Margot Paulino

Synthesis and anti-trypanosomal activity of novel 5-nitro-2-furaldehyde and 5-nitrothiophene-2-carboxaldehyde semicarbazone derivatives

Several novel semicarbazones derivatives were prepared from 5-nitro-2-furaldehyde or 5-nitrothiophene-2-carboxaldehyde, and tested in vitro as potential anti-trypanosomal agents. The compounds were prepared in good to excellent yields in 2-3 steps from readily available starting materials. Some derivatives were found to be active against Trypanosoma cruzi with an activity similar to that of Nifurtimox.

Theoretical studies of hydrogen-bonded complexes using semiempirical methods

Abstract Recently proposed modified MNDO methods (MNDO/M and MNDO-PM3) for the study of hydrogen-bonded complexes are tested in four simple systems: (H 2 O) 2 , NH 4 + · H 2 O, formamide water and formamide-methanol dimers. A comparison is made with AM1 since this method, intended to be a general improvement of MNDO, was already shown to fail in the former two dimers. Bifurcated hydrogen-bonded structures were always preferred over linear ones. AM1 also shows this artifact in the formamide-water dimer but not in the formamide-methanol one. This is due to the inability of -CH 3 to bind to the carbonyl oxygen. In this case the AM1 optimized global minimum is in excellent agreement with the Hartree-Fock ab initio DZP optimized structure. Moreover, when it is physically impossible to form bifurcated hydrogen bonds, AM1 is in good agreement with ab initio results. MNDO/M and MNDO-PM3 (an improvement on AM1 ) do not give any bifurcated structure either in the former two dimers or in the formamide complexes. MNDO/M and MNDO-PM3 structures are in agreement with ab initio and experimental ones in almost all cases, except for the global minima in both the formamide-water and the formamide- methanol dimers. Singly hydrogen-bonded structures are predicted instead of the experimentally determined and ab initio calculated correct doubly hydrogen-bonded ones. MNDO-PM3, although not entirely correct, gives structures which are more correct than the MNDO/M ones. In conclusion, neither of these semiempirical methods is entirely correct. Overall, MNDO-PM3 seems to be more trustworthy than AM1 or MNDO/M, although these methods may be used when no bifurcated hydrogen bonds (AM1) or cycles (MNDO//M) exist. Failures of MNDO-PM3 indicate that a better semiempirical method is still needed for studies of solvation and hydrogen- bonded clusters.

Computer assisted design of potentially active anti-trypanosomal compounds

Abstract A computer assisted molecular modeling was used to design molecules having a shape complementary to the active site of glutathione reductase (GR) and trypanothione reductase (TR). The designed 5-nitro compound derivatives, were obtained from structural knowledge gleaned on glutathione (GSSG), trypanothione (T[S]2) and GSP disulfide (glutathionylspermidine disulfide). These molecules form complexes with the enzymes GR and TR. The theoretical lead compound was: N1-[1-(5-nitro-2-furyl)methylidene]-N4-{4-[3-(2,2,2-trifluoroacetyl)hexahydro-1-1-pyrimidynil]butyl} semicarbazide (NPIPCO). A multi-disciplinary team developed around the efforts to synthesize this lead. In this work we report on eight compounds that were synthesized in the pathway to NPIPCO: 4-(2-methoxyethyl)-1-, 4-butyl-1-, 4-hexyl-1- and 2-methoxphenyl-1-(5-nitrofurfurilidene) semicarbazides and the corresponding 5-nitrothiophenes. These substances are expected to act as pro-transition state analogues. Enzymologic studies proved that many of these compounds are inhibitors of TR. Furthermore, they showed inhibitory activity on Tripanosoma cruzi growth in vitro.

Assaying phenothiazine derivatives as trypanothione reductase and glutathione reductase inhibitors by theoretical docking and molecular dynamics studies.

A theoretical docking study, conducted on a sample of previously reported phenothiazine derivatives, at the binding sites of Trypanosoma cruzi trypanothione reductase (TR) and human erythrocyte glutathione reductase (GR), examines interaction energies (affinities) towards the parasite enzyme to check for selectivity with respect to the human counterpart. Phenothiazine compounds were previously shown to be TR inhibitors. The analysis of data collected from the docking procedure was undertaken both from the numeric and graphical standpoints, including the comparison of force field, energies, molecular contacts and spatial location of the different orientations that ligands acquired at the binding sites. Molecular Dynamics simulations were also carried out for derivatives with known quantitative inhibition kinetics (K(i)). The results indicate that (positively) charged phenothiazines attain larger interaction energies at TR active site, in line with previous experimental information. Suitable molecular size and shape is also needed to complement the electrostatic effect, as clearly evidenced by graphical analysis of output docked conformations. Docking energies values are reasonably well correlated with those obtained by Molecular Dynamics as well as with the experimental K(i) values, confirming once again the validity of this type of scoring methods to rapidly assess ligand-receptor affinities. Alongside newly discovered classes of TR inhibitors, the promazine (N-alkylaminopropylphenothiazine) nucleus should still be considered when good candidates are sought as leaders for selective TR inhibition.

Toward the understanding of the molecular basis for the inhibition of COX-1 and COX-2 by phenolic compounds present in Uruguayan propolis and grape pomace

Propolis and grape pomace have significant amounts of phenols which can take part in anti-inflammatory mechanisms. As the cyclooxygenases 1 and 2 (COX-1 and COX-2) are involved in said mechanisms, the possibility for a selective inhibition of COX-2 was analyzed in vitro and in silico. Propolis and grape pomace from Uruguayan species were collected, extracted in hydroalcoholic mixture and analyzed. Based on phenols previously identified, and taking as reference the crystallographic structures of COX-1 and COX-2 in complex with the commercial drug Celecoxib, a molecular docking procedure was devised to adjust 123 phenolic molecular models at the enzyme-binding sites. The most important results of this work are that the extracts have an overall inhibition activity very similar in COX-1 and COX-2, i.e. they do not possess selective inhibition activity for COX-2. Nevertheless, 10 compounds of the phenolic database turned out to be more selective and 94 phenols resulted with similar selectivity than Celecoxib, an outcome that accounts for the overall experimental inhibition measures. Binding site environment observations showed increased polarity in COX-2 as compared with COX-1, suggesting that polarity is the key for selectivity. Accordingly, the screening of molecular contacts pointed to the residues: Arg106, Gln178, Leu338, Ser339, Tyr341, Tyr371, Arg499, Ala502, Val509, and Ser516, which would explain, at the atomic level, the anti-inflammatory effect of the phenolic compounds. Among them, Gln178 and Arg499 appear to be essential for the selective inhibition of COX-2.

Synthesis and Pharmacophore Modelling of 2,6,9-Trisubstituted Purine Derivatives and Their Potential Role as Apoptosis-Inducing Agents in Cancer Cell Lines

A series of 2,6,9-trisubstituted purine derivatives have been synthesized and investigated for their potential role as antitumor agents. Twelve compounds were obtained by a three step synthetic procedure using microwave irradiation in a pivotal step. All compounds were evaluated in vitro to determine their potential effect on cell toxicity by the MTT method and flow cytometry analysis on four cancer cells lines and Vero cells. Three out of twelve compounds were found to be promising agents compared to a known and effective anticancer drug, etoposide, in three out of four cancer cell lines assayed with considerable selectivity. Preliminary flow cytometry data suggests that compounds mentioned above induce apoptosis on these cells. The main structural requirements for their activity for each cancer cell line were characterized with a preliminary pharmacophore model, which identified aromatic centers, hydrogen acceptor/donor center and a hydrophobic area. These features were consistent with the cytotoxic activity of the assayed compounds.

Synthesis and biological characterization of new aryloxyindole-4,9-diones as potent trypanosomicidal agents

A new indole-4,9-dione and their phenoxy derivatives were synthesized and evaluated in vitro against the epimastigote form of Trypanosoma cruzi, Y strain. All of these novel compounds were found to be extremely potent and selective that the standard drug nifurtimox. Interestingly, phenoxyindole-4,9-dione 9d displayed excellent nanomolar inhibitory activity, IC50=20 nM, and high selectivity index, SI=625. In silico studies using MOE program were performed to generate a preliminary pharmacophore model.

Trypanothione Reductase: A Target for the Development of Anti- Trypanosoma cruzi Drugs

BACKGROUND & OBJECTIVE Chagas disease or American trypanosomiasis is a major parasitic disease in Latin America with restricted available treatment: nifurtimox and benznidazole. These two drugs are ineffective in the chronic phase of the disease; therefore, there is a need for the development of new, efficient and safe drugs for the treatment of this pathology. With this goal, one of the promising targets is trypanothione reductase (TR), a key enzyme in the metabolism of Trypanosoma cruzi. CONCLUSION In this review, we analyse the importance of TR as a drug target, as well as the well-known and new inhibitors reported in the last decade as potential therapeutic agents for Chagas disease.

In vitro and in silico analysis of the Aspergillus nidulans DNA–CreA repressor interactions

The CreA protein mediates carbon catabolite repression in the fungus Aspergillus nidulans. Its DNA-binding domain belongs to the Cys2-His2 class, binding specifically to a 5′ SYGGRG 3′ nucleotide sequence. EMSA experiments showed that the CreA(G27D) mutation resulted in a 30-fold increase of the Kdiss, and footprinting revealed a altered pattern of protein/DNA contacts. We modeled the CreA and the CreA(G27D) complexes in silico. A 15 ns molecular dynamics simulation of the solvated CreA(G27D) and CreA models was carried out using the MOE 2007.09 suite and the Amber99 force field. We have focused our analysis in residues Arg14, Glu16, His17, and Arg20 and Arg44, Asp46, and Arg50, previously, shown to be responsible for the specific contacts of the two Zn fingers. The electrostatic and the total potential energies showed the CreA(G27D) mutation to decrease the affinity of the complex, in agreement with the Kdiss′s values. The in silico approach highlighted the role of the inter-finger linker. We identified several differential structural characteristics of the CreA and CreA(G27D)/DNA complexes and observed that the latter resulted in a lower dynamic flexibility of the complex.

Analysis of cyclosporin A and a set of analogs as inhibitors of a T. cruzi cyclophilin by docking and molecular dynamics

Cyclophilins (CyPs) are enzymes involved in protein folding. In Trypanosoma cruzi (T. cruzi), the most abundantly expressed CyP is the isoform TcCyP19. It has been shown that TcCyP19 is inhibited by the immunosuppressive drug cyclosporin A (CsA) and analogs, which also proved to have potent trypanosomicidal activity in vitro. In this work, we continue and expand a previous study on the molecular interactions of CsA, and a set of analogs modeled in complexes with TcCyP19. The modeled complexes were used to evaluate binding free energies by molecular dynamics (MD), applying the Linear Interaction Energy (LIE) method. In addition, putative binding sites were identified by molecular docking. In our analysis, the binding free energy calculations did not correlate with experimental data. The heterogeneity of the non-bonded energies and the variation in the pattern of hydrogen bonds suggest that the systems may not be suitable for the application of the LIE method. Further, the docking calculations identified two other putative binding sites with comparable scoring energies to the active site, a fact that may also explain the lack of correlation found. Kinetic experiments are needed to confirm or reject the multiple binding sites hypothesis. In the meantime, MD simulations at the alternative sites, employing other methods to compute binding free energies, might be successful at finding good correlations with the experimental data.