Publications by authors named "Mercedes Campillo"

37 Publications

TMSNP: a web server to predict pathogenesis of missense mutations in the transmembrane region of membrane proteins.

NAR Genom Bioinform 2021 Mar 23;3(1):lqab008. Epub 2021 Feb 23.

Bioinformatics and Medical Statistics Group, Facultat de Ciències i Tecnologia, UVIC-UCC, 08500 Vic, Barcelona, Spain.

The massive amount of data generated from genome sequencing brings tons of newly identified mutations, whose pathogenic/non-pathogenic effects need to be evaluated. This has given rise to several mutation predictor tools that, in general, do not consider the specificities of the various protein groups. We aimed to develop a predictor tool dedicated to membrane proteins, under the premise that their specific structural features and environment would give different responses to mutations compared to globular proteins. For this purpose, we created TMSNP, a database that currently contains information from 2624 pathogenic and 196 705 non-pathogenic reported mutations located in the transmembrane region of membrane proteins. By computing various conservation parameters on these mutations in combination with annotations, we trained a machine-learning model able to classify mutations as pathogenic or not. TMSNP (freely available at http://lmc.uab.es/tmsnp/) improves considerably the prediction power of commonly used mutation predictors trained with globular proteins.
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http://dx.doi.org/10.1093/nargab/lqab008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902201PMC
March 2021

The mutational landscape of human olfactory G protein-coupled receptors.

BMC Biol 2021 Feb 5;19(1):21. Epub 2021 Feb 5.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, E-08193, Bellaterra, Spain.

Background: Olfactory receptors (ORs) constitute a large family of sensory proteins that enable us to recognize a wide range of chemical volatiles in the environment. By contrast to the extensive information about human olfactory thresholds for thousands of odorants, studies of the genetic influence on olfaction are limited to a few examples. To annotate on a broad scale the impact of mutations at the structural level, here we analyzed a compendium of 119,069 natural variants in human ORs collected from the public domain.

Results: OR mutations were categorized depending on their genomic and protein contexts, as well as their frequency of occurrence in several human populations. Functional interpretation of the natural changes was estimated from the increasing knowledge of the structure and function of the G protein-coupled receptor (GPCR) family, to which ORs belong. Our analysis reveals an extraordinary diversity of natural variations in the olfactory gene repertoire between individuals and populations, with a significant number of changes occurring at the structurally conserved regions. A particular attention is paid to mutations in positions linked to the conserved GPCR activation mechanism that could imply phenotypic variation in the olfactory perception. An interactive web application (hORMdb, Human Olfactory Receptor Mutation Database) was developed for the management and visualization of this mutational dataset.

Conclusion: We performed topological annotations and population analysis of natural variants of human olfactory receptors and provide an interactive application to explore human OR mutation data. We envisage that the utility of this information will increase as the amount of available pharmacological data for these receptors grow. This effort, together with ongoing research in the study of genetic changes in other sensory receptors could shape an emerging sensegenomics field of knowledge, which should be considered by food and cosmetic consumer product manufacturers for the benefit of the general population.
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http://dx.doi.org/10.1186/s12915-021-00962-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7866472PMC
February 2021

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

J Med Chem 2019 07 19;62(13):6035-6046. Epub 2019 Jun 19.

Departamento de Química Orgánica I, Facultad de Ciencias Químicas , Universidad Complutense de Madrid , E-28040 Madrid , Spain.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound [UCM-1336, IC = 2 μM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00145DOI Listing
July 2019

Inter-residue interactions in alpha-helical transmembrane proteins.

Bioinformatics 2019 08;35(15):2578-2584

Bioinformatics Area, School of International Studies, ESCI-UPF, Barcelona, Spain.

Motivation: The number of available membrane protein structures has markedly increased in the last years and, in parallel, the reliability of the methods to detect transmembrane (TM) segments. In the present report, we characterized inter-residue interactions in α-helical membrane proteins using a dataset of 3462 TM helices from 430 proteins. This is by far the largest analysis published to date.

Results: Our analysis of residue-residue interactions in TM segments of membrane proteins shows that almost all interactions involve aliphatic residues and Phe. There is lack of polar-polar, polar-charged and charged-charged interactions except for those between Thr or Ser sidechains and the backbone carbonyl of aliphatic and Phe residues. The results are discussed in the context of the preferences of amino acids to be in the protein core or exposed to the lipid bilayer and to occupy specific positions along the TM segment. Comparison to datasets of β-barrel membrane proteins and of α-helical globular proteins unveils the specific patterns of interactions and residue composition characteristic of α-helical membrane proteins that are the clue to understanding their structure.

Availability And Implementation: Results data and datasets used are available at http://lmc.uab.cat/TMalphaDB/interactions.php.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/bty978DOI Listing
August 2019

GPCR-SAS: A web application for statistical analyses on G protein-coupled receptors sequences.

PLoS One 2018 25;13(7):e0199843. Epub 2018 Jul 25.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain.

G protein-coupled receptors (GPCRs) are one of the largest protein families in mammals. They mediate signal transduction across cell membranes and are important targets for the pharmaceutical industry. The G Protein-Coupled Receptors-Sequence Analysis and Statistics (GPCR-SAS) web application provides a set of tools to perform comparative analysis of sequence positions between receptors, based on a curated structural-informed multiple sequence alignment. The analysis tools include: (i) percentage of occurrence of an amino acid or motif and entropy at a position or range of positions, (ii) covariance of two positions, (iii) correlation between two amino acids in two positions (or two sequence motifs in two ranges of positions), and (iv) snake-plot representation for a specific receptor or for the consensus sequence of a group of selected receptors. The analysis of conservation of residues and motifs across transmembrane (TM) segments may guide the design of more selective ligands or help to rationalize activation mechanisms, among others. As an example, here we analyze the amino acids of the "transmission switch", that initiates receptor activation following ligand binding. The tool is freely accessible at http://lmc.uab.cat/gpcrsas/.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199843PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059404PMC
December 2018

GPCRtm: An amino acid substitution matrix for the transmembrane region of class A G Protein-Coupled Receptors.

BMC Bioinformatics 2015 Jul 2;16:206. Epub 2015 Jul 2.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193, Bellaterra, Barcelona, Spain.

Background: Protein sequence alignments and database search methods use standard scoring matrices calculated from amino acid substitution frequencies in general sets of proteins. These general-purpose matrices are not optimal to align accurately sequences with marked compositional biases, such as hydrophobic transmembrane regions found in membrane proteins. In this work, an amino acid substitution matrix (GPCRtm) is calculated for the membrane spanning segments of the G protein-coupled receptor (GPCR) rhodopsin family; one of the largest transmembrane protein family in humans with great importance in health and disease.

Results: The GPCRtm matrix reveals the amino acid compositional bias distinctive of the GPCR rhodopsin family and differs from other standard substitution matrices. These membrane receptors, as expected, are characterized by a high content of hydrophobic residues with regard to globular proteins. On the other hand, the presence of polar and charged residues is higher than in average membrane proteins, displaying high frequencies of replacement within themselves.

Conclusions: Analysis of amino acid frequencies and values obtained from the GPCRtm matrix reveals patterns of residue replacements different from other standard substitution matrices. GPCRs prioritize the reactivity properties of the amino acids over their bulkiness in the transmembrane regions. A distinctive role is that charged and polar residues seem to evolve at different rates than other amino acids. This observation is related to the role of the transmembrane bundle in the binding of ligands, that in many cases involve electrostatic and hydrogen bond interactions. This new matrix can be useful in database search and for the construction of more accurate sequence alignments of GPCRs.
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http://dx.doi.org/10.1186/s12859-015-0639-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4489126PMC
July 2015

Does the S phase have an impact on the accuracy of comparative genomic hybridization profiles in single fibroblasts and human blastomeres?

Fertil Steril 2014 Feb 5;101(2):488-95. Epub 2013 Dec 5.

Unitat de Biologia Cel·lular i Genètica Mèdica. Facultat de Medicina, Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Bellaterra, Spain. Electronic address:

Objective: To investigate if there is an association between single-cell replicative stage and the segmental chromosome imbalances detected by comparative genomic hybridization (CGH).

Design: First, 135 fibroblasts from cell-line GM03184 (Coriell) at three cell stages (G0/G1, S, and G2/M) were amplified by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) or Sureplex and blindly analyzed by CGH. Second, 85 human blastomeres at the interphase and the metaphase stages, from 30 donated human cryopreserved embryos, were amplified by Sureplex and analyzed by CGH.

Setting: Academic center for reproductive medicine.

Patient(s): None.

Intervention(s): None.

Main Outcome Measure(s): Incidence of aneuploidy and segmental imbalances detected at the different cell stages.

Result(s): In DOP-PCR amplifications of fibroblasts, an increased incidence of segmental abnormalities was detected in the S phase. In Sureplex amplifications of fibroblasts and blastomeres, no differences were detected between the different cell stages. A significantly increased incidence of structural abnormalities was seen in the aneuploid blastomeres.

Conclusion(s): The segmental imbalances detected after Sureplex amplification in 73.3% of the cryopreserved embryos analyzed are mainly nontransitory. They correspond to segmental imbalances present in the cells due to chromosome instability, rather than to replicative DNA segments.
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http://dx.doi.org/10.1016/j.fertnstert.2013.10.031DOI Listing
February 2014

Non-meiotic chromosome instability in human immature oocytes.

Eur J Hum Genet 2014 Feb 22;22(2):202-7. Epub 2013 May 22.

1] Càtedra de Recerca Eugin-UAB, Universitat Autònoma de Barcelona, Bellaterra, Spain [2] Unitat de Biologia Cellular i Genètica Mèdica Facultat de Medicina Departament de Biologia Cellular, Fisiologia i Immunologia Universitat Autònoma de Barcelona, Bellaterra, Spain.

Aneuploidy has been a major issue in human gametes and is closely related to fertility problems, as it is known to be present in cleavage stage embryos and gestational losses. Pre-meiotic chromosome abnormalities in women have been previously described. The aim of this study is to assess the whole-chromosome complement in immature oocytes to find those abnormalities caused by mitotic instability. For this purpose, a total of 157 oocytes at the germinal vesicle or metaphase I stage, and discarded from IVF cycles, were analysed by CGH. Fifty-six women, between 18 and 45 years old (mean 32.5 years), including 32 IVF patients (25-45 years of age) and 24 IVF oocyte donors (18-33 years of age), were included in the study. A total of 25/157 (15.9%) of the oocytes analysed, obtained from three IVF clinics, contained chromosome abnormalities, including both aneuploidy (24/157) and structural aberrations (9/157). Independently of the maternal age, the incidence of abnormal oocytes which originated before meiosis is 15.9%, and these imbalances were found in 33.9% of the females studied. This work sheds light on the relevance of mitotic instability responsible for the generation of the abnormalities present in human oocytes.
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http://dx.doi.org/10.1038/ejhg.2013.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895626PMC
February 2014

The role of hydrophobic amino acids in the structure and function of the rhodopsin family of G protein-coupled receptors.

Methods Enzymol 2013 ;520:99-115

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain.

Recent advances in crystallization methods have permitted to resolve the molecular structure of several members of the rhodopsin family of G protein-coupled receptors (GPCRs). Comparison among these structures revealed a number of conserved polar and charged residues implicated in the receptor transduction pathways. These residues function as micro-switches in the process of receptor activation and has been the object of study of many research groups. However, hydrophobic forces, usually underappreciated, also play a major role in GPCR function. Conserved hydrophobic residues contribute significantly to receptor activation, G protein coupling, and oligomerization processes. This review focuses on the impact of the hydrophobic amino acids observed in the structure of class A GPCRs necessary for their function. This information represents a fundamental piece to complete a holistic view of the GPCR signal transduction machinery.
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http://dx.doi.org/10.1016/B978-0-12-391861-1.00005-8DOI Listing
June 2013

Risk, predictors, and clinical characteristics of lymphoma development in primary Sjögren's syndrome.

Semin Arthritis Rheum 2011 Dec 12;41(3):415-23. Epub 2011 Jun 12.

Internal Medicine Department of Vall d'Hebron University Hospital, Barcelona, Spain.

Objective: To assess the risk and predictors of lymphoma development in a large cohort of patients with primary Sjögren's syndrome (pSS).

Methods: Cox-regression analyses were used to study the predictive value of clinical and laboratory findings at pSS diagnosis, and Kaplan-Meier survival curves to compare survival probability between patients who developed lymphoma and the total cohort. Expected risk for lymphoma was calculated by comparison with the background population.

Results: Eleven (4.5%) from 244 patients developed a non-Hodgkin lymphoma (NHL). Diffuse large B-cell and mucosa-associated lymphoid tissue lymphomas occurred at a similar frequency. Three (27.3%) patients died: 2 due to transformation from mucosa-associated lymphoid tissue to diffuse large B-cell. Purpura (HR 8.04, 95% confidence interval [CI] 2.33-27.67), parotidomegaly (HR 6.75, 95%CI 1.89-23.99), anemia (HR 3.43, 95%CI 1.04-11.35), leukopenia (HR 8.70, 95%CI 2.38-31.82), lymphocytopenia (HR 16.47, 95%CI 3.45-78.67), hypergammaglobulinemia (HR 4.06, 95%CI 1.06-15.58), low C3 (HR 36.65, 95%CI 10.65-126.18), and low C4 (HR 39.70, 95%CI 8.85-126.18) levels at pSS diagnosis were significant predictors of NHL development, but only hypocomplementemia and lymphocytopenia were independent risk factors. Hypocomplementemia was related to earlier development of NHL and higher mortality. The cumulative risk of developing lymphoma ranged from 3.4% in the first 5 years to 9.8% at 15 years. Standardized incidence ratio (95%CI) for NHL development was 15.6 (95%CI 8.7-28.2).

Conclusions: Patients with pSS have a 16-fold increased risk of developing lymphoma. This risk increases with time. Hypocomplementemia and lymphocytopenia at pSS diagnosis are the strongest predictors. Survival is clearly reduced in patients with hypocomplementemia. Indolent lymphomas tend to evolve over time toward a more aggressive histologic type.
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http://dx.doi.org/10.1016/j.semarthrit.2011.04.006DOI Listing
December 2011

Sitamaquine overcomes ABC-mediated resistance to miltefosine and antimony in Leishmania.

Antimicrob Agents Chemother 2011 Aug 6;55(8):3838-44. Epub 2011 Jun 6.

Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas (IPBLN-CSIC), Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento s/n, 18100 Armilla, Granada, Spain.

Although oral miltefosine represented an important therapeutic advance in the treatment of leishmaniasis, the appearance of resistance remains a serious threat. LMDR1/LABCB4, a P-glycoprotein-like transporter included in the Leishmania ABC (ATP-binding cassette) family, was the first molecule shown to be involved in experimental miltefosine resistance. LMDR1 pumps drugs out of the parasite, thereby decreasing their intracellular accumulation. Sitamaquine, another promising oral drug for leishmaniasis, is currently in phase 2b clinical trials. The physicochemical features of this drug suggested to us that it could be considered for use as an LMDR1 inhibitor. Indeed, we report herein that nonleishmanicidal concentrations of sitamaquine reverse miltefosine resistance in a multidrug resistance Leishmania tropica line that overexpresses LMDR1. This reversal effect is due to modulation of the LMDR1-mediated efflux of miltefosine. In addition, sitamaquine is not a substrate of LMDR1, as this transporter does not affect sitamaquine accumulation or sensitivity in the parasite. Likewise, we show that ketoconazole, another oral leishmanicidal drug known to interact with ABC transporters, is also able to reverse LMDR1-mediated miltefosine resistance, although with a lower efficiency than sitamaquine. Molecular docking on a three-dimensional homology model of LMDR1 showed different preferential binding sites for each substrate-inhibitor pair, thus explaining this different behavior. Finally, we show that sitamaquine is also able to modulate the antimony resistance mediated by MRPA/LABCC3, another ABC transporter involved in experimental and clinical antimony resistance in this parasite. Taken together, these data suggest that the combination of sitamaquine with miltefosine or antimony could avoid the appearance of resistance mediated by these membrane transporters in Leishmania.
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http://dx.doi.org/10.1128/AAC.00065-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3147638PMC
August 2011

Development of non-peptide ligands of growth factor receptor-bound protein 2-SRC homology 2 domain using molecular modeling and NMR spectroscopy.

J Med Chem 2011 Feb 27;54(4):1096-100. Epub 2011 Jan 27.

Departamento de Química Orgánica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain.

We report a novel series of non-peptide ligands that inhibit the growth factor receptor-bound protein 2 (Grb2)-Src homology 2 (SH2) domain binding, designed using a combined computational and NMR-driven approach. We have identified a new lead compound, 1n (IC(50) = 56 μM), which is cytotoxic in HER2-positive breast cancer cells and disrupts the interaction between HER2 and Grb2. Thus, 1n can be used as a scaffold for the development of efficient Grb2-SH2 domain binding inhibitors.
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http://dx.doi.org/10.1021/jm101478nDOI Listing
February 2011

Age-related survival and clinical features in systemic sclerosis patients older or younger than 65 at diagnosis.

Rheumatology (Oxford) 2010 Jun 11;49(6):1112-7. Epub 2010 Mar 11.

Internal Medicine Department, Vall d'Hebron University Hospital and Universitat Autònoma de Barcelona, Barcelona, Spain.

Objective: To analyse the differences in SSc clinical features and survival in patients aged > or = 65 years compared with young SSc patients.

Methods: Of a total of 319 SSc patients, we identified 67 (21%) patients aged >65 years. Demographical data such as SSc subsets, the cutaneous complaint, internal organ involvement and the causes of morbidity and mortality were collected. Results of the elderly and young patients were compared.

Results: There were 61 (91%) women and 6 (9%) men aged > or = 65 years. The limited SSc (lSSc) subset was more prevalent in elderly than in young patients (74.6 vs 54%, P = 0.002). Pulmonary disease (86.6% in elderly vs 73.8% in young patients, P = 0.034) and cardiac involvement (70.1% in elderly vs 49.6% in young patients, P = 0.004) were significantly more prevalent in elderly patients. In contrast, signs of oesophageal involvement (43.3% in elderly vs 57.5% in young patients, P = 0.040) were less frequent in aged patients. In addition, pulmonary and heart disease appeared significantly earlier after the diagnosis in patients aged > or = 65 years. Mortality was significantly higher in elderly than in young patients (35.8 vs 19%, P = 0.005), but when standardized mortality ratios (SMRs) were analysed, there was no significant mortality increase in the elderly.

Conclusion: In elderly patients, the lSSc subset is more prevalent than the diffuse. Pulmonary and cardiac involvement are more prevalent in aged patients and appears sooner after the disease diagnosis. SSc is clearly related to increased mortality, although it is not significant in the elderly group.
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http://dx.doi.org/10.1093/rheumatology/keq046DOI Listing
June 2010

Benzimidazole derivatives as new serotonin 5-HT6 receptor antagonists. Molecular mechanisms of receptor inactivation.

J Med Chem 2010 Feb;53(3):1357-69

Departamento de Química Orgánica I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain.

On the basis of our previously described pharmacophore model for serotonin 5-HT(6) receptor (5-HT(6)R) antagonists, we have designed, synthesized, and pharmacologically characterized a series of benzimidazole derivatives 1-20 that represent a new family of potent antagonists at the human 5-HT(6)R. Site-directed mutagenesis and a beta(2)-adrenoceptor-based homology model of the 5-HT(6)R were used to predict the mode of binding of antagonist SB-258585 and the new synthesized ligands. Substitution of W6.48, F6.52, or N6.55 by Ala fully impedes compound 4 to block 5-HT-induced activation. Thus, we propose that D3.32 in TM 3 anchors the protonated piperazine ring, the benzimidazole ring expands parallel to EL 2 to hydrogen bond N6.55 in TM 6, and the aromatic ring is placed between TMs 3 and 5 in CH(2)-containing compounds and between TMs 3 and 6 in CO-containing compounds. This combined experimental and computational study has permitted to propose the molecular mechanisms by which the new benzimidazole derivatives act as 5-HT(6)R antagonists.
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http://dx.doi.org/10.1021/jm901672kDOI Listing
February 2010

Influence of the g- conformation of Ser and Thr on the structure of transmembrane helices.

J Struct Biol 2010 Jan 17;169(1):116-23. Epub 2009 Sep 17.

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain.

In order to study the influence of Ser and Thr on the structure of transmembrane helices we have analyzed a database of helix stretches extracted from crystal structures of membrane proteins and an ensemble of model helices generated by molecular dynamics simulations. Both complementary analyses show that Ser and Thr in the g- conformation induce and/or stabilize a structural distortion in the helix backbone. Using quantum mechanical calculations, we have attributed this effect to the electrostatic repulsion between the side chain Ogamma atom of Ser and Thr and the backbone carbonyl oxygen at position i-3. In order to minimize the repulsive force between these negatively charged oxygens, there is a modest increase of the helix bend angle as well as a local opening of the helix turn preceding Ser/Thr. This small distortion can be amplified through the helix, resulting in a significant displacement of the residues located at the other side of the helix. The crystal structures of aquaporin Z and the beta(2)-adrenergic receptor are used to illustrate these effects. Ser/Thr-induced structural distortions can be implicated in processes as diverse as ligand recognition, protein function and protein folding.
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http://dx.doi.org/10.1016/j.jsb.2009.09.009DOI Listing
January 2010

Comparative genomic hybridization analysis reveals new different subgroups in early-stage bladder tumors.

Urology 2010 Feb 3;75(2):347-55. Epub 2009 Aug 3.

Institut de Biotecnologia i Biomedicina and Department de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Barcelona, Spain.

Objectives: To classify bladder tumors according to their genomic imbalances and evaluate their association with patient's outcome.

Methods: Sixty-three superficially and minimally invasive bladder tumors were analyzed by conventional comparative genomic hybridization. Subtelomeric screening in 15 of these tumors was performed by multiplex ligation-dependent probe amplification.

Results: Losses of 9q and 9p (32% and 25% of all cases, respectively) as well as gains of chromosomes Xq and Xp (28% and 25%, respectively) were the most frequent chromosome imbalances. Losses of 8p and gains in 1q and 8q were detected in >20% of cases. Tumors were classified into 3 groups according to their individualized pattern of gains and losses. The largest group was characterized by few chromosome imbalances, presenting 77% and 49% of the Ta and T1 tumors, respectively. Another group characterized by chromosomal gains, was composed of equal number of Ta and T1 tumors, with +1q and +17q gains being the most common imbalances. A minority group was characterized by chromosomal losses on 11q, 5q, and 6q. The multiplex ligation-dependent probe amplification study showed good correlation with comparative genomic hybridization results. With regard to the biological significance of this classification, a remarkable fact is that this minority group composed mainly of T1 tumors, showed a significant decrease in patient overall survival.

Conclusions: Our data suggest that superficial carcinomas of the bladder can be subdivided into a larger number of subclasses than had previously been expected. Our results also demonstrate a decreased survival among patients whose tumors show more genomic losses than gains.
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http://dx.doi.org/10.1016/j.urology.2009.04.080DOI Listing
February 2010

Synthesis of new serotonin 5-HT7 receptor ligands. Determinants of 5-HT7/5-HT1A receptor selectivity.

J Med Chem 2009 Apr;52(8):2384-92

Departamento de Quimica Organica I, Facultad de Ciencias Quimicas, Universidad Complutense de Madrid, E-28040 Madrid, Spain.

We report the synthesis of a new set of compounds of general structure I (1-20) with structural modifications in the pharmacophoric elements of the previously reported lead UCM-5600. The new derivatives have been evaluated for binding affinity at 5-HT(7) and 5-HT(1A) receptors. The influence of the different structural features in terms of 5-HT(7)/5-HT(1A) receptor affinity and selectivity was analyzed by computational simulations of the complexes between compounds I and beta(2)-based 3-D models of these receptors. Compound 18 (HYD(1) = 1,3-dihydro-2H-indol-2-one; spacer = -(CH(2))(4)-; HYD(2) + HYD(3) = 3,4-dihydroisoquinolin-2(1H)-yl) exhibits high 5-HT(7)R affinity (K(i) = 7 nM) and selectivity over the 5-HT(1A)R (31-fold), and has been characterized as a partial agonist of the human 5-HT(7)R.
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http://dx.doi.org/10.1021/jm8014553DOI Listing
April 2009

Conformational toggle switches implicated in basal constitutive and agonist-induced activated states of 5-hydroxytryptamine-4 receptors.

Mol Pharmacol 2009 Apr 23;75(4):982-90. Epub 2009 Jan 23.

Centre National de Recherche Scientifique Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, Institut National de Sant et de Recherche Médicale U661, Universités Montpellier 1 and 2, Montpellier, France.

The extended classic ternary complex model predicts that a G protein-coupled receptor (GPCR) exists in only two interconvertible states: an inactive R, and an active R(*). However, different structural active R(*) complexes may exist in addition to a silent inactive R ground state (Rg). Here we demonstrate, in a cellular context, that several R(*) states of 5-hydroxytryptamine-4 (5-HT(4)) receptors involve different side-chain conformational toggle switches. Using site-directed mutagenesis and molecular modeling approaches, we show that the basal constitutive receptor (R(*)basal) results from stabilization of an obligatory double toggle switch (Thr3.36 from inactive g- to active g+ and Trp6.48 from inactive g+ to active t). Mutation of either threonine or tryptophan to alanine resulted in a lowering of the activity of the R(*)basal similar to the Rg. The T3.36A mutation shows that the Thr3.36 toggle switch plays a minor role in the stabilization of R(*) induced by 5-HT (R(*)-5-HT) and BIMU8 (R(*)-BIMU8) and is fully required in the stabilization of R(*) induced by (S)-zacopride, cisapride, and 1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-butyl-4-piperidinyl)-1-propanone (RS 67333) (R(*)-benzamides). Thus, benzamides stabilize R(*)-benzamides by forming a specific hydrogen bond with Thr3.36 in the active g+ conformation. Conversely, R(*)-BIMU8 was probably the result of a direct conformational transition of Trp6.48 from inactive g+ to active t by hydrogen bonding of this residue to a carboxyl group of BIMU8. We were surprised that the Trp6.48 toggle switch was not necessary for receptor activation by the natural agonist 5-HT. R(*)-5-HT is probably attained through other routes of activation. Thus, different conformational arrangements occur during stabilization of R(*)basal, R(*)-5-HT, R(*)-benzamides, and R(*)-BIMU8.
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http://dx.doi.org/10.1124/mol.108.053686DOI Listing
April 2009

Genomic imbalances in urothelial cancer: intratumor heterogeneity versus multifocality.

Diagn Mol Pathol 2008 Sep;17(3):134-40

Institut de Biotecnologia i Biomedicina, Department de Biologia Cellular, Universitat Autònoma de Barcelona, Bellaterra 08193, Barcelona, Spain.

Comparative genomic hybridization and fluorescence in situ hybridization were used to define genetic changes associated with multifocal bladder cancer and to investigate whether the genetic relationship between synchronous urothelial tumors is similar to that observed within different parts of the same tumor. We investigated 8 synchronous urothelial tumors from 3 patients and macroscopically different parts of the same tumor from 2 other patients. The most frequent imbalances were gains of 1q, 2p, and 17q, and losses in 4q. The high number of chromosome imbalances detected in the present report confirms that a high level of chromosome instability could be characteristic of multicentric bladder tumors. Comparative genomic hybridization profiles obtained from independent tumors belonging to the same patient allowed us to elaborate cytogenetic pedigrees portraying the accumulation of chromosome alterations as a form of clonal evolution from a single precursor cell. The analysis of different macroscopic parts of the same tumor allowed us to detect chromosomal heterogeneity and to delineate intratumor clonal evolution. Some chromosome regions that appeared as a gain in one subpopulation were amplified in others indicating a genetic evolution process. Identical processes were observed in different tumors of the same patient. Expansion of chromosome gains and losses between different parts of the same tumor as well as in different tumors of the same patient was also observed. Our results not only provide further evidence of a clonal relationship between different synchronous bladder tumors but also show that the intratumor heterogeneity present in different subpopulations of the same tumor reproduces the behavior of independent synchronous tumors in a same patient.
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http://dx.doi.org/10.1097/PDM.0b013e31815ce4e6DOI Listing
September 2008

Analysis of replication protein A (RPA) in human spermatogenesis.

Mol Hum Reprod 2007 Dec 2;13(12):837-44. Epub 2007 Nov 2.

Department of Medical Genetics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada.

Replication protein A (RPA) has been identified as a component of early recombination nodules. It is thought to stimulate homologous pairing and strand exchange reactions. The expression pattern of RPA in human spermatocytes has been analysed using immunocytogenetic techniques on testicular biopsies from adult male patients. What appears to be connecting RPA-filaments was observed between as yet unsynapsed homologous regions at early stages of zygotene. RPA foci were also observed in synaptic segments at zygotene and early pachytene, in numbers that peak at the end of zygotene. The presence of a localization pattern for RPA was also detected, but statistical analysis of distances between adjacent RPA foci shows that this pattern does not always follow a gamma distribution. Finally, it was determined that RPA is absent from non-centromeric heterochromatin in chromosome 9. The observed bridge-like structure could be the visualization of a proposed pre-synaptic RPA role in the strand invasion that precedes the formation of a Holliday Junction. These observations strengthen the original pre-synaptic model, although the visualization of post-synaptic RPA foci may indicate the presence of a different role for this protein during homologous recombination.
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http://dx.doi.org/10.1093/molehr/gam076DOI Listing
December 2007

Biological evaluation, structure-activity relationships, and three-dimensional quantitative structure-activity relationship studies of dihydro-beta-agarofuran sesquiterpenes as modulators of P-glycoprotein-dependent multidrug resistance.

J Med Chem 2007 Oct 12;50(20):4808-17. Epub 2007 Sep 12.

Instituto Universitario de Bio-OrgAnica Antonio GonzAlez, Universidad de La Laguna and Instituto Canario de Investigación del CAncer, Avenida Astrofísico Francisco SAnchez, 2, 38206 La Laguna, Tenerife, Spain.

Multidrug resistance (MDR) is one of the main challenges in the chemotherapy of cancer, malaria, and other important diseases. Here, we report the inhibitory activity of a series of 76 dihydro-beta-agarofuran sesquiterpenes, tested on NIH-3T3 cells expressing the human P-glycoprotein (Pgp) multidrug transporter, to establish quantitative comparisons of their respective abilities to block the drug transport activity. The screening was performed on the basis of the ability of sesquiterpenes to modulate the intracellular accumulation of the classical Pgp substrate daunorubicin. To understand the structural basis for inhibitory activity and guide the design of more potent Pgp inhibitors, we have performed a three-dimensional quantitative structure-activity relationship model using the comparative molecular similarity indices analysis (CoMSIA). The most salient features of these requirements are in the region of the substituents at the C-2, C-3, and C-8 positions, which seem to be critical for determining the overall effectiveness of sesquiterpenes as Pgp inhibitors.
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http://dx.doi.org/10.1021/jm070290vDOI Listing
October 2007

Structural models of class a G protein-coupled receptors as a tool for drug design: insights on transmembrane bundle plasticity.

Curr Top Med Chem 2007 ;7(10):991-8

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autonoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain.

G protein-coupled receptors (GPCRs) interact with an extraordinary diversity of ligands by means of their extracellular domains and/or the extracellular part of the transmembrane (TM) segments. Each receptor subfamily has developed specific sequence motifs to adjust the structural characteristics of its cognate ligands to a common set of conformational rearrangements of the TM segments near the G protein binding domains during the activation process. Thus, GPCRs have fulfilled this adaptation during their evolution by customizing a preserved 7TM scaffold through conformational plasticity. We use this term to describe the structural differences near the binding site crevices among different receptor subfamilies, responsible for the selective recognition of diverse ligands among different receptor subfamilies. By comparing the sequence of rhodopsin at specific key regions of the TM bundle with the sequences of other GPCRs we have found that the extracellular region of TMs 2 and 3 provides a remarkable example of conformational plasticity within Class A GPCRs. Thus, rhodopsin-based molecular models need to include the plasticity of the binding sites among GPCR families, since the "quality" of these homology models is intimately linked with the success in the processes of rational drug-design or virtual screening of chemical databases.
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http://dx.doi.org/10.2174/156802607780906799DOI Listing
July 2007

Synthesis and pharmacophore modeling of naphthoquinone derivatives with cytotoxic activity in human promyelocytic leukemia HL-60 cell line.

J Med Chem 2007 Feb 24;50(4):696-706. Epub 2007 Jan 24.

Instituto Universitario de Bio-OrgAnica "Antonio GonzAlez", Universidad de La Laguna, Avda. Astrofísico Fco. SAnchez 2, 38206 La Laguna, Tenerife, Spain.

Catalyst/HypoGen pharmacophore modeling approach and three-dimensional quantitative structure-activity relationship (3D-QSAR)/comparative molecular similarity indices analysis (CoMSIA) methods have been successfully applied to explain the cytotoxic activity of a set of 51 natural and synthesized naphthoquinone derivatives tested in human promyelocytic leukemia HL-60 cell line. The computational models have facilitated the identification of structural elements of the ligands that are key for antitumoral properties. The four most salient features of the highly active beta-cycled-pyran-1,2-naphthoquinones [0.1 microM < IC50 < 0.6 microM] are the hydrogen-bond interactions of the carbonyl groups at C-1 (HBA1) and C-2 (HBA2), the hydrogen-bond interaction of the oxygen atom of the pyran ring (HBA3), and the interaction of methyl groups (HYD) at the pyran ring with a hydrophobic area at the receptor. The moderately active 1,4-naphthoquinone derivatives accurately fulfill only three of these features. The results of our study provide a valuable tool in designing new and more potent cytotoxic analogues.
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http://dx.doi.org/10.1021/jm060849bDOI Listing
February 2007

The role of internal water molecules in the structure and function of the rhodopsin family of G protein-coupled receptors.

Chembiochem 2007 Jan;8(1):19-24

Laboratori de Medicina Computacional, Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.

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http://dx.doi.org/10.1002/cbic.200600429DOI Listing
January 2007

The importance of aneuploidy screening in reciprocal translocation carriers.

Reproduction 2006 Jun;131(6):1025-35

Departament de Biologia Cellular, Fisiologia i Immunologia, Unitat de Biologia, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain.

The purpose of this study is to investigate the aneuploidy rate and the mosaicism of chromosomes not involved in reciprocal translocations. Aneuploidy screening (AS) (13, 16, 18, 21 and 22) was performed as a re-analysis on fixed blastomeres from 126 embryos already analysed in preimplantation genetic diagnosis (PGD) cycles of eight female and five male reciprocal translocation carriers who had not achieved a pregnancy. A successful diagnosis for AS was achieved in 91.3% of embryos; 30.9% were euploid and 60.3% were aneuploid for the five chromosomes analysed. Of the embryos, 8.7% were euploid for AS and normal-balanced for the translocation and 22.2% were euploid for AS but unbalanced for the translocation; 8% of the embryos were aneuploid for AS but normal-balanced for the translocation and 52.4% were aneuploid for AS and also unbalanced for the translocation. At least 58.7% of the embryos were mosaic regarding mosaicism for the chromosomes involved and not involved in the translocations. Six of the 16 embryos transferred in the PGD cycles were aneuploid for the AS study; four of them were also mosaics. AS should be performed in reciprocal translocation carriers after segregation analysis in PGD.
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http://dx.doi.org/10.1530/rep.1.01063DOI Listing
June 2006

On the mechanism of interaction of potent surmountable and insurmountable antagonists with the prostaglandin D2 receptor CRTH2.

Mol Pharmacol 2006 Apr 17;69(4):1441-53. Epub 2006 Jan 17.

7TM Pharma A/S, Fremtidsvej 3, 2970 Hørsholm, Denmark.

Chemoattractant receptor-homologous molecule expressed on T helper 2 cells (CRTH2) has attracted interest as a potential therapeutic target in inflammatory diseases. Ramatroban, a thromboxane A2 receptor antagonist with clinical efficacy in allergic rhinitis, was recently found to also display potent CRTH2 antagonistic activity. Here, we present the pharmacological profile of three ramatroban analogs that differ chemically from ramatroban by either a single additional methyl group (TM30642), or an acetic acid instead of a propionic acid side chain (TM30643), or both modifications (TM30089). All three compounds bound to human CRTH2 stably expressed in human embryonic kidney 293 cells with nanomolar affinity. [3H]Prostaglandin D2 (PGD2) saturation analysis reveals that ramatroban and TM30642 decrease PGD2 affinity, whereas TM30643 and TM30089 exclusively depress ligand binding capacity (Bmax). Each of the three compounds acted as potent CRTH2 antagonists, yet the nature of their antagonism differed markedly. In functional assays measuring inhibition of PGD2-mediated 1) guanosine 5'-O-(3-thio)triphosphate binding, 2) beta-arrestin translocation, and 3) shape change of human eosinophils endogenously expressing CRTH2, ramatroban, and TM30642 produced surmountable antagonism and parallel rightward shifts of the PGD2 concentration-response curves. For TM30643 and TM30089, this shift was accompanied by a progressive reduction of maximal response. Binding analyses indicated that the functional insurmountability of TM30643 and TM30089 was probably related to long-lasting CRTH2 inhibition mediated via the orthosteric site of the receptor. A mechanistic understanding of insurmountability of CRTH2 antagonists could be fundamental for development of this novel class of anti-inflammatory drugs.
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http://dx.doi.org/10.1124/mol.105.017681DOI Listing
April 2006

The seventh transmembrane domains of the delta and kappa opioid receptors have different accessibility patterns and interhelical interactions.

Biochemistry 2005 Dec;44(49):16014-25

Department of Pharmacology and Center for Substance Abuse Research, Temple University School of Medicine, Philadelphia, PA 19140, USA.

We applied the substituted cysteine accessibility method (SCAM) to map the residues of the transmembrane helices (TMs) 7 of delta and kappa opioid receptors (deltaOR and kappaOR) that are on the water-accessible surface of the binding-site crevices. A total of 25 consecutive residues (except C7.38) in the TMs 7 were mutated to Cys, one at a time, and each mutant was expressed in HEK 293 cells. Most mutants displayed similar binding affinity for [(3)H]diprenorphine, an antagonist, as the wild types. Pretreatment with (2-aminoethyl)methanethiosulfonate (MTSEA) inhibited [(3)H]diprenorphine binding to eight deltaOR and eight kappaOR mutants. All mutants except deltaOR L7.52(317)C were protected by naloxone from the MTSEA effect, indicating that the side chains of V7.31(296), A7.34(299), I7.39(304), L7.41(306), G7.42(307), P7.50(315), and Y7.53(318) of deltaOR and S7.34(311), F7.37(314), I7.39(316), A7.40(317), L7.41(318), G7.42(319), Y7.43(320), and N7.49(326) of kappaOR are on the water-accessible surface of the binding pockets. Combining the SCAM data with rhodopsin-based molecular models of the receptors led to the following conclusions. (i) The residues of the extracellular portion of TM7 predicted to face TM1 are sensitive to MTSEA in kappaOR but are not in deltaOR. Thus, TM1 may be closer to TM7 in deltaOR than in kappaOR. (ii) MTSEA-sensitive mutants start at position 7.31(296) in deltaOR and at 7.34(311) in kappaOR, suggesting that TM7 in deltaOR may have an additional helical turn (from 7.30 to 7.33). (iii) There is a conserved hydrogen-bond network linking D2.50 of the NLxxxD motif in TM2 with W6.48 of the CWxP motif in TM6. (iv) The NPxxY motif in TM7 interacts with TM2, TM6, and helix 8 to maintain receptors in inactive states. To the best of our knowledge, this represents the first such comparison of the structures of two highly homologous GPCRs.
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http://dx.doi.org/10.1021/bi050938aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2688720PMC
December 2005

A three-dimensional pharmacophore model for 5-hydroxytryptamine6 (5-HT6) receptor antagonists.

J Med Chem 2005 Jun;48(13):4216-9

Departamento de Química Orgánica I, Facultad de Ciencias Químicas, Universidad Complutense, E-28040 Madrid, Spain.

Forty-five structurally diverse 5-hydroxytryptamine(6) receptor (5-HT(6)R) antagonists were selected to develop a 3D pharmacophore model with the Catalyst software. The structural features for antagonism at this receptor are a positive ionizable atom interacting with Asp(3.32), a hydrogen bond acceptor group interacting with Ser(5.43) and Asn(6.55), a hydrophobic site interacting with residues in a hydrophobic pocket between transmembranes 3, 4, and 5, and an aromatic-ring hydrophobic site interacting with Phe(6.52).
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http://dx.doi.org/10.1021/jm050247cDOI Listing
June 2005

Synthesis and structure-activity relationships of a new model of arylpiperazines. 8. Computational simulation of ligand-receptor interaction of 5-HT(1A)R agonists with selectivity over alpha1-adrenoceptors.

J Med Chem 2005 Apr;48(7):2548-58

Departamento de Química Orgánica I, Facultad de Ciencias Químicas, Universidad Complutense, E-28040 Madrid, Spain.

We have designed and synthesized a new series of arylpiperazines V exhibiting high 5-HT(1A)R affinity and selectivity over alpha(1)-adrenoceptors. The new selective 5-HT(1A)R ligands contain a hydantoin (m = 0) or diketopiperazine (m = 1) moiety and an arylpiperazine moiety separated by one methylene unit (n = 1). The aryl substituent of the piperazine moiety (Ar) consists of different benzofused rings mimicking the favorable voluminous substituents at ortho and meta positions predicted by 3D-QSAR analysis in the previously reported series I. In particular, (S)-2-[[4-(naphth-1-yl)piperazin-1-yl]methyl]-1,4-dioxoperhydropyrrolo[1,2-a]pyrazine [(S)-9, CSP-2503] (5-HT(1A), K(i) = 4.1 nM; alpha(1), K(i) > 1000 nM) has been pharmacologically characterized as a 5-HT(1A)R agonist at somatodendritic and postsynaptic sites, endowed with anxiolytic properties. Ligand (S)-9 is predicted, in computer simulations, to bind Asp(3.32) in TMH 3, Thr(5.39) and Ser(5.42) in TMH 5, and Trp(6.48) in TMH 6. We propose that agonists modify, by means of an explicit hydrogen bond, the conformation of Trp(6.48) from pointing toward TMH 7, in the inactive gauche+ conformation, to pointing toward the ligand binding site, in the active trans conformation.
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http://dx.doi.org/10.1021/jm048999eDOI Listing
April 2005

SAR studies of dihydro-beta-agarofuran sesquiterpenes as inhibitors of the multidrug-resistance phenotype in a Leishmania tropica line overexpressing a P-glycoprotein-like transporter.

J Med Chem 2004 Jan;47(3):576-87

Instituto de Parasitología y Biomedicina "López-Neyra", Consejo Superior de Investigaciones Científicas, c/Ventanilla 11, 18001 Granada, Spain.

A series of dihydro-beta-agarofuran sesquiterpenes isolated from the leaves of Maytenus cuzcoina (1-10) or semisynthetic derivatives (11-30) have been tested on a multidrug-resistant Leishmania tropica line overexpressing a P-glycoprotein-like transporter to determine their ability to revert the resistance phenotype and to modulate intracellular drug accumulation. Almost all natural compounds showed potent reversal activity with different degrees of selectivity. Compounds 2, 7, and 8 are the most effective reversal agents tested against the multidrug resistance phenotype of Leishmania. Three-dimensional quantitative structure-activity relationships using the comparative molecular similarity indices analysis (CoMSIA) were employed to characterize the steric (contribution of 5.4%), electrostatic (58.9%), lipophilic (10.0%), and hydrogen-bond-donor (13.3%) and -acceptor (7.5%) requirements of these sesquiterpenes as modulators at the P-glycoprotein-like transporter. The most salient features of these requirements are the H-bond interaction between the substituents at the C-2 and C-6 positions with the receptor.
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http://dx.doi.org/10.1021/jm0309699DOI Listing
January 2004