Structure based design, synthesis, and evaluation of potential inhibitors of steroid sulfatase.

The activity of the enzyme steroid sulfatase (STS) is high in breast tumors and elevated levels of STS mRNA expression have been associated with a poor prognosis. Potent STS irreversible inhibitors have been developed, paving the way to use this new type of therapy for the treatment of breast cancer. Several small molecules belonging to a natural products-inspired library of previously obtained inhibitors of tumor cell growth and new molecules planned to be reversible inhibitors of this enzyme were docked into STS. Some of the synthesized xanthone derivatives, which revealed high scores against STS, namely oxo-9H-xanthene-3,6-diyl bis(3-chlorobenzoate) (5), 9-oxo-9H-xanthene-3,6-diyl bis(4-tertbutylbenzoate) (6) and 9-oxo-9H-xanthene-3,6-diyl bis(4-methoxybenzoate) (7) showed poor water solubility. Therefore, formulations of these derivatives with cyclodextrins were prepared and characterized. The compounds were evaluated regarding their effect on the in vitro growth of various human tumor cell lines, as well as the effect in STS inhibition, for the compounds with the most favorable ΔG values. Additionally, the capacity of these derivatives and of some prenyl and acetoxy-benzophenone and xanthones to inhibit the in vitro growth of MCF-7 ER(+) and/or to inhibit STS in a micromolar range was also assessed. Some compounds developed in the present work were shown to be potential STS inhibitors.

Antifungal activity of tri- and tetra-thioureido amino derivatives against different Candida species.

The in vitro antifungal activity of six thioureido substituted amines (P1-P6) was evaluated against Candida species, including Candida albicans, C. glabrata, C. krusei and C. parapsilosis. These tri- and tetra-thioureido amino derivatives with different methylation levels were synthesised through easy synthetic routes to evaluate their antifungal properties against Candida species. Among all studied derivatives, the tri-(2-thioureido-ethyl)-amine (P1) was the most active compound inhibiting C. albicans and C. glabrata at a concentration of 0.49 µg ml(-1); P3, the N,N',N'',N'''-hexamethyl-derivative, also showed inhibitory activity against C. albicans and C. glabrata, but in higher concentrations (250 µg ml(-1) ). The N,N',N'',N'''-tetramethylated amine (P5) only inhibited the growth of C. glabrata, but its corresponding N,N',N'',N'''-octamethyl derivative (P6) was also active against C. glabrata (125 µg ml(-1)) and it was the only compound active against C. parapsilosis. P2 and P4 showed no significant antifungal activity. The structure-activity relationship of the thioureido-substituted derivatives indicates that the molecular branching and the alkylation levels can influence the antifungal activity. This study demonstrated that thioureido derivatives exhibited significant antifungal activity against Candida species and that they can be considered as a very promising bioactive lead compound to develop novel antifungal agents.

Integration of Ligand- and Target-Based Virtual Screening for the Discovery of Cruzain Inhibitors.

A myriad of methods are available for virtual screening of small organic compound databases. In this study we have successfully applied a quantitative model of consensus measurements, using a combination of 3D similarity searches (ROCS and EON), Hologram Quantitative Structure Activity Relationships (HQSAR) and docking (FRED, FlexX, Glide and AutoDock Vina), to retrieve cruzain inhibitors from collected databases. All methods were assessed individually and then combined in a Ligand-Based Virtual Screening (LBVS) and Target-Based Virtual Screening (TBVS) consensus scoring, using Receiving Operating Characteristic (ROC) curves to evaluate their performance. Three consensus strategies were used: scaled-rank-by-number, rank-by-rank and rank-by-vote, with the most thriving the scaled-rank-by-number strategy, considering that the stiff ROC curve appeared to be satisfactory in every way to indicate a higher enrichment power at early retrieval of active compounds from the database. The ligand-based method provided access to a robust and predictive HQSAR model that was developed to show superior discrimination between active and inactive compounds, which was also better than ROCS and EON procedures. Overall, the integration of fast computational techniques based on ligand and target structures resulted in a more efficient retrieval of cruzain inhibitors with desired pharmacological profiles that may be useful to advance the discovery of new trypanocidal agents.

Novel application of 2D and 3D-similarity searches to identify substrates among cytochrome P450 2C9, 2D6, and 3A4.

Cytochrome P450 (CYP450) is a class of enzymes where the substrate identification is particularly important to know. It would help medicinal chemists to design drugs with lower side effects due to drug-drug interactions and to extensive genetic polymorphism. Herein, we discuss the application of the 2D and 3D-similarity searches in identifying reference structures with higher capacity to retrieve substrates of three important CYP enzymes (CYP2C9, CYP2D6, and CYP3A4). On the basis of the complementarities of multiple reference structures selected by different similarity search methods, we proposed the fusion of their individual Tanimoto scores into a consensus Tanimoto score (T(consensus)). Using this new score, true positive rates of 63% (CYP2C9) and 81% (CYP2D6) were achieved with false positive rates of 4% for the CYP2C9-CYP2D6 data set. Extended similarity searches were carried out on a validation data set, and the results showed that by using the T(consensus) score, not only the area of a ROC graph increased, but also more substrates were recovered at the beginning of a ranked list.

Effects of organic solvents on the enzyme activity of Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase in calorimetric assays.

In drug discovery programs, dimethyl sulfoxide (DMSO) is a standard solvent widely used in biochemical assays. Despite the extensive use and study of enzymes in the presence of organic solvents, for some enzymes the effect of organic solvent is unknown. Macromolecular targets may be affected by the presence of different solvents in such a way that conformational changes perturb their active site structure accompanied by dramatic variations in activity when performing biochemical screenings. To address this issue, in this work we studied the effects of two organic solvents, DMSO and methanol (MeOH), in the isothermal titration calorimetry (ITC) kinetic assays for the catalyzed reaction of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Trypanosoma cruzi. The solvent effects on T. cruzi GAPDH had not yet been studied. This enzyme was shown here to be affected by the organic solvents content up to 5.0% for MeOH and up to 7.5% for DMSO. The results show that when GAPDH is assayed in the presence of DMSO (5%, v/v) using the ITC experiment, the enzyme exhibits approximately twofold higher activity than that of GAPDH with no cosolvent added. When MeOH (5%, v/v) is the cosolvent, the GAPDH activity is sixfold higher. The favorable effects of the organic solvents on the Michaelis-Menten enzyme-substrate complex formation ensure the consistency of the biological assays, structural integrity of the protein, and reproducibility over the measurement time. The reaction was also kinetically monitored by standard spectrophotometric assays to establish a behavioral performance of T. cruzi GAPDH when used for screening of potential inhibitors.

The role of molecular interaction fields on enantioselective and nonselective separation of chiral sulfoxides.

The separation of a series of 23 asymmetric sulfoxides, including the three proton pump inhibitors (PPI) omeprazole, lansoprazole and pantoprazole was investigated by HPLC, under reversed-phase elution with amylose tris(3,5-dimethylphenylcarbamate), amylose tris[(S)-1-phenylethylcarbamate] and amylose tris(3,5-dimethoxyphenylcarbamate) chiral stationary phases, CSP1-3, respectively. The whole set of sulfoxides showed better enantioselectivity and enantioresolution on CSP1. However, the three PPI were enantioseparated only when using CSP1 and CSP3. It was observed an improved enantioselectivity and enantioresolution on CSP3. The mechanisms of retention were evaluated by molecular interaction fields (MIF) generated via GRID force field, which yielded the geometric reasons leading to the scenario outlined. The enantioselective and nonselective interactions are discussed in terms of the reported selectivity. The steric structural outline of the CSP nonselective interaction sites is of major importance to deliver the sulfoxides to the chiral selective sites where the enantioselective interactions take place.

QSAR based on biological microcalorimetry.

In this paper we describe a QSAR based on biological microcalorimetry for a set of antimicrobial hydrazides acting against Saccharomyces cerivisiae and Escherichia coli. Results show that an extrathermodynamic relationship exists based upon partitioning (log P(TA)) and microcalorimetrically measured biopotencies using the same cell systems. Moreover, the extrathermodynamic relationship between drug potencies for these two cell systems shows that both cellular systems appear to behave in the same way with respect to the importance of partitioning. This means that the same set of congeneric compounds experience a similar environment in the two systems. This represents a lateral validation of the method and discloses the validity of the QSAR model.

Determination of receptor-bound drug conformations by QSAR using flexible fitting to derive a molecular similarity index.

Results are presented for a QSAR analysis of bisamidines, using a similarity index as descriptor. The method allows for differences in conformation of bisamidines at the receptor site to be taken into consideration. In particular, it has been suggested by others that pentamidine binds in the minor groove of DNA in a so-called isohelical conformation, and our QSAR supports this suggestion. The molecular similarity index for comparison of molecules can be used as a parameter for correlating and hence rationalising the activity as well as suggesting the design of bioactive molecules. The studied compounds had been evaluated for potency against Leishmania mexicana amazonensis, and this potency was used as a dependent variable in a series of QSAR analyses. For the calculation of similarity indexes, each analogue was in turn superimposed on a chosen lead compound in a reference conformation, either extended or isohelical, maximising overlap and hence similarity by flexible fitting.