In Silico Studies of Potential Selective Inhibitors of Thymidylate Kinase from Variola virus.

Continuing the work developed by our research group, in the present manuscript, we performed a theoretical study of 10 new structures derived from the antivirals cidofovir and ribavirin, as inhibitor prototypes for the enzyme thymidylate kinase from Variola virus (VarTMPK). The proposed structures were subjected to docking calculations, molecular dynamics simulations, and free energy calculations, using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, inside the active sites of VarTMPK and human TMPK (HssTMPK). The docking and molecular dynamic studies pointed to structures 2, 3, 4, 6, and 9 as more selective towards VarTMPK. In addition, the free energy data calculated through the MM-PBSA method, corroborated these results. This suggests that these compounds are potential selective inhibitors of VarTMPK and, thus, can be considered as template molecules to be synthesized and experimentally evaluated against smallpox.

Docking and molecular dynamics studies of peripheral site ligand-oximes as reactivators of sarin-inhibited human acetylcholinesterase.

In the present work, we performed docking and molecular dynamics simulations studies on two groups of long-tailored oximes designed as peripheral site binders of acetylcholinesterase (AChE) and potential penetrators on the blood brain barrier. Our studies permitted to determine how the tails anchor in the peripheral site of sarin-inhibited human AChE, and which aminoacids are important to their stabilization. Also the energy values obtained in the docking studies corroborated quite well with the experimental results obtained before for these oximes.

Association of the anti-tuberculosis drug rifampicin with a PAMAM dendrimer.

The association of the anti-tuberculosis drug rifampicin (RIF) with a 4th-generation poly(amidoamine) (G4-PAMAM) dendrimer was investigated by means of molecular dynamics simulations. The RIF load capacity was estimated to be around 20 RIF per G4-PAMAM at neutral pH. The complex formed by 20 RIF molecules and the dendrimer (RIF20-PAMAM) was subjected to 100 ns molecular dynamics (MD) simulations at two different pH conditions (neutral and acidic). The complex was found to be significantly more stable in the simulation at neutral pH compared to the simulation at low pH in which the RIF molecules were rapidly and almost simultaneously expelled to the solvent bulk. The high stability of the RIF-PAMAM complex under physiological pH and the rapid release of RIF molecules under acidic medium provide an interesting switch for drug targeting since the Mycobacterium resides within acidic domains of the macrophage. Altogether, these results suggest that, at least in terms of stability and pH-dependent release, PAMAM-like dendrimers may be considered suitable drug delivery systems for RIF and derivatives.

Design of inhibitors of thymidylate kinase from Variola virus as new selective drugs against smallpox.

Recently we constructed a homology model of the enzyme thymidylate kinase from Variola virus (VarTMPK) and proposed it as a new target to the drug design against smallpox. In the present work, we used the antivirals cidofovir and acyclovir as reference compounds to choose eleven compounds as leads to the drug design of inhibitors for VarTMPK. Docking and molecular dynamics (MD) studies of the interactions of these compounds inside VarTMPK and human TMPK (HssTMPK) suggest that they compete for the binding region of the substrate and were used to propose the structures of ten new inhibitors for VarTMPK. Further docking and MD simulations of these compounds, inside VarTMPK and HssTMPK, suggest that nine among ten are potential selective inhibitors of VarTMPK.

Molecular modeling toward selective inhibitors of dihydrofolate reductase from the biological warfare agent Bacillus anthracis.

In the present work, we applied docking and molecular dynamics techniques to study 11 compounds inside the enzymes dihydrofolate reductase (DHFR) from the biological warfare agent Bacillus anthracis (BaDHFR) and Homo sapiens sapiens (HssDHFR). Six of these compounds were selected for a study with the mutant BaF96IDHFR. Our results corroborated with experimental data and allowed the proposition of a new molecule with potential activity and better selectivity for BaDHFR.

Molecular modeling studies on nucleoside hydrolase from the biological warfare agent Brucella suis.

Brucella suis is a dangerous biological warfare agent already used for military purposes. This bacteria cause brucellosis, a zoonosis highly infective and difficult to fight. An important selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. We present here the first three-dimensional structure of B. suis NH (BsNH) and propose this enzyme as a molecular target to the drug design in the fight against brucellosis. In addition, we performed molecular docking studies, aiming to analyze the three-dimensional positioning of nine known inhibitors of Chritidia fasciculata NH (CfNH) in the active sites of BsNH and CfNH. We also analyzed the main interactions of some of these compounds inside the active site of BsNH and the relevant factors to biological activity. These results, together with further molecular dynamics (MD) simulations, pointed out to the most promising compound as lead for the design of potential inhibitors of BsNH. Most of the docking and MD results corroborated to each other and the docking results also suggested a good correlation with experimental data.

Cathechol-O-methyltransferase Val(158)Met polymorphism is associated with disruptive behavior disorders among children and adolescents with ADHD.

COMT Val(158)Met polymorphism has been associated with both symptoms of attention-deficit/hyperactivity disorder (ADHD) and disruptive behavior disorders (DBD): that is, oppositional defiant disorder (ODD) and conduct disorder (CD) often comorbid with ADHD. The aim of this study was to test the association between COMT Val(158)Met polymorphism and the presence of DBD in children with ADHD (n = 516). Homozygous Val/Val children showed a higher prevalence of ADHD comorbid with DBD (χ(2) = 5.762; p = 0.016; OR = 1.58; CI(95%) = 1.07-2.35). Our findings replicate previous results and suggest a role for COMT in the etiology of DBD in children and adolescents with ADHD.

Docking studies on the binding of quinoline derivatives and hematin to Plasmodium falciparum lactate dehydrogenase.

The literature has reported that ferriprotoporphyrin IX (hematin) intoxicates the malarial parasite through competition with NADH for the active site of the enzyme lactate dehydrogenase (LDH). In order to avoid this, the parasite polymerizes hematin to hemozoin. The quinoline derivatives are believed to form complexes with dimeric hematin, avoiding the formation of hemozoin and still inhibiting LDH. In order to investigate this hypothesis we calculated the docking energies of NADH and some quinoline derivatives (in the free forms and in complex with dimeric hematin) in the active site of the Plasmodium falciparum LDH (PfLDH). Ours results showed better docking score values to the complexes when compared to the free compounds, pointing them as more efficient inhibitors of Pf_LDH. Further we performed Molecular Dynamics (MD) simulations studies on the best docking conformation of the complex chloroquine-dimeric hematin with PfLDH. Our in silico results corroborate experimental data suggesting a possible action route for the quinoline derivatives in the inhibition of PfLDH.

Catechol-O-methyltransferase valine158methionine polymorphism moderates methylphenidate effects on oppositional symptoms in boys with attention-deficit/hyperactivity disorder.

BACKGROUND: The catechol-O-methyltransferase enzyme plays a key role in the function of prefrontal cortex, accounting for most of the degradation of dopamine. Previous studies have documented the improvement of oppositional symptoms in attention-deficit/hyperactivity disorder (ADHD) patients with methylphenidate (MPH) treatment. However, the effect of the COMT gene in the response to MPH on oppositional symptoms has not been investigated. METHODS: A total of 251 children with ADHD fulfilled inclusion criteria to participate in the study. Dosages of short-acting MPH were augmented until no further clinical improvement was detected or until there were significant adverse events (MPH dose always > .3 mg/kg/day). The outcome measure was the parent-rated oppositional subscale of the Swanson, Nolan and Pelham Scale-Version IV (SNAP-IV). The scale was applied by child psychiatrists blinded to genotype at baseline and in the first and third months. The COMT valine158methionine polymorphism was genotyped by polymerase chain reaction based methods. RESULTS: We detected significant improvement in SNAP-IV oppositional scores from baseline to the first and three months of treatment [n = 112; F(2,231) = 5.35, p = .005]. A significant effect of the presence of methionine allele in oppositional defiant disorder scores during treatment [F(1,148) = 5.02, p = .027] and a significant interaction between the methionine allele and treatment over time for the SNAP-IV oppositional scores during this period of treatment [F(2,229) = 6.40, p = .002] were both observed. CONCLUSIONS: These results suggest an effect of the COMT genotype on the trajectory of oppositional defiant disorder symptoms improvement with MPH treatment in boys with ADHD.

Analysis of Bacillus anthracis nucleoside hydrolase via in silico docking with inhibitors and molecular dynamics simulation.

As the enzyme nucleoside hydrolase (NH) is widely found in nature but has not yet been detected in mammals, it is considered an ideal target in the development of chemotherapy against parasitic diseases and bacterial infections like anthrax. Considering the risk that this biological warfare agent represents nowadays, the search for new drugs and new molecular targets in the development of chemotherapy against anthrax is imperative. On this basis, we performed docking studies of six known NH inhibitors at the active site of NH from Bacillus anthracis (BaNH). Subsequently, molecular dynamics (MD) simulations of these compounds inside BaNH were carried out in order to complement the docking studies and select the most promising compounds as leads for the design of potential BaNH inhibitors. Most of the docking and MD results obtained agreed well with each other and showed good correlation with experimental data.

Design of new chemotherapeutics against the deadly anthrax disease. Docking and molecular dynamics studies of inhibitors containing pyrrolidine and riboamidrazone rings on nucleoside hydrolase from Bacillus anthracis.

Anthrax is a disease caused by Bacillus anthracis, a dangerous biological warfare agent already used for both military and terrorist purposes. An important selective target for chemotherapy against this disease is nucleoside hydrolase (NH), an enzyme still not found in mammals. Having this in mind we have performed molecular docking studies, aiming to analyze the three-dimensional positioning of six known inhibitors of Trypanosoma vivax NH (TvNH) in the active site of B. anthracis NH (BaNH). We also analyzed the main interactions of these compounds with the active site residues of BaNH and the relevant factors to biological activity. These results, together with further molecular dynamics (MD) simulations, pointed out to the most promising compounds as lead for the design of potential inhibitors of BaNH. Most of the docking and MD results obtained corroborated to each other. Additionally, the docking results also suggested a good correlation with experimental data.

Development of new acetylcholinesterase reactivators: molecular modeling versus in vitro data.

In this work a theoretical methodology for evaluation of the association and kinetic reactivation constants of oximes using the Molegro and Spartan softwares was proposed and validated facing in vitro data previously reported in the literature. Results showed a very good agreement between the theoretical binding free energies of the reactivators and experimental data, suggesting that the proposed methodology could work well in the prediction of kinetic and thermodynamics parameters for oximes that might be helpful for the design and selection of new and more effective oximes.

Development of new acetylcholinesterase reactivators: molecular modeling versus in vitro data.

In this work a theoretical methodology for evaluation of the association and kinetic reactivation constants of oximes using the Molegro and Spartan softwares was proposed and validated facing in vitro data previously reported in the literature. Results showed a very good agreement between the theoretical binding free energies of the reactivators and experimental data, suggesting that the proposed methodology could work well in the prediction of kinetic and thermodynamics parameters for oximes that might be helpful for the design and selection of new and more effective oximes.

Further evidence for the association between attention deficit/hyperactivity disorder and the serotonin receptor 1B gene.

Several evidences suggested that the serotonin 5-HT1B receptor gene (HRT1B) might be involved in the susceptibility to attention deficit/hyperactivity disorder (ADHD). Prior studies reported excess transmissions of the HRT1B gene 861G allele to affected ADHD children and of a haplotype block containing this variant and two functional promoter SNPs to probands with ADHD-inattentive subtype. However, some investigations did not replicate these findings. Therefore, we tested for biased transmissions of haplotypes derived from the 861G > C, -161A > T, and -261T > G SNPs from parents to 343 families with ADHD children. We also sought to replicate findings from the literature that the association between HTR1B is preferentially with ADHD-Inattentive subtype. Using a transmission disequilibrium test we found evidence for an excess transmission of haplotype. -261G/-161T/861G (P = 0.014) for affected children in the total sample. When the analysis was repeated with 143 families with ADHD-Inattentive subtype no significant associations were observed. Our results provide additional evidence that HRT1B gene may be an important risk factor for the development of ADHD, but this effect seems not to be attributable to inattentive cases.

MAOA is associated with methylphenidate improvement of oppositional symptoms in boys with attention deficit hyperactivity disorder.

The monoamine oxidase A (MAOA) gene has been extensively related to aggressive, impulsive and violent behaviours. Previous studies have documented the improvement of oppositional symptoms in attention deficit hyperactivity disorder (ADHD) patients with methylphenidate (MPH). However, the effect of the MAOA gene in response to MPH has not been investigated. A sample of 85 boys from an ADHD outpatient service was genotyped for the MAOA-uVNTR polymorphism. The outcome measure was the parent-rated oppositional subscale of the Swanson, Nolan and Pelham Scale - version IV. The scale was applied by child psychiatrists blinded to genotype at baseline and in the first and third months of treatment. A significant interaction between the presence of MAOA high-activity genotype and treatment with MPH over time on oppositional scores was detected during the 3 months' treatment (n=85, F2,136=4.83, p=0.009). These results suggest an effect of the MAOA-uVNTR high-activity genotype on the improvement of oppositional symptoms with MPH treatment.

Association of the adrenergic alpha2A receptor gene with methylphenidate improvement of inattentive symptoms in children and adolescents with attention-deficit/hyperactivity disorder.

CONTEXT: Preclinical studies have demonstrated the relevance of adrenergic alpha2A receptor on the attentional process and the mechanism of action of methylphenidate hydrochloride. Several molecular genetic investigations suggest a role for the adrenergic alpha2A receptor gene (ADRA2A) in attention-deficit/hyperactivity disorder (ADHD), especially in the inattentive dimension. However, the effect of ADRA2A in the response to methylphenidate in humans has not been previously investigated, to our knowledge. OBJECTIVE: To evaluate the association between the ADRA2A -1291 C>G polymorphism and the clinical response to methylphenidate treatment in children and adolescents with ADHD. DESIGN: A pharmacogenomic study was undertaken between November 1, 2002, and May 1, 2004, using a nonrandom assignment, quasi-experimental design. SETTING: An ADHD outpatient program at a university hospital in Brazil. Patients One hundred six patients consecutively diagnosed as having ADHD were genotyped for the ADRA2A -1291 C>G polymorphism and were included in the analyses. Intervention Short-acting methylphenidate administered in increasing dosages until no further clinical improvement was detected or until limited adverse effects occurred. MAIN OUTCOME MEASURES: The primary outcome measure was the parent-rated inattentive subscale of the Swanson, Nolan, and Pelham Scale version IV. Secondary outcome measures included the Barkley Side Effect Rating Scale and the parent-rated hyperactivity-impulsivity subscale of the Swanson, Nolan, and Pelham Scale version IV. Scales were applied by child psychiatrists blinded to genotype at baseline and at 1 and 3 months of treatment. RESULTS: A significant interaction effect between the presence of the G allele and treatment with methylphenidate over time on inattentive scores was detected during the 3 months of treatment (n = 106; F(2,198) = 4.30; P = .02). CONCLUSIONS: We documented the effect of the G allele at the ADRA2A -1291 C>G polymorphism on the improvement of inattentive symptoms with methylphenidate treatment in children and adolescents with ADHD. Our findings provide clinical evidence for the involvement of the noradrenergic system in the modulation of methylphenidate action.