Raquel Álvarez

La leishmaniasis y tripanosomiasis (Enfermedad de Chagas y del Sueño), son zoonosis causadas por tripanosomátidos flagelados con elevada similitud filogenética, incluidas dentro de las enfermedades tropicales desatendidas. Por ello, la búsqueda de nuevos fármacos es fundamental. Este trabajo se centra en el diseño, síntesis y evaluación de compuestos que se unen a la tubulina parasitaria en el dominio de la colchicina. Los parásitos presentan diferentes sustituciones de aminoácidos en el sitio de unión a la colchicina con respecto a los mamíferos, lo que permite diseñar ligandos específicos que actúen frente a la tubulina parasitaria sin afectar al huésped. A partir de ligandos que han demostrado su potencial citotóxico frente a líneas tumorales, y las diferencias en el sitio de la colchicina, se ha desarrollado una familia de sulfonamidas diarílicas, que combinan un anillo de p-metoxifenilo con un anillo fenílico que incorpora sustituyentes polares como ácidos carboxílicos, ésteres o amidas en posición meta. Para la síntesis de los 18 compuestos obtenidos, se han realizado cinco tipos de reacciones diferentes y métodos de aislamiento y purificación determinados. Todos han sido caracterizados por resonancia magnética nuclear y espectroscopía de masas de alta resolución y han sido enviados para su ensayo frente a Leishmania infantum y Trypanosoma brucei.

Leishmaniasis is a group of neglected tropical diseases caused by at least 20 species of Leishmania protozoa, which are spread by the bite of infected sandflies. There are three main forms of the disease: cutaneous leishmaniasis (CL, the most common), visceral leishmaniasis (VL, also known as kala‐azar, the most serious), and mucocutaneous leishmaniasis. One billion people live in areas endemic to leishmaniasis, with an annual estimation of 30,000 new cases of VL and more than 1 million of CL. New treatments for leishmaniasis are an urgent need, as the existing ones are inefficient, toxic, and/or expensive. We have revised the experimental structure‐based drug design (SBDD) efforts applied to the discovery of new drugs against leishmaniasis. We have grouped the explored targets according to the metabolic pathways they belong to, and the key achieved advances are highlighted and evaluated. In most cases, SBDD studies follow high‐throughput screening campaigns and are secondary to pharmacokinetic optimization, due to the majoritarian belief that there are few validated targets for SBDD in leishmaniasis. However, some SBDD strategies have significantly contributed to new drug candidates against leishmaniasis and a bigger number holds promise for future development.

Antimitotic agents are one of the more successful types of anticancer drugs, but they suffer from toxicity and resistance. The application of approved drugs to new indications (i.e., drug repurposing) is a promising strategy for the development of new drugs. It relies on finding pattern similarities: drug effects to other drugs or conditions, similar toxicities, or structural similarity. Here, we recursively searched a database of approved drugs for structural similarity to several antimitotic agents binding to a specific site of tubulin, with the expectation of finding structures that could fit in it. These searches repeatedly retrieved frentizole, an approved nontoxic anti-inflammatory drug, thus indicating that it might behave as an antimitotic drug devoid of the undesired toxic effects. We also show that the usual repurposing approach to searching for targets of frentizole failed in most cases to find such a relationship. We synthesized frentizole and a series of analogs to assay them as antimitotic agents and found antiproliferative activity against HeLa tumor cells, inhibition of microtubule formation within cells, and arrest at the G₂/M phases of the cell cycle, phenotypes that agree with binding to tubulin as the mechanism of action. The docking studies suggest binding at the colchicine site in different modes. These results support the repurposing of frentizole for cancer treatment, especially for glioblastoma.

La Evaluación Clínica Objetiva y Estructurada (ECOE) es un tipo de examen de carácter práctico, orientado a valorar la adquisición de competencias profesionales del estudiante mediante la resolución de casos clínicos y habilidades, articulada en un circuito de estaciones o situaciones reales por las que los estudiantesrotarán de forma consecutiva en un tiempo determinado. Los resultados de su implementación en la asignatura de Prácticas tuteladas en el Grado en Farmacia de la Universidad de Salamanca confirman la idoneidad de este método de evaluación para medir la capacidad y habilidades de los futuros farmacéuticos. Los estudiantes encuentran adecuada la prueba, si bien muestran reticencias en cuanto a la novedad y al estrés que les supone enfrentarse a situaciones reales con un tiempo limitado, por lo que es necesario facilitarles recursos para su formación y entrenamiento.

Pan-Gyn cancers entail 1 in 5 cancer cases worldwide, breast cancer being the most commonly diagnosed and responsible for most cancer deaths in women. The high incidence and mortality of these malignancies, together with the handicaps of taxanes—first-line treatments—turn the development of alternative therapeutics into an urgency. Taxanes exhibit low water solubility that require formulations that involve side effects. These drugs are often associated with dose-limiting toxicities and with the appearance of multi-drug resistance (MDR). Here, we propose targeting tubulin with compounds directed to the colchicine site, as their smaller size offer pharmacokinetic advantages and make them less prone to MDR efflux. We have prepared 52 new Microtubule Destabilizing Sulfonamides (MDS) that mostly avoid MDR-mediated resistance and with improved aqueous solubility. The most potent compounds, N-methyl-N-(3,4,5-trimethoxyphenyl-4-methylaminobenzenesulfonamide 38, N-methyl-N-(3,4,5-trimethoxyphenyl-4-methoxy-3-aminobenzenesulfonamide 42, and N-benzyl-N-(3,4,5-trimethoxyphenyl-4-methoxy-3-aminobenzenesulfonamide 45 show nanomolar antiproliferative potencies against ovarian, breast, and cervix carcinoma cells, similar or even better than paclitaxel. Compounds behave as tubulin-binding agents, causing an evident disruption of the microtubule network, in vitro Tubulin Polymerization Inhibition (TPI), and mitotic catastrophe followed by apoptosis. Our results suggest that these novel MDS may be promising alternatives to taxane-based chemotherapy in chemoresistant Pan-Gyn cancers.

Colchicine site ligands suffer from low aqueous solubility due to the highly hydrophobic nature of the binding site. A new strategy for increasing molecular polarity without exposing polar groups—termed masked polar group incorporation (MPGI)—was devised and applied to nitrogenated combretastatin analogues. Bulky ortho substituents to the pyridine nitrogen hinder it from the hydrophobic pocket while increasing molecular polarity. The resulting analogues show improved aqueous solubilities and highly potent antiproliferative activity against several cancer cell lines of different origin. The more potent compounds showed moderate tubulin polymerization inhibitory activity, arrested the cell cycle of treated cells at the G₂/M phase, and subsequently caused apoptotic cell death represented by the cells gathered at the subG₀/G₁ population after 48 h of treatment. Annexin V/Propidium Iodide (PI) double-positive cells observed after 72 h confirmed the induction of apoptosis. Docking studies suggest binding at the colchicine site of tubulin in a similar way as combretastatin A4, with the polar groups masked by the vicinal substituents. These results validate the proposed strategy for the design of colchicine site ligands and open a new road to increasing the aqueous solubility of ligands binding in apolar environments.

Tubulin, the microtubules and their dynamic behavior are amongst the most successful antitumor, antifungal, antiparasitic, and herbicidal drug targets. Sulfonamides are exemplary drugs with applications in the clinic, in veterinary and in the agrochemical industry. This review summarizes the actual state and recent progress of both fields looking from the double point of view of the target and its drugs, with special focus onto the structural aspects. The article starts with a brief description of tubulin structure and its dynamic assembly and disassembly into microtubules and other polymers. Posttranslational modifications and the many cellular means of regulating and modulating tubulin’s biology are briefly presented in the tubulin code. Next, the structurally characterized drug binding sites, their occupying drugs and the effects they induce are described, emphasizing on the structural requirements for high potency, selectivity, and low toxicity. The second part starts with a summary of the favorable and highly tunable combination of physical–chemical and biological properties that render sulfonamides a prototypical example of privileged scaffolds with representatives in many therapeutic areas. A complete description of tubulin‐binding sulfonamides is provided, covering the different species and drug sites. Some of the antimitotic sulfonamides have met with very successful applications and others less so, thus illustrating the advances, limitations, and future perspectives of the field. All of them combine in a mechanism of action and a clinical outcome that conform efficient drugs.

A series of multinuclear heterometallic Cu–Zn complexes of molecular formula [(CuL)₂Zn(dca)₂] (1), [(CuL)₂Zn(NO₃)₂] (2), [(CuL)₂Zn₂(Cl)₄] (3), and [(CuL)₂Zn₂(NO₂)₄] (4) have been synthesized by reacting [CuL] as a “metalloligand (ML)” (where HL = N,N′-bis(5-chloro-2-hydroxybenzylidene)-2,2-dimethylpropane-1,3-diamine) and by varying the anions or coligands using the same molar ratios of the reactants. All of the four products including the ML have been characterized by infrared and UV–vis spectroscopies and elemental and single-crystal X-ray diffraction analyses. By varying the anions, different structures and topologies are obtained which we have tried to rationalize by means of thorough density functional theory calculations. All of the complexes (1–4) have now been applied for several biological investigations to verify their therapeutic worth. First, their cytotoxicity properties were assessed against HeLa human cervical carcinoma along with the determination of IC₅₀ values. The study was extended with extensive DNA and protein binding experiments followed by detailed fluorescence quenching study with suitable reagents to comprehend the mechanistic pathway. From all of these biological studies, it has been found that all of these heterometallic complexes show more than a few fold improvement of their therapeutic values as compared to the similar homometallic ones probably because of the simultaneous synergic effect of copper and zinc. Among all of the four heterometallic complexes, complex 3 exhibits highest binding constants and IC₅₀ values suggest for their better interaction toward the biological targets and hence have better clinical importance.