoxin (Rd) and rubredoxin:oxygen oxidoreductase (ROO) from Desulfovibrio gigas is studied by molecular modelling techniques. Experimental kinetic assays using recombinant proteins show that the Rd reoxidation by ROO displays a bell-shaped dependence on ionic strength, suggesting a non-trivial electrostatic dependence of the interaction between these two proteins. Rigid docking studies reveal a prevalence for Rd to interact, in a very specific way, with the surface of the ROO dimer near its FMN cofactors. The optimization of the lowest energy complexes, using molecular dynamics simulation, shows a very tight interaction between the surface of the two proteins, with a high probability for Rd residues (but not the iron centre directly) to be in direct contact with the FMN cofactors of ROO. Both electrostatics and van der Waals interactions contribute to the final energy of the complex. In these complexes, the major contributions for complex formation are polar interactions between acidic residues of Rd and basic residues of ROO, plus substantial non-polar interactions between different groups. Important residues for this process are identified. ET estimates (using the Pathways model), in the optimized lowest energy complexes, suggest that these configurations are efficient for transferring electrons. The experimental bell-shaped dependence of kinetics on ionic strength is analysed in view of the molecular modelling results, and hypotheses for the molecular basis of this phenomenon are discussed.

Amino Acid Sequence↗

PubMed2004

New analogues of amonafide and elinafide, containing aromatic heterocycles: synthesis, antitumor activity, molecular modeling, and DNA binding properties.

Amonafide- and elinafide-related mono and bisintercalators, modified by the introduction of a pi-excedent furan or thiophene ring fused to the naphthalimide moiety, have been synthesized. These compounds have shown an interesting antitumor profile. The best compound, 9, was 2.5-fold more potent than elinafide against human colon carcinoma cells (HT-29). Molecular dynamic simulations and physicochemical experiments have demonstrated that these compounds are capable of forming stable DNA complexes. These results, together with those previously reported by us for imidazo- and pyrazinonaphthalimide analogues, have prompted us to propose that the DNA binding process does not depend on the electronic nature of the fused heterocycle.

Adenine↗

PubMed2005

Pyrazolo[3,4-c]pyridazines as novel and selective inhibitors of cyclin-dependent kinases.

Pyrazolopyridazine 1a was identified in a high-throughput screening carried out by BASF Bioresearch Corp. (Worcester, MA) as a potent inhibitor of CDK1/cyclin B and shown to have selectivity for the CDK family. Analogues of the lead compound have been synthesized and their antitumor activities have been tested. A molecular model of the complex between the lead compound and the CDK2 ATP binding site has been built using a combination of conformational search and automated docking techniques. The stability of the resulting complex has been assessed by molecular dynamics simulations and the experimental results obtained for the synthesized analogues have been rationalized on the basis of the proposed binding mode for compound 1a. As a result of the SAR study, monofuryl 1o has been synthesized and is one of the most active compounds against CDK1 of this series.

Adenosine Triphosphate↗

PubMed1998

Cofilin and gelsolin segment-1: molecular dynamics simulation and biochemical analysis predict a similar actin binding mode.

An understanding of the actin-depolymerizing function attributed to members of the ADF/cofilin/destrin superfamily requires a structural model of these proteins in complex with actin. As a step toward defining actin-cofilin interactions, the complex of yeast cofilin with monomeric actin was predicted, starting with the actin-gelsolin segment-1 binding mode recently suggested for the actin-destrin complex. After refinement by molecular dynamics simulation, the structure of cofilin converged in a new binding mode that required only minimal changes induced in the actin-cofilin interface. The predicted complex exhibits strong interactions between the N termini of actin and cofilin, mediated by a salt bridge of cofilin Arg3 with actin Asp1. The forming of this salt bridge could be prevented by the phosphorylation of cofilin Ser4, which is believed to inhibit cofilin depolymerization activity. Recent mutagenesis studies, crosslinking experiments and peptide binding studies are consistent with the predicted model of the actin-cofilin complex. The structural homology between cofilin and gelsolin segment-1 binding to actin was confirmed experimentally by two types of competitive binding assays.

Actin Depolymerizing Factors↗