The natural flavone fukugetin as a mixed-type inhibitor for human tissue kallikreins.

The human tissue kallikreins (KLK1-KLK15) comprise a family of 15 serine peptidases detected in almost every tissue of the human body and that actively participate in many physiological and pathological events. Some kallikreins are involved in diseases for which no effective therapy is available, as for example, epithelial disorders, bacterial infections and in certain cancers metastatic processes. In recent years our group have made efforts to find inhibitors for all kallikreins, based on natural products and synthetic molecules, and all the inhibitors developed by our group presented a competitive mechanism of inhibition. Here we describe fukugetin, a natural product that presents a mixed-type mechanism of inhibition against KLK1 and KLK2. This type of inhibitor is gaining importance today, especially for the development of exosite-type inhibitors, which present potential to selectively inhibit the enzyme activity only against specific substrate.

Isomannide-based peptidomimetics as inhibitors for human tissue kallikreins 5 and 7.

Human kallikrein 5 (KLK5) and 7 (KLK7) are potential targets for the treatment of skin inflammation and cancer. Previously, we identified isomannide derivatives as potent and competitive KLK7 inhibitors. The introduction of N-protected amino acids into the isomannide-based scaffold was studied. Some KLK5 inhibitors with submicromolar affinity (K i values of 0.3-0.7 µM) were identified, and they were 6- to 13-fold more potent than our previous hits. Enzyme kinetics studies and the determination of the mechanism of inhibition confirmed that the new isomannide-based derivatives are competitive inhibitors of both KLK5 and KLK7. Molecular docking and MD simulations of selected inhibitors into the KLK5 binding site provide insight into the molecular mechanism by which these compounds interact with the enzyme. The promising results obtained in this study open new prospects on the design and synthesis of highly specific KLK5 and KLK7 inhibitors.

Integration of methods in cheminformatics and biocalorimetry for the design of trypanosomatid enzyme inhibitors.

BACKGROUND: The enzyme gapdh, which acts in the glycolytic pathway, is seen as a potential target for pharmaceutical intervention of chagas disease. RESULTS: Herein, we report the discovery of new Trypanosoma cruzi GAPDH (TcGAPDH) inhibitors from target- and ligand-based virtual screening protocols using isothermal titration calorimetry (ITC) and molecular dynamics. Molecular dynamics simulations were used to gain insight on the binding poses of newly identified inhibitors acting at the TcGAPDH substrate (G3P) site. CONCLUSION: Nequimed125, the most potent inhibitor to act upon TcGAPDH so far, which sits on the G3P site without any contact with the co-factor (NAD(+)) site, underpins the result obtained by ITC that it is a G3P-competitive inhibitor. Molecular dynamics simulation provides biding poses of TcGAPDH inhibitors that correlate with mechanisms of inhibition observed by ITC. Overall, a new class of dihydroindole compounds that act upon TcGAPDH through a competitive mechanism of inhibition as proven by ITC measurements also kills T. cruzi.

Isomannide derivatives as new class of inhibitors for human kallikrein 7.

Human kallikrein 7 (KLK7) is a potential target for the treatment of skin inflammation and cancer. Despite its potential, few KLK7-specific small-molecule inhibitors have been reported in the literature. As an extension of our program to design serine protease inhibitors, here we describe the in vitro assays and the investigation of the binding mechanism by molecular dynamics simulation of a novel class of pseudo-peptide inhibitors derived from isomannide. Of the inhibitors tested, two inhibited KLK7 with K(i) values in the low micromolar range (9g=1.8µM; 9j=3.0µM). Eadie-Hofstee and Dixon plots were used to evaluate the competitive mechanism of inhibition for the molecules. Calculated binding free energies using molecular MM/PB(GB)SA approach are in good agreement with experimental results, suggesting that the inhibitors share the same binding mode, which is stabilized by hydrophobic interactions and by a conserved network of hydrogen bonds. The promising results obtained in this study make these compounds valid leads for further optimization studies aiming to improve the potency of this new class of kallikrein inhibitors.

Biological evaluation and docking studies of natural isocoumarins as inhibitors for human kallikrein 5 and 7.

Human kallikrein 5 and 7 (KLK5 and KLK7) are trypsin-like and chymotrypsin-like serine proteases, respectively, and promising targets for the treatment of skin desquamation, inflammation and cancer. In an effort to develop new inhibitors for these enzymes, we carried out enzymatic inhibition assays and docking studies with three isocoumarin compounds. Some promising inhibitors were uncovered, with vioxanthin and 8,8'-paepalantine being the most potent competitive inhibitors of KLK5 (K(i)=22.9 µM) and KLK7 (K(i)=12.2 µM), respectively. Our docking studies showed a good correlation with the experimental results, and revealed a distinct binding mode for the inhibitors at the binding sites of KLK5 and KLK7. In addition, the docking results suggested that the formation of hydrogen bonds at the oxyanion hole is essential for a good inhibitor.

Expression, purification and kinetic characterization of His-tagged glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi.

Trypanosomes are flagellated protozoa responsible for serious parasitic diseases that have been classified by the World Health Organization as tropical sicknesses of major importance. One important drug target receiving considerable attention is the enzyme glyceraldehyde-3-phosphate dehydrogenase from the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas disease (T. cruzi Glyceraldehyde-3-phosphate dehydrogenase (TcGAPDH); EC 1.2.1.12). TcGAPDH is a key enzyme in the glycolytic pathway of T. cruzi and catalyzes the oxidative phosphorylation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphosphoglycerate (1,3-BPG) coupled to the reduction of oxidized nicotinamide adenine dinucleotide, (NAD(+)) to NADH, the reduced form. Herein, we describe the cloning of the T. cruzi gene for TcGAPDH into the pET-28a(+) vector, its expression as a tagged protein in Escherichia coli, purification and kinetic characterization. The His(6)-tagged TcGAPDH was purified by affinity chromatography. Enzyme activity assays for the recombinant His(6)-TcGAPDH were carried out spectrophotometrically to determine the kinetic parameters. The apparent Michaelis-Menten constant (K(M)(app)) determined for D-glyceraldehyde-3-phosphate and NAD(+) were 352±21 and 272±25 µM, respectively, which were consistent with the values for the untagged enzyme reported in the literature. We have demonstrated by the use of Isothermal Titration Calorimetry (ITC) that this vector modification resulted in activity preserved for a higher period. We also report here the use of response surface methodology (RSM) to determine the region of optimal conditions for enzyme activity. A quadratic model was developed by RSM to describe the enzyme activity in terms of pH and temperature as independent variables. According to the RMS contour plots and variance analysis, the maximum enzyme activity was at 29.1°C and pH 8.6. Above 37°C, the enzyme activity starts to fall, which may be related to previous reports that the quaternary structure begins a process of disassembly.

The GRID/CPCA approach in drug discovery.

IMPORTANCE OF THE FIELD: Bimolecular recognition is the basis for almost all processes in biological systems. The geometrical and chemical complementarity of small molecule ligands and their macromolecular biological targets, matching paired interacting parts, can result in binding that will eventually yield a biological response. AREAS COVERED IN THIS REVIEW: The topics covered include the integration of molecular interaction fields and chemometrics, via the GRID/CPCA (consensus principal component analysis) method that is actively contributing to the optimization of potency and selectivity of ligands towards a chosen target. Key applications that hallmark the usefulness of the method are critically presented. By comparison of the GRID/CPCA and GRID/PCA, the breakthroughs and challenges are highlighted. WHAT THE READER WILL GAIN: Molecular recognition studies support the development of pharmacophore-based descriptors, which provide the means to identify new ligand templates ('scaffold-hopping'). The GRID/CPCA approach can simultaneously reveal common trends in more than one block of data for more than two target proteins, with several three-dimensional structures per protein. It offers the benefit of improving the weighting between different interaction energy probes within the GRID parameterization. An important consequence is that hydrophobic interactions can be assessed for selectivity between proteins. TAKE HOME MESSAGE: Molecular-field-based methods along with CPCA analysis are extremely powerful to understand bimolecular interactions. Because drug discovery and development is a costly, time consuming and high-risk activity and GRID/CPCA is at the forefront of the computer-aided design, it should be used as early as possible for discovering new drugs.

Discovery of novel Trypanosoma cruzi glyceraldehyde-3-phosphate dehydrogenase inhibitors.

Based on its essential role in the life cycle of Trypanosoma cruzi, the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been considered a promising target for the development of novel chemotherapeutic agents for the treatment of Chagas' disease. In the course of our research program to discover novel inhibitors of this trypanosomatid enzyme, we have explored a combination of structure and ligand-based virtual screening techniques as a complementary approach to a biochemical screening of natural products using a standard biochemical assay. Seven natural products, including anacardic acids, flavonoid derivatives, and one glucosylxanthone were identified as novel inhibitors of T. cruzi GAPDH. Promiscuous inhibition induced by nonspecific aggregation has been discarded as specific inhibition was not reversed or affected in all cases in the presence of Triton X-100, demonstrating the ability of the assay to find authentic inhibitors of the enzyme. The structural diversity of this series of promising natural products is of special interest in drug design, and should therefore be useful in future medicinal chemistry efforts aimed at the development of new GAPDH inhibitors having increased potency.

2D QSAR and similarity studies on cruzain inhibitors aimed at improving selectivity over cathepsin L.

Hologram quantitative structure-activity relationships (HQSAR) were applied to a data set of 41 cruzain inhibitors. The best HQSAR model (Q(2)=0.77; R(2)=0.90) employing Surflex-Sim, as training and test sets generator, was obtained using atoms, bonds, and connections as fragment distinctions and 4-7 as fragment size. This model was then used to predict the potencies of 12 test set compounds, giving satisfactory predictive R(2) value of 0.88. The contribution maps obtained from the best HQSAR model are in agreement with the biological activities of the study compounds. The Trypanosoma cruzi cruzain shares high similarity with the mammalian homolog cathepsin L. The selectivity toward cruzain was checked by a database of 123 compounds, which corresponds to the 41 cruzain inhibitors used in the HQSAR model development plus 82 cathepsin L inhibitors. We screened these compounds by ROCS (Rapid Overlay of Chemical Structures), a Gaussian-shape volume overlap filter that can rapidly identify shapes that match the query molecule. Remarkably, ROCS was able to rank the first 37 hits as being only cruzain inhibitors. In addition, the area under the curve (AUC) obtained with ROCS was 0.96, indicating that the method was very efficient to distinguishing between cruzain and cathepsin L inhibitors.

Computational study of the interaction between TIBO inhibitors and Y181 (C181), K101, and Y188 amino acids.

The non-nucleoside inhibitors of HIV-1 reverse transcriptase (NNRTIs) are an important class of drugs employed in anti-HIV chemotherapy. TIBO compounds, which belong to the NNRTIs class, are potent inhibitors of the HIV-1 reverse transcriptase enzyme (HIV-1 RT). However, mutations in the amino acids present in the active site of these inhibitors limit their clinical use. In this work, the intermolecular interactions taking place between compounds of the TIBO family and Y181 (C181), K101, and Y188 amino acids are investigated. For this purpose the coordinates of three RT crystalline structures complexed with TIBO were taken from PDB database, and were analyzed by means of the B3LYP/6-31+G(d,p) model. The natural bond orbital (NBO) and atoms in molecules (AIM) methods indicate that not only does the Y181C mutation lead to loss of favorable interactions between the TIBO side chains and tyrosine, but it also affects the interaction between the inhibitor and K101 and Y188. Results also revealed that the interaction between TIBO and K101 is stabilized by N-H...O and N-H...S hydrogen bonds. This is the first time that the presence of the latter hydrogen bond (N-H...S) is reported to play an important role in the stabilization of the interaction between TIBO and K101. In addition the NBO and natural population analyses (NPA) indicate that the 8 Cl-TIBO inhibitor presents a more effective interaction with the Y181, K101, and Y188 than that of 9 Cl-TIBO.