Structural and thermodynamic analysis of thrombin:suramin interaction in solution and crystal phases

Suramin is a hexasulfonated naphthylurea which has been recently characterized as a non-competitive inhibitor of human alpha-thrombin activity over fibrinogen, although its binding site and mode of interaction with the enzyme remain elusive. Here, we determined two X-ray structure of the thrombin: suramin complex, refined at 2.4 angstrom resolution. While a single thrombin: suramin complex was found in the asymmetric unit cell of the crystal, some of the crystallographic contacts with symmetrically related molecules are mediated by both the enzyme and the ligand. Molecular dynamics simulations with the 1:1 complex demonstrate a large rearrangement of suramin in the complex, but with the protein scaffold and the more extensive protein-ligand regions keep unchanged. Small-angle X-ray scattering measurements at high micromolar concentration demonstrate a suramin-induced dimerization of the enzyme. These data indicating a dissimilar binding mode in the monomeric and oligomeric states, with a monomeric, 1:1 complex to be more likely to exist at the thrombin physiological, nanomolar concentration range. Collectively, close understanding on the structural basis for interaction is given which might establish a basis for design of suramin analogues targeting thrombin. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Characterization of suramin binding sites on the human group IIA secreted phospholipase A(2) by site-directed mutagenesis and molecular dynamics simulation

Suramin is a polysulphonated naphthylurea with inhibitory activity against the human secreted group IIA phospholipase A(2) (hsPLA2GIIA), and we have investigated suramin binding to recombinant hsPLA2GIIA using site-directed mutagenesis and molecular dynamics (MD) simulations. The changes in suramin binding affinity of 13 cationic residue mutants of the hsPLA2GIIA was strongly correlated with alterations in the inhibition of membrane damaging activity of the protein. Suramin binding to hsPLA2GIIA was also studied by MD simulations, which demonstrated that altered intermolecular potential energy of the suramin/mutant complexes was a reliable indicator of affinity change. Although residues in the C-terminal region play a major role in the stabilization of the hsPLA2GIIA/suramin complex, attractive and repulsive hydrophobic and electrostatic interactions with residues throughout the protein together with the adoption of a bent suramin conformation, all contribute to the stability of the complex. Analysis of the h5PLA2GIIA/suramin interactions allows the prediction of the properties of suramin analogues with improved binding and higher affinities which may be candidates for novel phospholipase A(2) inhibitors. (C) 2012 Elsevier Inc. All rights reserved.

The Effect of Prostaglandin Analogs and Prostamide on Central Corneal Thickness

Objective: The aim of this study was to verify the influence of prostaglandin analogs and prostamide on central corneal thickness (CCT). Methods: A prospective analysis was done of CCT in glautomatous patients submitted to monotherapy with prostaglandin analogs (latanoprost 0.005% or travoprost 0.004%) or prostamide (bimatoprost 0.03%) during an 8-week period. A control group of patients without any ocular medication was also evaluated. CCT measurements were performed with a commercially available ultrasound pachymeter. A total of 73 patients were included in this study. Mean age was 68.5 +/- 9.2 (range, 48-85) years old. Results: A statistically significant reduction in CCT was observed in all groups, except the control group (n = 21): Bimatoprost 0.03% group (n = 21): 544.41 +/- 35.4 vs. 540.35 +/- 35.9 mu m (P = 0.039); travoprost 0.004% group (n = 17): 538.47 +/- 32.0 vs. 532.25 +/- 30.4 mu m (P = 0.009); latanoprost 0.005% group (n = 14): 548.57 +/- 32.4 vs. 543.88 +/- 35.6 mu m (P = 0.036). Conclusion: Topical therapy with prostaglandin analogs and bimatoprost is associated with CCT reduction over a period of at least 8 weeks.

Novel and facile solution-phase synthesis of 2,5-diketopiperazines and O-glycosylated analogs

This work describes the synthesis in Solution of a series of related diketopiperazines with potential biological activities: cyclo(L-Pro-L-Ser), cyclo(L-Phe-L-Ser), cyclo(D-Phe-L-Ser) and the corresponding glycosylated analogs of the latter, cyclo[D-Phe-L-Ser(alpha GlcNAc)] and cyclo[D-Phe-L-Ser(beta GlcNAc)]. The synthetic approach involved coupling reactions of -OH or O-glycosylated serine benzyl esters with NFmoc-protected amino acids (Pro or Phe), followed by one-pot deprotection-cyclization reaction in the presence of 20% piperidine in DMF. (C) 2009 Elsevier Ltd. All rights reserved.

Suramin inhibits macrophage activation by human group IIA phospholipase A(2), but does not affect bactericidal activity of the enzyme

Suramin is a polysulphonated napthylurea antiprotozoal and anthelminitic drug, which also presents inhibitory activity against a broad range of enzymes. Here we evaluate the effect of suramin on the hydrolytic and biological activities of secreted human group IIA phospholipase A(2) (hsPLA(2)GIIA). The hsPLA(2)GIIA was expressed in E. coli, and refolded from inclusion bodies. The hydrolytic activity of the recombinant enzyme was measured using mixed dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) liposomes. The activation of macrophage cell line RAW 264.7 by hsPLA(2) GIIA was monitored by NO release, and bactericidal activity against Micrococcus luteus was evaluated by colony counting and by flow cytometry using the fluorescent probe Sytox Green. The hydrolytic activity of the hsPLA(2) GIIA was inhibited by a concentration of 100 nM suramin and the activation of macrophages by hsPLA(2) GIIA was abolished at protein/suramin molar ratios where the hydrolytic activity of the enzyme was inhibited. In contrast, both the bactericidal activity of hsPLA(2) GIIA against Micrococcus luteus and permeabilization of the bacterial inner membrane were unaffected by suramin concentrations up to 50 mu M. These results demonstrate that suramin selectively inhibits the activity of the hsPLA(2) GIIA against macrophages, whilst leaving the anti-bacterial function unchanged.

Mapping of suramin binding sites on the group IIA human secreted phospholipase A(2)

Suramin is a polysulphonated napthylurea used as an antiprotozoal/anthelminitic drug, which also inhibits a broad range of enzymes. Suramin binding to recombinant human secreted group IIA phospholipase A(2) (hsPLA(2)GIIA) was investigated by molecular dynamics simulations (MD) and isothermal titration calorimetry (ITC). MD indicated two possible bound suramin conformations mediated by hydrophobic and electrostatic interactions with amino-acids in three regions of the protein. namely the active-site and residues located in the N- and C-termini, respectively. All three binding sites are located on the phospholipid membrane recognition surface, suggesting that suramin may inhibit the enzyme, and indeed a 90% reduction in hydrolytic activity was observed in the presence of 100 nM suramin. These results correlated with ITC data, which demonstrated 2.7 suramin binding sites on the hsPLA(2)GIIA, and indicates that suramin represents a novel class of phosphohpase A(2) inhibitor. (C) 2009 Elsevier Inc. All rights reserved.

Factors determining normal adult height in girls with gonadotropin-dependent precocious puberty treated with depot gonadotropin-releasing hormone analogs

Context: Several factors can affect adult height (AH) of patients with gonadotropin-dependent precocious puberty (GDPP) treated with depot GnRH analogs. Objective: Our objective was to determine factors influencing AH in patients with GDPP treated with depot GnRH analogs. Patients: A total of 54 patients (45 girls) with GDPP treated with depot GnRH analog who reached AH was included in the study. Design: Univariate and multivariate analyses of the factors potentially associated with AH were performed in all girls with GDPP. In addition, clinical features of the girls who attained target height (TH) range were compared with those who did not. Predicted height using Bayley and Pinneau tables was compared with attained AH. Results: In girls the mean AH was 155.3 +/- 6.9 cm (-1.2 +/- 1 SD) with TH range achieved by 81% of this group. Multiple regression analysis revealed that the interval between chronological age at onset of puberty and at the start of GnRH analog therapy, height SD scores (SDSs) at the start and end of therapy, and TH explained 74% of AH variance. The predicted height at interruption of GnRH therapy, obtained from Bayley and Pinneau tables for average bone age, was more accurate than for advanced bone age in both sexes. In boys the mean AH was 170.6 +/- 9.2 cm (-1 +/- 1.3 SDS), whereas TH was achieved by 89% of this group. Conclusions: The major factors determining normal AH in girls with GDPP treated with depot GnRH analogs were shorter interval between the onset of puberty and start of therapy, higher height SDS at the start and end of therapy, and TH. Therefore, prompt depot GnRH analog therapy in properly selected patients with GDPP is critical to obtain normal AH.

Immunohistochemical Expression of HLA-DR in the Conjunctiva of Patients Under Topical Prostaglandin Analogs Treatment

Purpose: Subclinical inflammation may be observed in patients, using topical antiglaucomatous drugs. The objective,e of this study was to investigate inflammation in conjunctiva of glaucoma patients using prostaglandin analogs, by the detection of a immunogenetic marker (HLA-DR) and compare the effect of 3 different drugs: latanoprost, bimatoprost, and travoprost in the induction of this inflammation. Subjects and Methods: Thirty-three patients with primary open-angle glaucoma were evaluated without and with prostaglandin analogs topical therapy. Imprints of conjunctival cells were obtained, fixed on glass slides. and Prepared for immunohistochemical analysis. Results: Before the use of prostaglandin analogs, 4 of the 33 patients evaluated presented expression (of HLA-DR in the conjunctiva (mild). After 1 month oil prostaglandin analog treatment, all but 1 patient presented HLA-DR staining. HLA-DR expression of these 32 patients was scored as mild (19 patients), medium (11 patients), or intense (2 patients). The differences were statistically significant both when the presence and the increased expression of HLA-DR were considered (P<0.001). When the 3 different groups were analyzed (latanoprost, bimatoprost, and travoprost) no statistically significant difference was round (P 0.27). Conclusions: The use of prostaglandin analogs eye drops provokes, a reaction, observed by HLA-DR subclinical inflammatory expression, even after a short period of treatment, independently of the class of the prostaglandin analogs used.

In vitro wear, surface roughness and hardness of propanal-containing and diacetyl-containing novel composites and copolymers based on bis-GMA analogs

Objective. To evaluate the effect of two additives, aldehyde or diketone, on the wear, roughness and hardness of bis-GMA-based composites/copolymers containing TEGDMA, propoxylated bis-GMA (CH(3)bis-GMA) or propoxylated fluorinated bis-GMA (CF(3)bis-GMA). Methods. Fifteen experimental composites and 15 corresponding copolymers were prepared combining bis-GMA and TEGDMA, CH3bis-GMA or CF3bis-GMA, with aldehyde (24mol% and 32 mol%) or diketone (24 mol% and 32 mol%) totaling 30 groups. For composites, hybrid treated filler (barium aluminosilicate glass/pyrogenic silica; 60 wt%) was added to monomer mixtures. Photopolymerization was affected by 0.2 wt% each of camphorquinone and N,N-dimethyl-p-toluidine. Wear (W) test was conducted in a toothbrushing abrasion machine (n = 6) and quantified using a profilometer. Surface roughness (R) changes, before and after abrasion test, were determined using a rugosimeter. Microhardness (H) measurements were performed for dry and wet samples using a Knoop microindenter (n = 6). Data were analyzed by one-way ANOVA and Tukey`s test (alpha = 0.05). Results. Incorporation of additives led to improved W and H values for bis-GMA/TEGDMA and bis-GMA/CH(3)bis-GMA systems. Additives had no significant effect on the W and H changes of bis-GMA/CF(3)bis-GMA. With regard to R changes, additives produced decreased values for bis-GMA/CH3bis-GMA and bis-GMA/CF3bis-GMA composites. Bis-GMA/TEGDMA and bis-GMA/CH(3)bis-GMA copolymers with additives became smoother after abrasion test. Significance. The findings correlate with additives ability to improve degree of conversion of some composites/copolymers thereby enhancing mechanical properties. Published by Elsevier Ltd on behalf of Academy of Dental Materials

Procedures for monitoring recombinant erythropoietin and analogs in doping.

Hemoglobin concentration is one of the principal factors of aerobic power and, consequently, of performance in many types of physical activities. The use of recombinant human erythropoietin is, therefore, particularly powerful for improving the physical performances of patients, and, more generally, improving their quality of life. This article discusses procedures for monitoring recombinant erythropoietin and its analogues in doping for athletic performance.

Analogs of Cuntz algebras on Lp-spaces

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Clinical and Virological Efficacy of Etravirine Plus Two Active Nucleos(t)ide Analogs in an Heterogeneous HIV-Infected Population.

Etravirine (ETV) is recommended in combination with a boosted protease inhibitor plus an optimized background regimen for salvage therapy, but there is limited experience with its use in combination with two nucleos(t)ide reverse-transcriptase inhibitors (NRTIs). This multicenter study aimed to assess the efficacy of this combination in two scenarios: group A) subjects without virologic failure on or no experience with non-nucleoside reverse-transcriptase inhibitors (NNRTIs) switched due to adverse events and group B) subjects switched after a virologic failure on an efavirenz- or nevirapine-based regimen. The primary endpoint was efficacy at 52 weeks analysed by intention-to-treat. Virologic failure was defined as the inability to suppress plasma HIV-RNA to <50 copies/mL after 24 weeks on treatment, or a confirmed viral load >200 copies/mL in patients who had previously achieved a viral suppression or had an undetectable viral load at inclusion. Two hundred eighty seven patients were included. Treatment efficacy rates in group A and B were 88.0% (CI95, 83.9-92.1%) and 77.4% (CI95, 65.0-89.7%), respectively; the rates reached 97.2% (CI95, 95.1-99.3%) and 90.5% (CI95, 81.7-99.3), by on-treatment analysis. The once-a-day ETV treatment was as effective as the twice daily dosing regimen. Grade 1-2 adverse events were observed motivating a treatment switch in 4.2% of the subjects. In conclusion, ETV (once- or twice daily) plus two analogs is a suitable, well-tolerated combination both as a switching strategy and after failure with first generation NNRTIs, ensuring full drug activity. TRIAL REGISTRATION ClinicalTrials.gov NCT01437241.

Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.