Molecular dynamics simulations of the free and inhibitor-bound cruzain systems in aqueous solvent: insights on the inhibition mechanism in acidic pH.

The major cysteine protease of Trypanosoma cruzi, cruzain (CRZ), has been described as a therapeutic target for Chagas' disease, which affects millions of people worldwide. Thus, a series of CRZ inhibitors has been studied, including a new competitive inhibitor, Nequimed176 (NEQ176). Nevertheless, the structural and dynamic basis for CRZ inhibition remains unclear. Hoping to contribute to this ever-growing understanding of timescale dynamics in the CRZ inhibition mechanism, we have performed the first study using 100 ns of molecular dynamics (MD) simulations of two CRZ systems in an aqueous solvent under pH 5.5: CRZ in the apo form (ligand free) and CRZ complexed to NEQ176. According to the MD simulations, the enzyme adopts an open conformation in the apo form and a closed conformation in the NEQ176-CRZ complex. We also suggest that this closed conformation is related to the hydrogen-bonding interactions between NEQ176 and CRZ, which occurs through key residues, mainly Gly66, Met68, Asn69, and Leu160. In addition, the cross-correlation analysis shows evidence of the correlated motions among Ala110-Asp140, Leu160-Gly189, and Glu190-Gly215 subdomains, as well as, the movements related to Ala1-Thr59 and Asp60-Pro90 regions seem to be crucial for CRZ activity.

Segregated flux balance analysis constrained by population structure/function data: the case of PHA production by mixed microbial cultures.

In this study we developed a segregated flux balance analysis (FBA) method to calculate metabolic flux distributions of the individual populations present in a mixed microbial culture (MMC). Population specific flux data constraints were derived from the raw data typically obtained by the fluorescence in situ hybridization (FISH) and microautoradiography (MAR)-FISH techniques. This method was applied to study the metabolic heterogeneity of a MMC that produces polyhydroxyalkanoates (PHA) from fermented sugar cane molasses. Three populations were identified by FISH, namely Paracoccus sp., Thauera sp., and Azoarcus sp. The segregated FBA method predicts a flux distribution for each of the identified populations. The method is shown to predict with high accuracy the average PHA storage flux and the respective monomeric composition for 16 independent experiments. Moreover, flux predictions by segregated FBA were slightly better than those obtained by nonsegregated FBA, and also highly concordant with metabolic flux analysis (MFA) estimated fluxes. The segregated FBA method can be of high value to assess metabolic heterogeneity in MMC systems and to derive more efficient eco-engineering strategies. For the case of PHA-producing MMC considered in this work, it becomes apparent that the PHA average monomeric composition might be controlled not only by the volatile fatty acids (VFA) feeding profile but also by the population composition present in the MMC.

Mixed culture polyhydroxyalkanoate (PHA) production from volatile fatty acid (VFA)-rich streams: effect of substrate composition and feeding regime on PHA productivity, composition and properties.

In this study, the possibility of manipulating biopolymer composition in mixed culture polyhydroxyalkanoate (PHA) production from fermented molasses was assessed by studying the effects of substrate volatile fatty acid (VFA) composition and feeding regime (pulse wise versus continuous). It was found that the use of a continuous feeding strategy rather than a pulse feeding strategy can not only help mitigate the process constraints of the pulse-feeding strategy (resulting in higher specific and volumetric productivities) but also be used as means to broaden the range of polymer structures. Continuous feeding increased the hydroxyvalerate content by 8% relatively to that obtained from the same feedstock using pulse wise feeding. Therefore, the feeding strategy can be used to manipulate polymer composition. Furthermore, the range of PHA compositions, copolymers of P(HB-co-HV) with HV fraction ranging from 15 to 39%, obtained subsequently resulted in different polymer properties. Increasing HV content resulted in a decrease of the average molecular weight, the glass transition and melting temperatures and also in a reduction in the crystallinity degree from a semi-crystalline material to an amorphous matrix.

Antiplatelet activity and structure-activity relationship study of Pyrazolopyridine Derivatives as potential series for treating thrombotic diseases.

AIM: Platelets plays a central role in hemostatic processes and consequently are similarly involved in pathological processes, such as arterial thrombosis and atherosclerosis. Herein we described the synthesis, antiplatelet profile and structure-activity relationship (SAR) of a new series of N'-substitutedphenylmethylene-1H-pyrazolo[3,4-b]pyridine-carbohydrazide derivatives (3a-3k). METHODS: These compounds were synthesized in good yield and tested in platelet aggregation assays using collagen, ADP and arachidonic acid as agonists. We also performed a SAR studies using SPARTAN' 08 program, in silico ADMET screening and the Lipinski " rule of five " using Osiris Property Explorer and molinspiration on-line programs. RESULTS: Interestingly, the new compounds were active against collagen and arachidonic acid (AA) with the two most actives compounds (3a and 3c - IC(50)=61 microM and 68 microM respectively) almost 5-fold more potent than aspirin (IC(50)=300 microM). These derivatives showed low theoretical toxicity risks in in silico ADMET screening and fulfilled the Lipinski rule of five, suggesting good oral biodisponibility. CONCLUSION: This work showed carbohydrazide group as potential for designing new antiplatelets. On that purpose, 3a and 3c may act as prototypes to generate more efficient and safe molecules for treating thrombotic diseases.

Mixed culture polyhydroxyalkanoates production from sugar molasses: the use of a 2-stage CSTR system for culture selection.

Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers. The use of mixed microbial cultures (MMC) and low cost feedstocks have a positive impact on the cost-effectiveness of the process. It has typically been carried out in Sequencing Batch Reactors (SBR). In this study, a 2-stage CSTR system (under Feast and Famine conditions) was used to effectively select for PHA-storing organisms using fermented molasses as feedstock. The effect of influent substrate concentration (60-120 Cmmol VFA/L) and HRT ratio between the reactors (0.2-0.5h/h) on the system's selection efficiency was assessed. It was shown that Feast reactor residual substrate concentration impacted on the selective pressure for PHA storage (due to substrate-dependent kinetic limitation). Moreover, a residual substrate concentration coming from the Feast to the Famine reactor did not jeopardize the physiological adaptation required for enhanced PHA storage. The culture reached a maximum PHA content of 61%. This success opens new perspectives to the use of wastewater treatment infrastructure for PHA production, thus valorizing either excess sludge or wastewaters.

Polyhydroxyalkanoate (PHA) production by a mixed microbial culture using sugar molasses: effect of the influent substrate concentration on culture selection.

In Polyhydroxyalkanoate (PHA) production processes using Mixed Microbial Culture (MMC), the success of the culture selection step determines, to a great extent, the PHA accumulation performance obtained in the final PHA production stage. In this study, the effect of the influent substrate concentration (30-60Cmmol VFA/L) on the selection of a PHA-storing culture using a complex feedstock, fermented sugar molasses, was assessed. At 30 and 45Cmmol VFA/L, substrate concentration impacted on the process kinetics through a substrate dependent kinetic limitation effect. However, further increasing the carbon substrate concentration to 60Cmmol VFA/L, resulted in an unforeseen growth limitation effect associated with a micronutrient deficiency of the fermented feedstock (magnesium) and high operating pH. Struvite precipitation caused a nutrient limitation which prevented biomass concentration increase, thus causing the feast to famine length ratio to vary in the selection reactor, with subsequent impact on the selective pressure for PHA-storing organisms. A highly dynamic response of the selected population to transient conditions of feast to famine ratio, in the range of 0.21-1.1, was observed. Kinetic (limiting concentration of carbon source) and physiological (loss of internal growth limitation due to the shorter length of famine phase) effects, resulting from variation of the influent substrate concentration, were subsequently demonstrated in batch studies. The culture selected at an influent substrate concentration of 45Cmmol VFA/L showed the best PHA-storing capacity since neither substrate concentration nor feast to famine ratio were limiting factors. This culture, highly enriched in PHA-storing organisms (88%), reached a maximum PHA content of 74.6%.

Strategies for the development of a side stream process for polyhydroxyalkanoate (PHA) production from sugar cane molasses.

A three-stage process was developed to produce polyhydroxyalkanoates (PHAs) from sugar cane molasses. The process includes (1) molasses acidogenic fermentation, (2) selection of PHA-accumulating cultures, (3) PHA batch accumulation using the enriched sludge and fermented molasses. In the fermentation step, the effect of pH (5-7) on the organic acids profile and productivity was evaluated. At higher pH, acetic and propionic acids were the main products, while lower pH favoured the production of butyric and valeric acids. PHA accumulation using fermented molasses was evaluated with two cultures selected either with acetate or fermented molasses. The effect of organic acids distribution on polymer composition and yield was evaluated with the acetate selected culture. Storage yields varied from 0.37 to 0.50Cmmol HA/Cmmol VFA. A direct relationship between the type of organic acids used and the polymers composition was observed. Low ammonia concentration (0.1Nmmol/l) in the fermented molasses stimulated PHA storage (0.62Cmmol HA/Cmmol VFA). In addition, strategies of reactor operation to select a PHA-accumulating culture on fermented molasses were developed. The combination of low organic loading with high ammonia concentration selected a culture with a stable storage capacity and with a storage yield (0.59Cmmol HA/Cmmol VFA) similar to that of the acetate-selected culture.

HIV-1 reverse transcriptase: a therapeutical target in the spotlight.

Human Immunodeficiency Virus type 1 Reverse Transcriptase (HIV-1 RT) is one of the most important targets for treatment of Acquired Immune Deficiency Syndrome (AIDS). It catalyzes the reverse transcription of HIV-RNA into a double stranded DNA, and the knowledge of its substrate specificity and catalytic mechanism has guided the development of several inhibitors widely used on current HIV/AIDS therapy. However, mutations in HIV-1 RT structure can lead to the emergence of drug-resistant virus strains. The goal of this review is to summarize relevant structural features of HIV-1 RT and its inhibitors in such a way that this cost-effective target in the development of new antiretroviral drugs is particularly highlighted.

Pseudo-peptides derived from isomannide as potential inhibitors of serine proteases.

Hepatitis C, Dengue and West Nile virus are among of the most important flaviviruses that share one important serine protease enzyme. Serine proteases belong to the most studied class of proteolytic enzymes, and are a primary target in the drug development field. In this paper, we describe the synthesis and preliminary molecular modeling studies of a novel class of N-t-Boc amino acid amides derived of isomannide as potential serine proteases inhibitors.

N- t-Boc-amino acid esters of isomannide. Potential inhibitors of serine proteases.

Hepatitis C, Dengue and West Nile virus are some of the most important flaviviruses, that share one important serine protease enzyme. Serine proteases are the most studied class of proteolytic enzyme and, in these cases, a primary target for drug discovery. In this paper, we describe the synthesis and preliminary molecular modeling studies of a novel class of N- t-Boc amino acid esters derived of isomannide as potential serine proteases inhibitors.

Snake venom thrombin-like enzymes: from reptilase to now.

The snake venom thrombin-like enzymes (SVTLEs) comprise a number of serine proteases functionally and structurally related to thrombin. Until recently, only nine complete sequences of this subgroup of the serine protease family were known. Over the past 5 years, the primary structure of several SVTLEs has been characterized, and now this family includes several members. Of particular interest is their possible use in pathologies such as thrombosis. The aim of the present review is to summarize the state of the art concerning the evolutionary, structural and biological features of the SVTLEs.

Four-dimensional quantitative structure-activity relationship analysis of a series of interphenylene 7-oxabicycloheptane oxazole thromboxane A2 receptor antagonists.

A series of 39 (a training set of 29 and a test set of 10) interphenylene 7-oxabicyclo [2.2.1]heptane oxazole thromboxane A2 (TXA2) receptor antagonists were studied using four-dimensional quantitative structure-activity relationship (4D-QSAR) analysis. Two thousand conformations of each analogue were sampled to generate a conformational energy profile (CEP) from a molecular dynamic simulation (MDS) of 100,000 trajectory states. Each conformation was placed in a grid cell lattice for each of six trial alignments. Cubic grid cell sizes of 1 and 2 A were considered. The frequency of occupation of each grid cell was computed for each of seven types of pharmacophoric group classes of atoms of each compound. These grid cell occupancy descriptors (GCODs) were then used as independent variables in constructing three-dimensional (3D)-QSAR models after data reduction. The types of data reduction included doing no reducing, reduction based on individual GCOD correlation with activity, and reduction from minimum variance constraints over the GCOD population. The 3D-QSAR models were generated and evaluated by a scheme that combines a genetic algorithm (GA) optimization with partial least squares (PLS) regression. The 3D-QSAR models were evaluated by cross-validation using the leave-one-out technique. The cross-validated correlation coefficient, Q2, ranged from 0.27 to 0.86. The models are not from chance correlation because a scrambled data set was generated and evaluated (Q2 = 0.25-0.37). A composite 3D-QSAR model was constructed using the best models derived from GCODs of both 1 and 2 A grid cell size lattices. The 3D-QSAR models provide detailed 3D pharmacophore requirements in terms of atom types and corresponding locations needed for high TXA2 inhibition activity. Specific sites in space that should not be occupied by an active inhibitor are also specified. The GCOD measures for the compounds in the training set permit reference points regarding which pharmacophore sites can provide the largest boosts in inhibition activity relative to the existing analogues.

Derivaçäo fêmoro-axilar direita para rrevascularizaçäo cerebral e do membro superior direito: relato de um caso / Femoral-axillary bypass for cerebral and right upper limb revascularization - report of a case

Com o objetivo de aumentar a irrigaçäo sanguínea cerebral e do membro superior direito, foi rrealizada uma derivaçäo fêmoro-axilar direita em uma paciente portadora de oclusöes e estenoses dos ramos supra-aórticos outras doenças associadas que näo permitiam a toracotomia. Embora os resultados näo tenham sido plenamente satisfatórios, este procedimento mostrou-se hemodinamicamente viável e uma alternativa válida para o tratamento da insuficiência vascular cerebral e de membros superiores, em casos eecíficos.