A critical assessment of force field accuracy against NMR data for cyclic peptides containing ß-amino acids.

Hybrid cyclic α/ß-peptides, in which one or more ß-amino acids are incorporated into the backbone, are gaining increasing interest as potential therapeutics, thanks to their ability to achieve enhanced binding affinities for a biological target through pre-organization in solution. The in silico prediction of their three dimensional structure through strategies such as MD simulations would substantially advance the rational design process. However, whether the molecular mechanics force fields are accurate in sampling highly constrained cyclopeptides containing ß-amino acids remains to be verified. Here, we present a systematic assessment of the ability of 8 widely used force fields to reproduce 79 NMR observables (including chemical shifts and 3J scalar couplings) on five cyclic α/ß-peptides that contain the integrin recognition motif isoDGR. Most of the investigated force fields, which include force fields from AMBER, OPLS, CHARMM and GROMOS families, display very good agreement with experimental 3J(HN,Hα), suggesting that MD simulations could be an appropriate tool in the rational design of therapeutic cyclic α-peptides. However, for NMR observables directly related to ß-amino acids, we observed a poor agreement with experiments and a remarkable dependence of our evaluation on the choice of Karplus parameters. The force field weaknesses herein unveiled might constitute a source of inspiration for further force field optimization.

Thermodynamically-Weighted Conformational Ensemble of Cyclic RGD Peptidomimetics from NOE Data.

In the case of flexible molecules, the standard approach of transforming NOE intensities into spatial restraints and of building conformational models minimizing these restraints greatly neglects the richness of molecular conformations. Making use of NOE intensities measured in triplicate and of an iterative molecular-dynamics scheme, we optimized a force field to generate a set of conformations whose ensemble is compatible with the experimental data, and is weighted according to the Boltzmann distribution. This scheme is applied to two cyclic peptidomimetic ligands of integrins. Their difference in binding affinity is recapitulated in terms of their difference in conformational fluctuations.

New potent αvß3 integrin ligands based on azabicycloalkane (γ,α)-dipeptide mimics.

We have designed a new synthetic strategy for the preparation of a new class of cyclic RGD integrin ligands in which the azabicycloalkane scaffold can be envisaged as a (γ,α) dipeptide mimic. The synthesis and in vitro biological evaluation of these RGD derivatives, as well as the computational study of their conformational properties and binding modes to αVß3 integrin are described. Compound has shown to be a promising candidate as αVß3 integrin antagonist able to interfere with both cell adhesion and movement on vitronectin with no evidence of cytotoxic effects.

Epidemiology of hepatitis D virus (HDV) infection in an urban area of northern Italy.

OBJECTIVES: The introduction of vaccination against hepatitis B initially reduced the number of HBV (hepatitis B virus) and HDV (hepatitis delta virus) infections, but the decreasing trend of HDV infection seems to have stopped. The aim of this study was to assess the prevalence of HDV infection in the general population living in the catchment area of Legnano Hospital in northern Italy. METHODS: Of the 22,758 subjects tested in 2007-2008, the 488 who were HBsAg (hepatitis B surface antigen)-positive [including 107 (21.9%) of non-Italian origin] were subsequently tested for anti-HDV antibodies. RESULTS: Of the 488 subjects who tested positive for HBsAg, 24 (4.9%) were anti-HDV positive, all aged between 30 and 60 years. The difference in prevalence between males (7.1%) and females (1.9%) was statistically significant (p < 0.05), but not that between Italian (5.0%) and non-Italian patients (4.7%). The differences in anti-HDV seropositivity between the patients with acute (0%) and chronic infections (6.3%), and between the incident (2.5%) and prevalent cases (7.4%), were not statistically significant, but there was a significant difference (p < 0.01) between those with asymptomatic (2.1%) and clinically symptomatic infections (10.3%). Intravenous drug abuse was the main source of infection. CONCLUSIONS: In the catchment area of our hospital, the prevalence of HDV infection does not seem to be due to patients of non-Italian origin, but to Italian patients who are not vaccinated against HBV and who survived the HDV epidemic of the 1970s and 1980s. Nevertheless, the increase in the number of immigrants from non-EU countries in recent years is soon likely to lead to a change in the epidemiology of HDV.

Structural investigations of isomeric oxidised forms of hyperforin by HPLC-NMR and HPLC-MSn.

The prenylated phloroglucinol hyperforin, thought to be an essential component for the anti-depressant activity of St. John's Wort (Hypericum perforatum), is unstable. The facile oxidative degradation of hyperforin poses serious problems for standardisation, and may also dramatically affect the pharmacological activity of the extracts. Hyperforin was dissolved in hexane and stored at room temperature for 3 days and yielded various closely related degradation products which, although difficult to isolate on the preparative scale, have been analysed by on-flow and stop-flow HPLC-NMR and HPLC-MS/MS. From on-line spectroscopic data, and with the aid of complementary in-mixture standard NMR two-dimensional correlation experiments, the different oxidised forms of hyperforin were found to be phloroglucinol derivatives in which a hydroxy-dihydrofuran ring is formed involving the enol OH at C-7 or C-9 (tautomeric form) and the prenyl chain at C-8 of the core nucleus of hyperforin. The strategy followed for the on-line identification of these constituents is discussed.

Potent integrin antagonists from a small library of RGD-including cyclic pseudopeptides.

[structure: see text]. A small library of cyclic RGD pseudopentapeptides incorporating stereoisomeric 6,5- and 7,5-fused bicyclic lactams was synthesized with the aim of developing active and selective integrin antagonists. The solid-phase synthesis and activity of these RGD derivatives is described. The approach led to two of the most active known inhibitors of alpha(V)beta3 receptor.

Simulation of carbohydrate-protein interactions: computer-aided design of a second generation GM1 mimic.

The oligosaccharide of ganglioside GM1 [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)Galbeta1-4Glcbeta1-1Cer] is the cellular target of two bacterial enterotoxins: the cholera toxin (CT) and the heat-labile toxin of E. coli (LT). We recently reported that the pseudosaccharide 2 [Galbeta1-3GalNAcbeta1-4(NeuAcalpha2-3)DCCHD] is a high-affinity ligand for CT. and thus a functional mimic of GM1 (Bernardi, A., Checchia, A., Brocca, P., Sonnino, S. and Zuccotto, F., J. Am. Chem. Soc., 121 (1999) 2032-2036). In this paper we describe the design of a second-generation mimic, formally obtained from 2 by inverting the configuration of a single stereocenter, thus transforming a N-acetyl galactosamine into a N-acetyl glucosamine. The design process involved modeling of the free ligand and its LT complex, followed by qualitative and quantitative comparison with the corresponding structures of 2. The protocol employed relied on both conformational search and molecular dynamics methodologies to account for the flexibility of both the ligand and the protein receptor. The conformational search of the LT:inhibitor complex showed that, compared to 2, the new compound can insert one more hydroxy group within the protein binding site. Molecular dynamics simulations showed that, in turn, this may trigger a series of rearrangements and reorientations of side chains and crystallographic water molecules in the toxin, leading to new H-bond contacts which may result in enhanced affinity of the new inhibitor. FEP calculations were performed by mutating the structure of 2 in solution and in the protein complex, and the prediction was made that the second-generation mimic should be a stronger binder than its parent compound.

Premature ovarian failure: steroid synthesis and autoimmunity.

The androgen biosynthesis and autoimmunity of 25 patients with premature ovarian failure (POF) and 18 control subjects with normal cycles were examined. Serum levels of dehydroepiandrosterone sulfate (DHEAS), 17-hydroxyprogesterone (17-OHP), androstenedione, and testosterone were analyzed in POF patients with or without organ-specific autoimmunity, and the results compared with those of women with normal ovarian function. The comparative analysis of DHEAS, 17-OHP, androstenedione and testosterone showed that POF patients had significantly lower values than normal women (DHEAS, androstenedione and testosterone p < 0.01, 17-OHP p < 0.05). Furthermore, we found one or more organ-specific autoantibodies in 11 patients with POF (44%), while only one woman in the control group showed autoimmunity (antithyroid microsome) (5.5%). Only one patient had both anti-ovarian and anti-adrenal antibodies (4%). The comparison of androgen levels in POF patients with or without autoimmunity revealed a statistically significant reduction of DHEAS levels in POF patients with organ-specific autoimmunity (p < 0.01). These data reveal the reduction in androgen synthesis in POF patients, particularly in those with organ-specific autoimmunity.

Design and synthesis of nonpeptide angiotensin II receptor antagonists featuring acyclic imidazole-mimicking structural units.

Extensive molecular modelling studies, including conformational analysis and the comparison of molecular electrostatic potential distributions, wee used to evaluate structural parameters of new antagonists containing acyclic replacements of the N = C-N imidazole region. The synthesis and the biological screening of a series of acyl biphenyltetrazole derivatives were planned and realized to gain an insight into the structure-activity relationships of this unusual class of Angiotensin II antagonists.

A 3D QSAR CoMFA study of non-peptide angiotensin II receptor antagonists.

A series of non-peptide angiotensin II receptor antagonists was investigated with the aim of developing a 3D QSAR model using comparative molecular field analysis descriptors and approaches. The main goals of the study were dictated by an interest in methodologies and an understanding of the binding requirements to the AT1 receptor. Consistency with the previously derived activity models was always checked to contemporarily test the validity of the various hypotheses. The specific conformations chosen for the study, the procedures invoked to superimpose all structures, the conditions employed to generate steric and electrostatic field values and the various PCA/PLS runs are discussed in detail. The effect of experimental design techniques to select objects (molecules) and variables (descriptors) with respect to the predictive power of the QSAR models derived was especially analysed.

Expression of 17 alpha-hydroxylase and aromatase in the syndrome of androgen resistance: a case report.

A complete form of testicular feminization with normal gonadotropin and high testosterone levels is described. The testicular histology of tubular atrophy and hyperplastic Leydig cells accords with the high testosterone levels. We evaluated the expression of the enzymes involved in the production of testosterone and estrogens. An increase in P450c17 (17 alpha-hydroxylase/17,20-lyase) messenger RNA expression has been shown in testis with androgen resistance compared with in normal testis. More P450 aromatase was expressed in normal testis than in testis with androgen resistance.

N-3-substituted pyrimidinones as potent, orally active, AT1 selective angiotensin II receptor antagonists.

A novel series of nonpeptide angiotensin II (A II) antagonists containing a pyrimidinone ring which carries a C-linked biphenyltetrazole moiety and a carboxyheteroaryl group on the 3-position have been prepared. Their affinity for the AT1 receptor was determined in a binding assay on rat adrenal cortical membranes. The in vivo antihypertensive properties were tested by evaluating the inhibition of the pressor response to A II followed by iv and id administration. Extensive molecular modeling studies, including comparison of molecular electrostatic potential distributions, conformational analysis, and overlays on a computational pharmacophore model of A II, were used to evaluate structural parameters of the new compounds, in comparison to other known A II antagonists (e.g., DUP-753 and SK&F 108566). According to the modeling studies, the introduction of a (carboxyheteroaryl)methyl moiety at the 3-position of the pyrimidinone ring led to derivatives with increased potency. Methyl 2-[[4-butyl-2-methyl-6-oxo-5-[[2'-(1H-tetrazol-5-yl)[1,1'-biphenyl ]- 4-yl]methyl]-1-(6H)-pyrimidinyl]methyl]-3-thiophenecarboxylate (3k, LR-B/081), one of the most potent compounds in the series (Ki = 1.4 nM), exhibited a marked antihypertensive activity on oral administration to conscious renal hypertensive rats, with long duration of action. It was selected for clinical evaluation in the treatment of hypertension in man.

Nonpeptide angiotensin II receptor antagonists. Synthesis, in vitro activity, and molecular modeling studies of N-[(heterobiaryl)methyl] imidazoles.

With the aim of explaining the influence of the structural changes on the biphenylic moiety on the activity, a series of N-[(heterobiaryl)methyl]imidazoles (I), constructed on the model of DuPont compounds by replacing either the central or terminal phenyl ring with a heteroaromatic one, such as furan, thiophene, thiazole, and pyridine, was synthesized. Compared to the reference DuPont compound (EXP-7711), all the heterobiaryl derivatives showed a reduced potency both in receptor binding (rat adrenal capsular membranes) and in the functional assay (angiotensin II-induced contraction of rabbit aorta strips). The lower activity was justified by the extensive molecular modeling studies, which took into consideration the conformational and electrostatic features of several heterobiaryl derivatives. On the basis of the results obtained, it was hypothesized that the central aromatic ring of the biarylic portion works as a spacer, orienting in the right way the terminal phenyl ring, whose electronic distribution is, instead, crucial to its fitting well with a lipophilic pocket at the receptor site.

A 3D QSAR approach to the search for geometrical similarity in a series of nonpeptide angiotensin II receptor antagonists.

A 3D QSAR methodology based on the combined use of conformational analysis and chemometrics was applied to perform a comparative analysis of the 3D conformational features of 13 nonpeptide angiotensin II receptor antagonists showing different levels of binding affinity. Conformational analysis by using a molecular mechanics MM2 method was carried out for each of these structures to obtain conformational minima. These minima were described by ten interatomic distances which define the relative spatial disposition of five significant atoms belonging to relevant functional groups present in all the 13 molecules. The structure-activity relationship between the interatomic distances and the biological activity was then assessed by using chemometric methods (cluster analysis, principal component analysis, classification methods). With our indirect approach based on the search for geometrical similarity it was possible, even though structural information on the receptor active site was lacking, to identify the 3D geometrical requirements for the binding affinity of nonpeptide angiotensin II receptor inhibitors.

Organ-specific autoimmunity in patients with premature ovarian failure.

The diagnosis of premature ovarian failure (POF) was made in 45 women (aged 14 to 39 yr) with high concentrations of FSH and LH. Sera from these patients and a control group of 28 women were used to detect organ-specific autoantibodies. Eighteen patients with POF (40%) were positive for at least one autoantibody. In the control group only one woman (3.6%) showed autoimmunity. Antithyroid was the most representative autoimmunity (20%) in the POF group. By indirect immunofluorescence, only one patient was positive for antiovarian and antiadrenal antibodies. In summary, the results of this study are consistent with previous immunohistochemical data which indicate that autoimmune disorders are common in patients with POF. Furthermore, the detection of antiovarian antibodies in only one patient confirms that they are probably detectable in a short period of the disease.

Structure-activity relationship of Ca2+ channel blockers: a study using conformational analysis and chemometric methods.

A structure-activity relationship study has been done on 8 compounds with the activity known as 'Ca2+ channel blockers'. Conformational analysis was carried out using a molecular mechanics method. The 3D-QSAR approach was used and the most polar functional groups present in all the molecules were considered. Eight interatomic distances are necessary to define the relative spatial disposition of these relevant molecular fragments. The structure-activity relationship between interatomic distances and biological activity was performed using statistic and chemometric methods. In particular, with Principal Component Analysis, it was possible to reduce the number of interatomic distances: only six of the eight distances are sufficient to describe the system in a useful way. A classification method was iteratively used to select the most probable conformations linked to the biological activity and to build a model able to classify conformations according to their biological behaviour. Cluster analysis on the active selected conformations subsequently allowed the identification of two different geometrical patterns for the active compounds. Finally the validity of the model was verified by correctly predicting the activity of other molecules not used in the construction of the model but possessing known activity.

[Large colpohematometra with bilateral hematosalpinx resulting from progressive obstruction caused by incomplete vaginal septum]. / Voluminoso colpoematometra con ematosalpinge bilaterale da ostruzione progressiva di setto vaginale incompleto.

Complete or incomplete transverse vaginal septum is a rare malformation of the female genital tract. Usually the complete congenital type occurs at puberty because of the collection of menstrual blood above the septum with amenorrhea and cyclic lower abdominal pain as presenting symptoms. On the contrary, in the case shown by the authors, the subacute epilogue occurred in the perimenopausal phase: a very large colpohematometra is reported in a 49 years old woman, with an incomplete vaginal septum resulting in progressive obstruction. The association between this malformation and the presence of endometriotic localizations in the genital tract, as reported by other authors, is interesting. In this case, endometriosis can be secondary to the presence of the septum or could have determined the impairment of the obstruction in consequence of the associated status of chronic flogosis.