Modulation of the functional interfaces between retroviral intasomes and the human nucleosome.

Infection by retroviruses as HIV-1 requires the stable integration of their genome into the host cells. This process needs the formation of integrase (IN)-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes within cell chromatin. To provide new tools to analyze this association and select drugs, we applied the AlphaLISA technology to the complex formed between the prototype foamy virus (PFV) intasome and nucleosome reconstituted on 601 Widom sequence. This system allowed us to monitor the association between both partners and select small molecules that could modulate the intasome/nucleosome association. Using this approach, drugs acting either on the DNA topology within the nucleosome or on the IN/histone tail interactions have been selected. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, in silico molecular simulations and cellular approaches. These drugs were shown to inhibit both PFV and HIV-1 integration in vitro. Treatment of HIV-1-infected PBMCs with the selected molecules induces a decrease in viral infectivity and blocks the integration process. Thus, in addition to providing new information about intasome-nucleosome interaction determinants, our work also paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring. IMPORTANCE In this work, we report the first monitoring of retroviral intasome/nucleosome interaction by AlphaLISA. This is the first description of the AlphaLISA application for large nucleoprotein complexes (>200 kDa) proving that this technology is suitable for molecular characterization and bimolecular inhibitor screening assays using such large complexes. Using this system, we have identified new drugs disrupting or preventing the intasome/nucleosome complex and inhibiting HIV-1 integration both in vitro and in infected cells. This first monitoring of the retroviral/intasome complex should allow the development of multiple applications including the analyses of the influence of cellular partners, the study of additional retroviral intasomes, and the determination of specific interfaces. Our work also provides the technical bases for the screening of larger libraries of drugs targeting specifically these functional nucleoprotein complexes, or additional nucleosome-partner complexes, as well as for their characterization.

Assessing the effects of PMM2 variants on protein stability.

Phosphomannomutase 2 deficiency, PMM2-CDG, is the most frequent disorder of protein N-glycosylation. It is an autosomal recessive disease with a broad clinical and biochemical phenotype. Trying to predict the impact of novel variants is often a challenge due to the high number of variants and the difficulty to establish solid genotype-phenotype correlations. A potential useful strategy is to use computational chemistry calculations as a tool from which relevant information on the structural impact of novel variants may be deduced. Here we present our analyses based on four well-known PMM2 deleterious variants (p.(Leu32Arg), p.(Asp65Tyr), p.(Phe119Leu), p.(Arg141His)) and the polymorphic p.(Glu197Ala) for which we have predicted the effect on protein stability. Our work predicts the effect of different amino acid residues on the conformation and stability of PMM2. These computational simulations are, therefore, an extremely useful methodology which, in combination with routinely used in silico methods of pathogenicity prediction, may help to reveal the structural impact of novel variants at the protein level, potentially leading to a better understanding of target biological molecules.

Primary melanoma of the oral cavity: A multi-institutional retrospective analysis in Brazil.

BACKGROUND: Melanoma is an aggressive malignant tumor, rarely observed in the oral cavity. The aim of this study was to describe the clinicopathologic features of a series of oral melanomas. MATERIAL AND METHODS: A retrospective descriptive study was performed. A total of 15,482 biopsy records from two oral and maxillofacial pathology services in Brazil were analyzed. All cases of oral melanomas were reviewed, and clinical, demographic, histopathological data, treatment, and follow-up status were collected. In addition, immunohistochemistry stains (pan-cytokeratin AE1/AE3, vimentin, α-SMA, CD45, S-100 protein, HMB-45, Melan A, and Ki-67) were performed. RESULTS: The series comprised of 5 males (71.4%) and 2 females (28.6%), with a mean age of 58.0 ± 9.2 years (range: 45-69 years) and a 2.5:1 male-to-female ratio. The gingiva (n = 3, 42.8%) and hard palate (n = 2, 28.6%) were the most common affected sites, presenting clinically as ulcerated swellings with a brown to black color. Cervical lymph node metastasis was detected in three patients during the first examination. Microscopically, 6 cases (85.7%) were melanotic, and one (14.3%) was amelanotic. Most cases (n = 4, 57.1%) presented a predominance of epithelioid cells. S-100 and HMB-45 were positive in all cases (n = 7, 100.0%). In contrast, only 4 cases (57.1%) were positive for Melan-A. The proliferative index with Ki-67 was high, with labeling index ranging from 70.0% to more than 90% of positive cells. Five patients died from complications of the tumors after a mean follow-up period of 7.8 months. CONCLUSIONS: Melanoma is an aggressive malignant tumor that rarely occurs in the oral cavity. It occurs mainly in adult and elderly patients and often is diagnosed in advanced stages. The current findings were similar to previous studies and reflected the characteristics of the services from where lesions were retrieved.

Epidemiologic analysis of salivary gland tumors over a 10-years period diagnosed in a northeast Brazilian population.

BACKGROUND: Salivary gland tumors (SGT) correspond to a heterogeneous group of lesions with variable biological behavior. The present study aimed to determine the distribution and demographic findings of salivary gland neoplasms in a northeast Brazilian population. MATERIAL AND METHODS: A retrospective descriptive cross-sectional study was performed. A total of 588 cases of SGT were diagnosed between 2006 and 2016 of 4 pathology services in the state of Sergipe, Brazil. All cases were reviewed, and data such as sex, age, anatomical location, and histopathological diagnosis were collected. RESULTS: A total of 470 (79.9%) tumors were benign and 118 (20.1%) were malignant. The majority of the patients were females (n=328, 55.8%) with an overall female:male ratio of 1.2:1. The major salivary glands were affected more than the minor glands (69.5% vs. 30.5%). Pleomorphic adenoma (n=419, 71.3%) and mucoepidermoid carcinoma (n=29, 4.9%) were the most frequent benign and malignant tumors, respectively. In addition, both benign and malignant tumors occurred more frequently in the parotid gland (n=300, 51%, p<0.05). CONCLUSIONS: The epidemiologic profile and clinical characteristics of SGT were similar to those described in other countries and other regions of Brazil. Epidemiological studies of SGT help to understand their clinical and pathological features and are essential to establish the proper management and prognosis.

Allelic loss in amalgam-associated oral lichenoid lesions compared to oral lichen planus and mucosa.

BACKGROUND: The amalgam-associated oral lichenoid lesion (AAOLL) shows clinical and histopathological features similar to oral lichen planus (OLP). Molecular researches to improve knowledge of pathogenesis and clinical behavior of AAOLL are still scarce. OBJECTIVE: We investigated for the first time the use of loss of heterozygosity (LOH) as a molecular approach for genetic characterization of AAOLL in comparison with OLP and evaluated the cell proliferation index. MATERIALS AND METHODS: The sample comprised nine AAOLLs, 10 OLPs, and eight NOMs matched by patients' gender and age. LOH was assessed using polymorphic microsatellite markers at chromosomes 9p (D9S157, D9S162, D9S171), 11q (D11S1369), and 17p (TP53, AFM238WF2). Cell proliferation was assessed by immunohistochemical expression of Ki-67 (MIB-1). The association between LOH and Ki-67 was investigated. RESULTS: Loss of heterozygosity occurred in 5/9 AAOLLs and in 2/10 OLPs in at least one marker each, while NOM showed no LOH. Cell proliferation index in AAOLL ranged from 2 to 23%. There was no association between cell proliferation and LOH, independent of the marker. CONCLUSION: Our study shows that the profile of molecular changes in AAOLL and OLP, evaluated by LOH and Ki-67 expression, is similar. Additional studies including larger samples should be performed to confirm or to refute our findings.

The photophysics of fac-[Re(CO)3(NN)(bpa)](+) complexes: a theoretical/experimental study.

The influence of the polypyridyl ligand on the photophysics of fac-[Re(CO)3(NN)(bpa)](+), bpa = 1,2-bis-(4-pyridyl)ethane and NN = 1,10-phenanthroline (phen), pyrazino[2,3-f][1,10]-phenanthroline (dpq), and dipyrido[3,2-a:2'3'-c]phenazine (dppz) has been investigated by steady state and time-resolved emission spectroscopy combined with theoretical calculations using time-dependent density functional theory (TD-DFT). The fac-[Re(CO)3(phen)(bpa)](+) is a typical MLCT emitter in acetonitrile with ϕ = 0.11 and τ = 970 ns. The emission lifetime and quantum yield decrease significantly in fac-[Re(CO)3(dpq)(bpa)](+) (ϕ = 0.05; τ = 375 ns) due to the presence of a close lying dark charge transfer state located at the pyrazine ring of dpq, as indicated by TD-DFT data. The luminescence of these complexes is quenched by hydroquinone with kq = (2.9 ± 0.1) × 10(9) and (2.6 ± 0.1) × 10(9) L mol(-1) s(-1), respectively, for NN = phen or dpq. These values are increased respectively to (4.6 ± 0.1) × 10(9) and (4.2 ± 0.1) × 10(9) L mol(-1) s(-1) in the 1 : 1 H2O-CH3CN mixture. In this medium Stern-Volmer constants determined by steady-state and time-resolved measurements differ from each other, which is indicative of static quenching, i.e. the pre-association of hydroquinone and the complexes through hydrogen bonding between the remote N-atom in the bpa ligand (KA ≅ 1-2 × 10(1) L mol(-1)), followed by a concerted proton-electron transfer. In contrast to other investigated complexes, fac-[Re(CO)3(dppz)(bpa)](+) is weakly emissive in acetonitrile at room temperature (ϕ ≅ 10(-4)) and does not exhibit a rigidochromic effect. This photophysical behaviour as well as TD-DFT data indicate that the lowest lying triplet excited state can be described as (3)ILdppz. The results provide additional insight into the influence of the polypyridyl ligand on the photophysical properties of Re(I) complexes.

Structural and dynamics analysis of matrix metalloproteinases MMP-2 complexed with chemically modified tetracyclines (CMTs).

Matrix metalloproteinases (MMPs) play a critical role in physiological processes and pathological conditions such tumor invasion and metastasis. In recent years, a number of MMP inhibitors have been proposed, including the chemically modified tetracyclines (CMTs), which have been evaluated in preclinical cancer models showing promising results. This work provides insights into the structure and dynamics of the MMP-2 catalytic domain complexed with seven CMT (CMT-n), based on the analysis of molecular dynamics trajectories in solution. The comparative analysis of various relevant molecular aspects of the different complexes of MMP-2 and CMT-n derivatives was performed aiming to elucidate the effect of ligands on the enzyme structure. These include the radial distribution function of the water molecules around the catalytic zinc, the solvent accessible surface area for the inhibitors and the root-mean-square fluctuation for all amino acid residues. The results help to understand the differences in the binding modes of related compounds and, therefore, add to further design of novel tetracycline-based inhibitors for MMP enzymes.

Farnesyltransferase inhibitors: a comprehensive review based on quantitative structural analysis.

Farnesyltransferase inhibitors (FTIs) have mainly been used in cancer therapy. However, more recently, investigations on these inhibitors revealed that FTIs can be used for the treatment of other diseases such as Progeria, P. falciparum resistant malaria, Trypnosomatid, etc. Hence the development of novel FTIs is an important task for the drug discovery program. Initially, numerous peptidomimetic FTIs were developed from the template of CAAX (CVIM was the first pharmacophore model used as a peptidomimetic). Later, many non-peptidomimetic FTIs have been discovered with the structural modification of the peptidomimetics. The structural analysis of those developed FTIs by various researchers suggested that the presence of a heterocycle or a polar group in place of the thiol group is required for interaction with the Zn(2+) ion. The bulky naphthyl, quinolinyl, phenyl, phenothazine, etc in this position provide better hydrophobicity to the molecules which interact with the aromatic amino acid moieties in the hydrophobic pocket. A hydrophilic region with polar groups is necessary for the polar or hydrogen bonding interactions with the amino acids or water molecules in the active site. Many FTIs have been isolated from natural products, which possessed inhibitory activity against farnesyltransferase (FTase). Among them, pepticinnamin E (9R), fusidienol (9T), gliotoxin (9V), cylindrol A (9X), etc possessed potential FTase inhibitory activities and their structural features are comparable to those of the synthetic molecules. The clinical studies progressing on FTIs showed that tipifarnib in combination with bortezomib is used for the treatment of patients with advanced acute leukemias. Successful phase I and II studies are undergoing for tipifarnib alone or in combination with other drugs/radiation for the treatment of multiple myeloma, AML, breast cancer, mantle cell lymphoma, solid tumors, non-small cell lung cancer (NSCLC), pancreatic cancer, glioblastoma, etc. Phase I pharmacokinetic (maximum tolerated dose, toxicity) and pharmacodynamic studies of AZD3409 (an orally active double prodrug) is progressing on patients with solid malignancies taking 500 mg once a day. A phase II study is undergoing on lonafarnib alone and in combination with zoledronic acid and pravastatin for the treatment of Hutchinson-Gilford Progeria syndrome (HGPS) and progeroid laminopathies. Lonafarnib therapy improved cardiovascular status of children with HGPS, by improved peripheral arterial stiffness, bone structure and audiological status in the patients. Other important FTIs such as BMS-214662, LB42908, LB42708, etc are under clinical studies for the treatment of various cancers. This review concluded that the quantitative structural analysis report with an elaborative study on the natural product compounds provides ideas for development of novel molecules for the FTase inhibitory activity. The fragment based analysis is also needed to select the substituents, which provides significant inhibitory activities and can also have good pharmacokinetic properties in the clinical studies.

Protein-ligand docking in the new millennium--a retrospective of 10 years in the field.

Protein-ligand docking is currently an important tool in drug discovery efforts and an active area of research that has been the subject of important developments over the last decade. These are well portrayed in the rising number of available protein-ligand docking software programs, increasing level of sophistication of its most recent applications, and growing number of users. While starting by summarizing the key concepts in protein-ligand docking, this article presents an analysis of the evolution of this important field of research over the past decade. Particular attention is given to the massive range of alternatives, in terms of protein-ligand docking software programs currently available. The emerging trends in this field are the subject of special attention, while old established docking alternatives are critically revisited. Current challenges in the field of protein-ligand docking such as the treatment of protein flexibility, the presence of structural water molecules and its effect in docking, and the entropy of binding are dissected and discussed, trying to anticipate the next years in the field.

New designs for MRI contrast agents.

New designs for Magnetic Resonance Imaging contrast agents are presented. Essentially, they all are host-guest inclusion complexes between y-cyclodextrins and polyazamacrocycles of gadolinium (III) ion. Substitutions have been made to the host to optimise the host-guest association. Molecular mechanics calculations have been performed, using the UFF force field for metals, to decide on the suitability of the substitutions, and to evaluate the host-guest energies of association. Interesting general conclusions have been obtained, concerning the improvement of Magnetic Resonance Imaging contrast agents; namely, a set of rational methodologies have been deduced to improve the association between the gadolinium (III) chelates and the cyclodextrins, and their efficiency is demonstrated with a large set of substituted complexes, opening new doors to increase the diagnostic capabilities of Magnetic Resonance Imaging.