Could chroman-4-one derivative be a better inhibitor of PTR1? - Reason for the identified disparity in its inhibitory potency in Trypanosoma brucei and Leishmania major.

Most notable Kinetoplastids are of the genus Trypanosoma and Leishmania, affecting several millions of humans in Africa and Latin America. Current therapeutic options are limited by several drawbacks, hence the need to develop more efficacious inhibitors. An investigation to decipher the mechanism behind greater inhibitory potency of a chroman-4-one derivative (compound 1) in Trypanosoma brucei pteridine reductase 1 (TbPTR1) and Leishmania major pteridine reductase 1 (LmPTR1) was performed. Estimation of ΔGbind revealed that compound 1 had a greater binding affinity in TbPTR1 with a ΔGbind value of -49.0507 Kcal/mol than -29.2292 Kcal/mol in LmPTR1. The ΔGbind in TbPTR1 were predominantly contributed by "strong" electrostatic energy compared to the "weak" van der Waals in LmPTR1. In addition to this, the NADPH cofactor contributed significantly to the total energy of TbPTR1. A characteristic weak aromatic π interaction common in PTR1 was more prominent in TbPTR1 than LmPTR1. The consistent occurrence of high-affinity conventional hydrogen bond interactions as well as a steady interaction of crucial active site residues like Arg14/Arg17, Ser95/Ser111, Phe97/Phe113 in TbPTR1/LmPTR1 with chroman-4-one moiety equally revealed the important role the moiety played in the activity of compound 1. Overall, the structural and conformational analysis of the active site residues in TbPTR1 revealed them to be more rigid than LmPTR1. This could be the mechanism of interaction TbPTR1 employs in exerting a greater potency than LmPTR1. These findings will further give insight that will be assistive in modifying compound 1 for better potency and the design of novel inhibitors of PTR1.

Biochemical characterization and inhibition of the alternative oxidase enzyme from the fungal phytopathogen Moniliophthora perniciosa.

Moniliophthora perniciosa is a fungal pathogen and causal agent of the witches' broom disease of cocoa, a threat to the chocolate industry and to the economic and social security in cocoa-planting countries. The membrane-bound enzyme alternative oxidase (MpAOX) is crucial for pathogen survival; however a lack of information on the biochemical properties of MpAOX hinders the development of novel fungicides. In this study, we purified and characterised recombinant MpAOX in dose-response assays with activators and inhibitors, followed by a kinetic characterization both in an aqueous environment and in physiologically-relevant proteoliposomes. We present structure-activity relationships of AOX inhibitors such as colletochlorin B and analogues which, aided by an MpAOX structural model, indicates key residues for protein-inhibitor interaction. We also discuss the importance of the correct hydrophobic environment for MpAOX enzymatic activity. We envisage that such results will guide the future development of AOX-targeting antifungal agents against M. perniciosa, an important outcome for the chocolate industry.

Astaxanthin from shrimp by-products ameliorates nephropathy in diabetic rats.

AIM: This study investigated the hypoglycemic and antioxidant effects of shrimp astaxanthin on the kidney of alloxan-induced diabetic rats. METHODS: Animals were distributed into four groups of six rats each: a control group (C), a diabetic group (D), a diabetic group supplemented with Astaxanthin (D+As) dissolved in olive oil and a diabetic group supplemented with olive oil (D+OO). In vitro antidiabetic effect was tested in plasma and kidney tissue. RESULTS: The group D of rats showed significant (P < 0.05) increase of glycemia, creatinine, urea and uric acid levels compared to those of the control group (C). Moreover, plasma and kidney malondialdehyde (MDA) and protein carbonyl (PCO) levels for the rats of the group D were significantly increased compared to the control group. Contrariwise, antioxidant enzyme activities, such as catalase (EC 1.11.1.6), superoxide dismutase (EC 1.15.1.1) and non-enzymatic levels of reduced glutathione, were significantly (P < 0.05) decreased in the plasma and kidney of diabetic rats compared to the control ones. The astaxanthin supplementation in rats diet improved the antioxidant enzyme activities and significantly decreased the MDA and PCO levels compared to diabetic rats. Indeed, no significant (P ≥ 0.05) improvement was observed for the fourth group (D+OO) compared to the control group (C). Histological analysis of kidney showed glomerular hypertrophy and tubular dilatation for the diabetic rats. For D+As rats, these histopathological changes were less prominent. CONCLUSIONS: Our results suggest that shrimp astaxanthin may play an important role in reduction of oxidative damage and could prevent pathological changes in diabetic rats suggesting promising application of shrimp astaxanthin in diabet treatment.

Toxic effects induced by mycotoxin fumonisin B1 on equine spermatozoa: assessment of viability, sperm chromatin structure stability, ROS production and motility.

Fumonisin B1 (FB1) is a mycotoxin produced by Fusarium species that exerts its toxic effect through interference with the sphingolipid pathway by inhibiting ceramide synthase. A FB1-dependent sperm toxicity was reported in boars. No information on FB1-related reproductive toxicity in stallions, the most sensitive animal species, has been reported. The aim of the present study was to assess the in vitro toxicity of FB1 on fresh and frozen-thawed equine spermatozoa by analyzing sperm viability, chromatin stability (SCSA) and reactive oxygen species (ROS) production by flow cytometry and sperm motility by CASA system. Fumonisin B1 did not affect viability of fresh spermatozoa after 2h exposure up to 25 µM. Damage on sperm chromatin structure was observed only in one frozen sample after exposure up to 2.5 x 10⁻5 µM FB1 without associated increase of ROS. Increase of ROS, at FB1 levels up to 2.5 x 10⁻4 µM, was found on another frozen-thawed sperm sample, may be as a consequence of seminal plasma removal. At 7.5 and 15 µM, FB1 induced reduction of total and progressive motility.

High-throughput screen for inhibitors of 1-deoxy-d-xylulose 5-phosphate reductoisomerase by surrogate ligand competition.

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (Dxr) is a key enzyme in a biosynthetic pathway for isoprenoids that is unique to eubacteria and plants. Dxr catalyzes the rearrangement and NADPH-dependent reduction of 1-deoxy-D-xylulose 5-phosphate to 2-C-methyl-D-erythritol 4-phosphate. The authors have purified Escherichia coli Dxr and devised a high-throughput screen (HTS) for compounds that bind to this enzyme at a functional site. Evidence is presented that the surrogate ligand directly binds or allosterically affects both the D-1-deoxyxylulose 5-phosphate (DXP) and NADPH binding sites. Compounds that bind at either or both sites that compete for binding with the surrogate ligand register as hits. The time-resolved fluorescence-based assay represents an improvement over the Dxr enzyme assay that relies on relatively insensitive measurements of NADPH oxidation. Screening 32,000 compounds from a diverse historical library, the authors obtained 89 potent inhibitors in the surrogate ligand competition assay. The results presented here suggest that peptide surrogate ligands may be useful in formatting HTS for proteins with difficult biochemical assays or targets of unknown function.

QSARs for 6-azasteroids as inhibitors of human type 1 5alpha-reductase: prediction of binding affinity and selectivity relative to 3-BHSD.

Quantitative structure-activity relationships (QSARs) are developed to describe the ability of 6-azasteroids to inhibit human type 1 5alpha-reductase. Models are generated using a set of 93 compounds with known binding affinities (K(i)) to 5alpha-reductase and 3beta-hydroxy-Delta(5)-steroid dehydrogenase/3-keto-Delta(5)-steroid isomerase (3-BHSD). QSARs are generated to predict K(i) values for inhibitors of 5alpha-reductase and to predict selectivity (S(i)) of compound binding to 3-BHSD relative to 5alpha-reductase. Log(K(i)) values range from -0.70 log units to 4.69 log units, and log(S(i)) values range from -3.00 log units to 3.84 log units. Topological, geometric, electronic, and polar surface descriptors are used to encode molecular structure. Information-rich subsets of descriptors are identified using evolutionary optimization procedures. Predictive models are generated using linear regression, computational neural networks (CNNs), principal components regression, and partial least squares. Compounds in an external prediction set are used for model validation. A 10-3-1 CNN is developed for prediction of binding affinity to 5alpha-reductase that produces root-mean-square error (RMSE) of 0.293 log units (R(2) = 0.97) for compounds in the external prediction set. Additionally, an 8-3-1 CNN is generated for prediction of inhibitor selectivity that produces RMSE = 0.513 log units (R(2) = 0.89) for the external prediction set. Models are further validated through Monte Carlo experiments in which models are generated after dependent variable values have been scrambled.

Improving 5-FU with a novel dihydropyrimidine dehydrogenase inactivator.

GW776C85 is a new drug that has been shown to be an effective inactivator of dihydropyrimidine dehydrogenase (DPD). Preclinical studies demonstrated that administration of GW776C85 with 5-fluorouracil (5-FU) resulted in several desirable pharmacologic effects. Initial clinical data on 5-FU combined with GW776C85 suggest potentially increased antitumor activity in at least some malignancies with tolerable toxicity, as well as several distinct economic and quality-of-life advantages including the following: (1) The possibility of administering 5-FU as an oral drug due to excellent bioavailability of 5-FU following inactivation of DPD; (2) a cost-effective alternative to continuous or protracted infusion of 5-FU without the need for hospitalization or surgical placement of an intravenous access and availability of an ambulatory pump; and (3) potential for less interpatient variation of 5-FU toxicity (e.g., in host tissues, such as bone marrow and gastrointestinal mucosa cells) due to inactivation of DPD in essentially all patients treated, permitting better 5-FU dosing guidelines. Finally, because tumors may theoretically become resistant to 5-FU by increased levels of DPD, the use of GW776C85 to inactivate DPD may provide a potential means by which tumor resistance can be reversed.

The medical management of lower urinary tract symptoms and benign prostatic hyperplasia.

Prostatism is a widely used term assigned to the symptom complex of older men with voiding dysfunction. The cause of the syndrome has routinely been ascribed to an enlarged prostate. More recent thinking recognizes that many men with such symptoms do not, in fact, have prostate enlargement or benign prostatic hyperplasia (BPH) and that such symptoms are not a surrogate for BPH. Such recognition is essential if cost effective medical management of lower urinary tract symptoms (LUTS) is to be achieved. Prostate volume has emerged as a key factor in the selection of medical therapy of LUTS and BPH not only regarding symptom relief but also to the newer concept of the prevention of disease progression and the avoidance of future adverse events in those men with true BPH. In the United States, medical management is now first line therapy for LUTS. The proper selection of therapy based on the patient's individual pathophysiologic characteristics is now made possible by many new recent studies within the medical literature.

Substrate-based inhibitors of lanosterol 14 alpha-methyl demethylase: I. Assessment of inhibitor structure-activity relationship and cholesterol biosynthesis inhibition properties.

A series of 15-, 32-, and 15,32-substituted lanost-8-en-3 beta-ols is described which function as inhibitors of cholesterol biosynthesis. These agents inhibit lanosterol 14 alpha-methyl demethylase activity as well as suppress HMG-CoA reduction activity in cultured cells. Several of these agents are extremely potent as both demethylase inhibitors and reductase suppressors, while others are more selective in their activities. Selected regio double bond isomers show preference for demethylase inhibition with the following order: delta 8 > delta 7 > delta 6 = unsaturated sterols. Comparisons also show that 4,4-dimethyl sterols are always more potent demethylase inhibitors and reductase suppressors than their 4,4-bisnomethyl counterparts. However, evaluation of an extensive oxylanosterol series leads us to conclude that demethylase inhibition and reductase suppression are not parallel in the same molecule. In addition, the oxylanosterols, but not the oxycholesterols, are able to disrupt coordinate regulation of HMG-CoA reductase from the LDL receptor. Thus, oxylanosterol treatment at levels which suppress reductase activity enhances LDL receptor activity. These results demonstrate that compounds can be made which (1) are selective reductase suppressors enabling dissection of the dual inhibitor nature of these compounds and (2) maximize reductase suppression and LDL receptor induction without demethylase inhibition which could lead to novel agents for serum cholesterol lowering.

Succinate-cytochrome c reductase: assessment of its value in the investigation of defects of the respiratory chain.

Defects of the respiratory chain are important causes of human disease and one of the most commonly used assays in the investigation of these patients is the measurement of succinate-cytochrome c reductase. However, this assay measures several components of the respiratory chain and the ability to detect a partial defect in one enzyme complex will depend on the amount of control exerted by that enzyme step on overall electron flux. We show that measurement of succinate-cytochrome c reductase activity may fail to detect partial defects of complex III and therefore is of limited diagnostic value in the identification of complex III defects. However, complex II is a major point of control of flux through succinate-cytochrome reductase and it is likely that measurement of the latter will detect defects of complex II.