Glicoproteína-P, resistência a múltiplas drogas (MDR) e relação estrutura-atividade de moduladores

Multidrug resistance, MDR is a major obstacle for cancer chemotherapy. MDR can be reversed by drugs that vary in their chemical structure and main biological activity. Many efforts have been done to overcome MDR based on studies of structure-activity relationships and in this review we summarize some aspects of MDR mediated by P-glycoprotein (P-gp), as the most experimentally and clinically tested form of drug resistance. The most significant MDR mechanisms revealed until now are shortly discussed. Physicochemical and structural properties of MDR modulators, measures of the MDR reversal, and QSAR studies are included.

3D-Pharmacophore mapping of thymidine-based inhibitors of TMPK as potential antituberculosis agents

Tuberculosis (TB) is the primary cause of mortality among infectious diseases. Mycobacterium tuberculosis monophosphate kinase (TMPKmt) is essential to DNA replication. Thus, this enzyme represents a promising target for developing new drugs against TB. In the present study, the receptor-independent, RI, 4D-QSAR method has been used to develop QSAR models and corresponding 3D-pharmacophores for a set of 81 thymidine analogues, and two corresponding subsets, reported as inhibitors of TMPKmt. The resulting optimized models are not only statistically significant with r (2) ranging from 0.83 to 0.92 and q (2) from 0.78 to 0.88, but also are robustly predictive based on test set predictions. The most and the least potent inhibitors in their respective postulated active conformations, derived from each of the models, were docked in the active site of the TMPKmt crystal structure. There is a solid consistency between the 3D-pharmacophore sites defined by the QSAR models and interactions with binding site residues. Moreover, the QSAR models provide insights regarding a probable mechanism of action of the analogues.

Rational Design and 3D-Pharmacophore Mapping of 5 `-Thiourea-Substituted alpha-Thymidine Analogues as Mycobacterial TMPK Inhibitors

Thymidine monophosphate kinase (TMPK) has emerged as an attractive target for developing inhibitors of Mycobacterium tuberculosis growth. In this study the receptor-independent (RI) 4D-QSAR formalism has been used to develop QSAR models and corresponding 3D-pharmacophores for a set of 5`-thiourea-substituted alpha-thymidine inhibitors. Models were developed for the entire training set and for a subset of the training set consisting of the most potent inhibitors. The optimized (RI) 4D-QSAR models are statistically significant (r(2) = 0.90, q(2) = 0.83 entire set, r(2) = 0.86, q(2) = 0.80 high potency subset) and also possess good predictivity based on test set predictions. The most and least potent inhibitors, in their respective postulated active conformations derived from the models, were docked in the active site of the TMPK crystallographic structure. There is a solid consistency between the 3D-pharmacophore sites defined by the QSAR models and interactions with binding site residues. This model identifies new regions of the inhibitors that contain pharmacophore sites, such as the sugar-pyrimidine ring structure and the region of the 5`-arylthiourea moiety. These new regions of the ligands can be further explored and possibly exploited to identify new, novel, and, perhaps, better antituberculosis inhibitors of TMPKmt. Furthermore, the 3D-pharmacophores defined by these models can be used as a starting point for future receptor-dependent antituberculosis drug design as well as to elucidate candidate sites for substituent addition to optimize ADMET properties of analog inhibitors.

Generation and Analysis of Interaction Energy Maps of p-Substituted Benzoic Acid [(5-Nitro-thiophen-2-yl)-methylene]-Hydrazides Active against MRSA

In this preliminary study eighteen p-substituted benzoic acid [(5-nitro-thiophen-2-yl)-methylene]-hydrazides with antimicrobial activity were evaluated against multidrug-resistant Staphylococcus aureus, correlating the three-dimensional characteristics of the ligands with their respective bioactivities. The computer programs Sybyl and CORINA were used, respectively, for the design and three-dimensional conversion of the ligands. Molecular interaction fields were calculated using GRID program. Calculations using Volsurf resulted in a statistically consistent model with 48 structural descriptors showing that hydrophobicity is a fundamental property in the analyzed biological response.

In vitro Antitumour Activity of Orsellinates

Lichen phenolic compounds exhibit antioxidant, antimicrobial, antiproliferative. and cytotoxic activities. The purpose of this study was to evaluate the anticancer activity of lecanoric acid, a secondary metabolite of the lichen Parmotrema tinctorum, and its derivatives, orsellinates, obtained by structural modification. A cytotoxicity assay was carried out hi vitro with sulforhodamine B (SRB) using HEp-2 larynx carcinoma, MCF7 breast carcinoma, 786-0 kidney carcinoma, and B16-F10 murine melanoma cell lines, in addition to a normal (Vero) cell line in order to calculate the selectivity index of the compounds. n-Butyl orsellinate was the most active compound, with IC(50) Values (the concentration that inhibits 50% of growth) ranging from 7.2 to 14.0 mu g/ml, against all the cell lines tested. The compound was more active (IC(50), = 11.4 mu g/mL) against B16-F10 cells than was cisplatin (12.5 mu g/mL). Conversely, lecanoric acid and methyl orsellinate were less active against all cell lines, having an IC(50) value higher than 50 mu g/mL. Ethyl orsellinate was more active against HEp-2 than against MCF7, 786-0, or B16-F10 cells. The same pattern was observed for n-propyl and n-butyl orsellinates. n-Pentyl orsellinate was less active than n-propyl or n-butyl orsellinates against HEp-2 cells. The orsellinate activity increased with chain elongation (from methyl to n-butyl), a likely consequence of an increase in lipophilicity. The results revealed that the structural modification of lecanoric acid increases the cytotoxic activity of the derivatives tested.

Design, synthesis and biologival evaluation of novel isoniazid derivatives with potente antitubercular activity

The disturbing emergence of multidrug-resistant strains of Mycobacterium tuberculosis (Mtb) has been driving the scientific community to urgently search for new and efficient antitubercular drugs. Despite the various drugs currently under evaluation, isoniazid is still the key and most effective component in all multi-therapeutic regimens recommended by the WHO. This paper describes the QSAR-oriented design, synthesis and in vitro antitubercular activity of several potent isoniazid derivatives (isonicotinoyl hydrazones and isonicotinoyl hydrazides) against H37Rv and two resistant Mtb strains. QSAR studies entailed RFs and ASNNs classification models, as well as MLR models. Strict validation procedures were used to guarantee the models' robustness and predictive ability. Lipophilicity was shown not to be relevant to explain the activity of these derivatives, whereas shorter N-N distances and lengthy substituents lead to more active compounds. Compounds I, 2, 4, 5 and 6, showed measured activities against H37Rv higher than INH (i.e., MIC <= 0.28 mu M), while compound 9 exhibited a six fold decrease in MIC against the katG (S315T) mutated strain, by comparison with INH (Le., 6.9 vs. 43.8 mu M). All compounds were ineffective against H37Rv(INH) (Delta katG), a strain with a full deletion of the katG gene, thus corroborating the importance of KatG in the activation of INH-based compounds. The most potent compounds were also shown not to be cytotoxic up to a concentration 500 times higher than MIC. (C) 2014 Elsevier Masson SAS. All rights reserved.

Estudios de compuestos heterocíclicos y modificaciones químicas de membranas con fines farmacéuticos 4127

Teniendo en cuenta las propiedades bactericidas y tripanosas de algunos de los derivados sintetizados en nuestro laboratorio, se continuará con la obtención de nuevos derivados naftoquinónicos con sustituyentes hetericíclicos, derivados del isoxazol y del pirazol. A los compuestos obtenidos se le estudiarán sus propiedades físico-químicas y su estabilidad, tanto en solución como en estado sólido. Por otro lado se prepararán y caracterizarán membranas de polisacáridos naturales o semisintéticos con el objeto de obtener materiales farmacéuticos con propiedades especiales. Los estudios comprenderán: I. Síntesis de nuevos análogos nitrogenados de naftoquinonas. Se obtendrán derivados con sustituyentes carboxílicos, y con la posterior formación de sales se intentarán mejorar las propiedades hidrofílicas de derivados de isoxazolilnaftoquinonas. Se continuará con la síntesis de las pirazolilnaftoquinonas. II. Estabilidad de isoxazolilnaftoquinonas. Se realizarán estudios hidrolíticos, utilizando espectrofotometría UV-visible y cromatografía líquida de alta presión, y térmicos, por DSC, TG y DTA, a diversas isoxazolilnaftoquinonas con finalidad de establecer las mejores condiciones para su formulación. III. Se aplicarán los conceptos de QSAR a fin de establecer los parámetros que mejor se correlacionen con la actividad biológica. También se determinarán las modificaciones moleculares que incrementen la actividad biológica mediante cálculos utilizando métodos semiempíricos. IV. Preparación de membranas asimétricas de polisacáridos naturales o semisintéticos y sus modificaciones químicas. Estas investigaciones comprenderán cuatro etapas: 1- preparación de membranas; 2- reacciones de superficie; 3- estudios de liberación de los principios activos en diferentes medios; 4- caracterización de las membranas.

Síntesis de derivados nucleosídicos con potencial actividad antiviral

La infección por el Virus de Inmunodeficiencia Humana tipo 1 (HIV-1), agente causal del Síndrome de Inmunodeficiencia Adquirida (SIDA) está descripta como la epidemia más importante de los '90 y tal vez del siglo. A pesar de los esfuerzos a nivel mundial, no ha sido exitosa la obtención de compuestos efectivos para la profilaxis y tratamiento del SIDA con un índice terapéutico óptimo. Dado que el HIV-1 es un retrovirus, la transcriptasa reversa se ha convertido en un objetivo importante en la quimioterapia de esta enfermedad. Clínicamente se utiliza como inhibidor de dicha enzima, la zidovudina (3'-azido-3'-deoxitimidina, AZT, 1) y como drogas alternativas en pacientes infectados con el virus HIV, que no responden a la monoterapia con AZT, la zalcitabina (2',3'-dideoxicitidina, ddC, 2) y la didanosina (2',3'-dideoxiinosina, ddl, 3). Estos nucleósidos, al igual que el AZT, actúan como finalizadores de la cadena proviral e inhibidores competitivos de la transcriptasa reversa. Nuestro grupo de trabajo está abocado a la búsqueda de nuevas alternativas terapéuticas y, por lo tanto, encaró la síntesis de análogos de AZT por introducción de halógenos (3'-azido-6-bromo-3'-deoxitimidina, AZT-Br) y por asociación molecular con derivados del ácido isonicotínico (3'-azido-3'-deoxi-5'-O-isonicotinoiltimidina, AZT-ISO). Estos compuestos demostraron poseer una muy importante actividad biológica frente a HIV-1 y una moderada (pero significativa desde el punto de vista terapéutico) actividad frente a infecciones bacterianas. Basándonos en estos alentadores resultados hemos continuado la síntesis de derivadores de AZT con la finalidad de realizar estudios sobre Relación Estructura-Actividad Biológica (QSAR), para lo cual es necesario tener una base de datos lo más completa posible. El trabajo tiene como objetivo la obtención de nuevos fármacos que modulen la conducta farmacocinética de los principales agentes utilizados actualmente en la terapia contra el Virus de la Inmunodeficiencia Humana (AZT, ddC, ddl). Para tal fin, planificamos la síntesis de derivados de AZT (1), ddC (2) y ddl (3), por modificación de la posición-5'. Dicha modificación favorecerá la farmacocinética de esas drogas ya que puede disminuir la glucoronidación de la posición-5' y, por lo tanto, la velocidad de metabolización. Además, dado que actualmente la terapia para el SIDA involucra el tratamiento de las infecciones oportunistas (principalmente la tuberculosis), se propone la asociación de 1-3 con ácido p-aminosalicílico (PAS, 4).

Diseño y Desarrollo de Drogas y Medicamentos Huérfanos

Nuestro grupo de investigación trabaja desde hace unos años en plantear soluciones a problemas de medicamentos con conflictos de disponibilidad. Esta situación generalmente está asocada a las enfermedades huérfanas (raras y olvidadas). Desde la investigación básica y aplicada se intenta lograr una interacción entre la Farmacoquímica y la Farmacoepidemiología, estrategia que justamente usa la industria farmacéutica cuando quiere desarrollar un nuevo medicamento. Esto nos ha permitido consolidar un grupo de investigación con la experiencia suficiente para encarar actividades de campo y experimentales. Para los primeros se utilizan métodos descriptivos cualitativos y cuantitativos, de observación y de intervención. Se trabaja en colaboración con los 14 Hospitales Públicos de la ciudad de Córdoba y con algunas clínicas privadas (6), con ANMAT y con las bases de datos de Agencias Oficiales de Medicamentos de países de referencia (EEUU y Europa), entre otros. A continuación, se analizan los factores que causan la “orfandad” de los medicamentos, la significancia clínica y el impacto sanitario, social y económico originado por su falta de disponibilidad. Respecto al diseño y desarrollo de nuevas entidades químicas farmacológicas, se decidió enfocar el estudio en fármacos para enfermedades huérfanas, principalmente las parasitarias (malaria, Chagas, leishmaniasis). Estas enfermedades son raras en países desarrollados y olvidadas o desatendidas en países pobres o menos desarrollados como el nuestro. En este sentido, se utilizan las metodologías propias de la Química Medicinal, como son el descubrimiento y optimización de prototipos. Se recurre a las estrategias más racionales actualmente utilizadas, como son el diseño y preparación de una quimioteca focalizada, su evaluación biológica, la identificación de líderes y su optimización utilizando la farmacomodulación, el diseño directo e indirecto asistido por computadoras y el estudio de las REA, QSAR y 3D-QSAR. La quimioteca actual está compuesta de derivados bencenosulfonilos de heterocíclicos, y cuenta actualmente con 105 compuestos, muchos de los cuales demostraron actividad antiparasitaria. La quimioteca se la diseñó utilizando la estrategia denominada "diseño de fármacos basada en fragmentos". La hipótesis es que tanto la fracción bencenosulfonilo como la heterocíclica han probado ser bioactivas. El objetivo general del proyecto es contribuir al mejoramiento de la salud humana, al avance científico y tecnológico y a la formación de recursos humanos por medio del diseño y desarrollo de Drogas y Medicamentos Huérfanos. Proponemos los siguientes objetivos específicos: 1) Incrementar el número de compuestos de la quimioteca de N-bencenosulfonilos de heterociclos. 2) Evaluar la actividad antiparasitaria in vitro. 3) Estudiar las propiedades del estado sólido de los derivados. 4) Usar la información adquirida en los puntos 1-3 para estudios de cribado virtual y mejorar la solubilidad de los compuestos seleccionados. 4) Continuar con los estudios farmacoepidmiológicos sobre medicamentos huérfanos o no disponibles en nuestro país, que retroalimenta y complementa los estudios farmacoquímicos. El proyecto presenta un impacto científico y socio-económico importante, ya que abarca varios aspectos de la problemática de medicamentos no disponibles o huérfanos, desde el diagnóstico de la situación en nuestro medio, pasando por la identificación de nuevas entidades químicas y el desarrollo de posibles soluciones para su transferencia a la industria. Esto se debe a su enfoque original (en academias) al plantearse la retroalimentación entre la farmacoquímica y la farmacoepidemiología. Esta experiencia es muy estimulante, el contacto con los profesionales del equipo de salud enriquece el intercambio de opiniones y la consolidación de proyectos multidisciplinarios, permitiendo abordar el problema de un modo integral.

Obtenció de nous anàlegs amb activitat brassinoesteroide mitjançant modelització molecular i síntesi

Els brassinoesteroides són productes naturals que actuen com a potents reguladors del creixement vegetal. Presenten aplicacions prometedores en l’agricultura degut a que, aplicats exògenament, augmenten la qualitat i la quantitat de les collites. Ara bé, el seu ús s’ha vist restringit degut a la seva costosa obtenció. Aquest fet ha motivat la recerca de nous compostos actius més assequibles. En aquest projecte es planteja el disseny i obtenció de nous anàlegs seguint diferents estratègies que impliquen tant l’ús de mètodes de modelització molecular com de síntesi orgànica. La primera d’aquestes estratègies consisteix en buscar compostos actius en bases de dades de compostos comercials a través de processos de Virtual Screening desenvolupats amb mètodes computacionals basats en Camps d’Interacció Molecular. Així, es van establir i interpretar models de Relacions Quantitatives Estructura-Activitat (QSAR) emprant descriptors independents de l’alineament (GRIND) i, amb col•laboració amb la Universitat de Perugia, aquest criteri de cerca es va ampliar amb l’aplicació de descriptors FLAP de nova generació. Una altra estratègia es va basar en intentar substituir l’esquelet esteroide dels brassinoesteroides per una estructura equivalent, fixant com a cadena lateral el grup (R)-hexahidromandelil. S’han aplicat dos criteris: mètodes computacionals basats en models QSAR establerts amb descriptors GRIND i també en la metodologia SHOP (scaffold hopping), i, per altra banda, anàlegs proposats racionalment a partir d’un estudi efectuat sobre disruptors endocrins no esteroïdals. Sobre les estructures trobades s’hi va unir la cadena lateral comercial esmentada per via sintètica, en la qual s’ha hagut de fer un èmfasi especial en grups protectors. En total, 49 estructures es proposen per a ser obtingudes sintèticament. També s’ha treballat en l’obtenció un agonista derivat de l’hipotètic antagonista KM-01. Totes les molècules candidates, ja siguin comercials o obtingudes sintèticament, estant sent avaluades en el test d’inclinació de la làmina d’arròs (RLIT).

Comparison of indices proposed as criteria for assigning skin notation

OBJECTIVES: Skin notations are used as a hazard identification tool to flag chemicals associated with a potential risk related to transdermal penetration. The transparency and rigorousness of the skin notation assignment process have recently been questioned. We compared different approaches proposed as criteria for these notations as a starting point for improving and systematizing current practice. METHODS: In this study, skin notations, dermal acute lethal dose 50 in mammals (LD(50)s) and two dermal risk indices derived from previously published work were compared using the lists of Swiss maximum allowable concentrations (MACs) and threshold limit values (TLVs) from the American Conference of Governmental Industrial Hygienists (ACGIH). The indices were both based on quantitative structure-activity relationship (QSAR) estimation of transdermal fluxes. One index compared the cumulative dose received through skin given specific exposure surface and duration to that received through lungs following inhalation 8 h at the MAC or TLV. The other index estimated the blood level increase caused by adding skin exposure to the inhalation route at kinetic steady state. Dermal-to-other route ratios of LD(50) were calculated as secondary indices of dermal penetrability. RESULTS: The working data set included 364 substances. Depending on the subdataset, agreement between the Swiss and ACGIH skin notations varied between 82 and 87%. Chemicals with a skin notation were more likely to have higher dermal risk indices and lower dermal LD(50) than chemicals without a notation (probabilities between 60 and 70%). The risk indices, based on cumulative dose and kinetic steady state, respectively, appeared proportional up to a constant independent of chemical-specific properties. They agreed well with dermal LD(50)s (Spearman correlation coefficients -0.42 to -0.43). Dermal-to-other routes LD(50) ratios were moderately associated with QSAR-based transdermal fluxes (Spearman correlation coefficients -0.2 to -0.3). CONCLUSIONS: The plausible but variable relationship between current skin notations and the different approaches tested confirm the need to improve current skin notations. QSAR-based risk indices and dermal toxicity data might be successfully integrated in a systematic alternative to current skin notations for detecting chemicals associated with potential dermal risk in the workplace. [Authors]

Descripció de propietats moleculars i activitats biològiques emprant l'energia de repulsió electró-electró

En aquest treball es descriu l'ús de les mesures de semblança molecular quàntica (MSMQ) per a caracteritzar propietats i activitats biològiques moleculars, i definir descriptors emprables per a construir models QSAR i QSPR. L'estudi que es presenta consisteix en la continuació d'un treball recent, on es descrivien relacions entre el paràmetre log P i MSMQ, donant així una alternativa a aquest parimetre hidrofòbic empíric. L'actual contribució presenta una nova mesura, capaç d'estendre l'ús de les MSMQ, que consisteix en l'energia de repulsió electró-electró (Vee). Aquest valor, disponible normalment a partir de programari de química quàntica, considera la molècula com una sola entitat, i no cal recórrer a l'ús decontribucions de fragments. La metodologia s'ha aplicat a cinc tipus diferents de compostos on diferents propietats moleculars i activitats biològiques s'han correlacionat amb Vee com a únic descriptor molecular. En tots els casos estudiats, s'han obtingut correlacions satisfactòries.

Internal test sets studies in a group of antimalarials

Topological indices have been applied to build QSAR models for a set of 20 antimalarial cyclic peroxy cetals. In order to evaluate the reliability of the proposed linear models leave-n-out and Internal Test Sets (ITS) approaches have been considered. The proposed procedure resulted in a robust and consensued prediction equation and here it is shown why it is superior to the employed standard cross-validation algorithms involving multilinear regression models

Prediction of cross-recognition of peptide-HLA A2 by Melan-A-specific cytotoxic T lymphocytes using three-dimensional quantitative structure-activity relationships.

The cross-recognition of peptides by cytotoxic T lymphocytes is a key element in immunology and in particular in peptide based immunotherapy. Here we develop three-dimensional (3D) quantitative structure-activity relationships (QSARs) to predict cross-recognition by Melan-A-specific cytotoxic T lymphocytes of peptides bound to HLA A*0201 (hereafter referred to as HLA A2). First, we predict the structure of a set of self- and pathogen-derived peptides bound to HLA A2 using a previously developed ab initio structure prediction approach [Fagerberg et al., J. Mol. Biol., 521-46 (2006)]. Second, shape and electrostatic energy calculations are performed on a 3D grid to produce similarity matrices which are combined with a genetic neural network method [So et al., J. Med. Chem., 4347-59 (1997)] to generate 3D-QSAR models. The models are extensively validated using several different approaches. During the model generation, the leave-one-out cross-validated correlation coefficient (q (2)) is used as the fitness criterion and all obtained models are evaluated based on their q (2) values. Moreover, the best model obtained for a partitioned data set is evaluated by its correlation coefficient (r = 0.92 for the external test set). The physical relevance of all models is tested using a functional dependence analysis and the robustness of the models obtained for the entire data set is confirmed using y-randomization. Finally, the validated models are tested for their utility in the setting of rational peptide design: their ability to discriminate between peptides that only contain side chain substitutions in a single secondary anchor position is evaluated. In addition, the predicted cross-recognition of the mono-substituted peptides is confirmed experimentally in chromium-release assays. These results underline the utility of 3D-QSARs in peptide mimetic design and suggest that the properties of the unbound epitope are sufficient to capture most of the information to determine the cross-recognition.

Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents.

The study reports a set of forty proteinogenic histidine-containing dipeptides as potential carbonyl quenchers. The peptides were chosen to cover as exhaustively as possible the accessible chemical space, and their quenching activities toward 4-hydroxy-2-nonenal (HNE) and pyridoxal were evaluated by HPLC analyses. The peptides were capped at the C-terminus as methyl esters or amides to favor their resistance to proteolysis and diastereoisomeric pairs were considered to reveal the influence of configuration on quenching. On average, the examined dipeptides are less active than the parent compound carnosine (βAla + His) thus emphasizing the unfavorable effect of the shortening of the βAla residue as confirmed by the control dipeptide Gly-His. Nevertheless, some peptides show promising activities toward HNE combined with a remarkable selectivity. The results emphasize the beneficial role of aromatic and positively charged residues, while negatively charged and H-bonding side chains show a detrimental effect on quenching. As a trend, ester derivatives are slightly more active than amides while heterochiral peptides are more active than their homochiral diastereoisomer. Overall, the results reveal that quenching activity strongly depends on conformational effects and vicinal residues (as evidenced by the reported QSAR analysis), offering insightful clues for the design of improved carbonyl quenchers and to rationalize the specific reactivity of histidine residues within proteins.