Carolina Carrillo

Computer-guided drug repurposing: Identification of trypanocidal activity of clofazimine, benidipine and saquinavir

In spite of remarkable advances in the knowledge on Trypanosoma cruzi biology, no medications to treat Chagas disease have been approved in the last 40 years and almost 8 million people remain infected. Since the public sector and non-profit organizations play a significant role in the research efforts on Chagas disease, it is important to implement research strategies that promote translation of basic research into the clinical practice. Recent international public-private initiatives address the potential of drug repositioning (i.e. finding second or further medical uses for known-medications) which can substantially improve the success at clinical trials and the innovation in the pharmaceutical field. In this work, we present the computer-aided identification of approved drugs clofazimine, benidipine and saquinavir as potential trypanocidal compounds and test their effects at biochemical as much as cellular level on different parasite stages. According to the obtained results, we discuss biopharmaceutical, toxicological and physiopathological criteria applied to decide to move clofazimine and benidipine into preclinical phase, in an acute model of infection. The article illustrates the potential of computer-guided drug repositioning to integrate and optimize drug discovery and preclinical development; it also proposes rational rules to select which among repositioned candidates should advance to investigational drug status and offers a new insight on clofazimine and benidipine as candidate treatments for Chagas disease. One Sentence Summary: We present the computer-guided drug repositioning of three approved drugs as potential new treatments for Chagas disease, integrating computer-aided drug screening and biochemical, cellular and preclinical tests.

Application of Computer-Aided Drug Repurposing in the Search of New Cruzipain Inhibitors: Discovery of Amiodarone and Bromocriptine Inhibitory Effects

Cruzipain (Cz) is the major cystein protease of the protozoan Trypanosoma cruzi , etiological agent of Chagas disease. From a 163 compound data set, a 2D-classifier capable of identifying Cz inhibitors was obtained and applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. Fifty-four approved drugs were selected as candidates, four of which were acquired and tested on Cz and T. cruzi epimastigotes. Among them, the antiparkinsonian and antidiabetic drug bromocriptine and the antiarrhythmic amiodarone showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.

Polyamine depletion inhibits the autophagic response modulating Trypanosoma cruzi infectivity

Autophagy is a cell process that in normal conditions serves to recycle cytoplasmic components and aged or damaged organelles. The autophagic pathway has been implicated in many physiological and pathological situations, even during the course of infection by intracellular pathogens. Many compounds are currently used to positively or negatively modulate the autophagic response. Recently it was demonstrated that the polyamine spermidine is a physiological inducer of autophagy in eukaryotic cells. We have previously shown that the etiological agent of Chagas disease, the protozoan parasite Trypanosoma cruzi, interacts with autophagic compartments during host cell invasion and that preactivation of autophagy significantly increases host cell colonization by this parasite. In the present report we have analyzed the effect of polyamine depletion on the autophagic response of the host cell and on T. cruzi infectivity. Our data showed that depleting intracellular polyamines by inhibiting the biosynthetic enzyme ornithine decarboxylase with difluoromethylornithine (DFMO) suppressed the induction of autophagy in response to starvation or rapamycin treatment in two cell lines. This effect was associated with a decrease in the levels of LC3 and ATG5, two proteins required for autophagosome formation. As a consequence of inhibiting host cell autophagy, DFMO impaired T. cruzi colonization, indicating that polyamines and autophagy facilitate parasite infection. Thus, our results point to DFMO as a novel autophagy inhibitor. While other autophagy inhibitors such as wortmannin and 3-methyladenine are nonspecific and potentially toxic, DFMO is an FDA-approved drug that may have value in limiting autophagy and the spread of the infection in Chagas disease and possibly other pathological settings.

Trypanosoma cruzi Coexpressing Ornithine Decarboxylase and Green Fluorescence Proteins as a Tool to Study the Role of Polyamines in Chagas Disease Pathology.

Polyamines are essential for Trypanosoma cruzi, the causative agent of Chagas disease. As T. cruzi behaves as a natural auxotrophic organism, it relies on host polyamines biosynthesis. In this paper we obtained a double-transfected T. cruzi parasite that expresses the green fluorescent protein (GFP) and a heterologous ornithine decarboxylase (ODC), used itself as a novel selectable marker. These autotrophic and fluorescent parasites were characterized; the ODC presented an apparent Km for ornithine of 0.51 ± 0.16 mM and an estimated Vmax value of 476.2 nmoles/h/mg of protein. These expressing ODC parasites showed higher metacyclogenesis capacity than the auxotrophic counterpart, supporting the idea that polyamines are engaged in this process. This double-transfected T. cruzi parasite results in a powerful tool—easy to follow by its fluorescence—to study the role of polyamines in Chagas disease pathology and in related processes such as parasite survival, invasion, proliferation, metacyclogenesis, and tissue spreading.

Trypanosoma cruzi Polyamine Transporter: Its Role on Parasite Growth and Survival Under Stress Conditions

Trypanosoma cruzi is the etiological agent of Chagas disease, a major health problem in Latin America. Polyamines are polycationic compounds that play a critical role as regulators of cell growth and differentiation. In contrast with other protozoa, T. cruzi is auxotrophic for polyamines because of its inability to synthesize putrescine due to the lack of both, arginine and ornithine decarboxylase; therefore, the intracellular availability of polyamines depends exclusively on transport processes. In this work, the polyamine transporter TcPAT12 was overexpressed in T. cruzi epimastigotes demonstrating that growth rates at different concentrations of polyamines strongly depend on the regulation of the polyamine transport. In addition, parasites overexpressing TcPAT12 showed a highly increased resistance to hydrogen peroxide and the trypanocidal drugs nifurtimox and benznidazole, which act by oxidative stress and interfering the synthesis of polyamine derivatives, respectively. Finally, the presence of putative polyamine transporters was analyzed in T. cruzi, Trypanosoma brucei, and Leishmania major genomes identifying 3–6 genes in these trypanosomatids.

High-throughput Drug Repositioning for the Discovery of New Treatments for Chagas Disease

Despite affecting around 8 million people worldwide and representing an economic burden above

Identification of Levothyroxine Antichagasic Activity through Computer-Aided Drug Repurposing

7 billion/ year, currently approved medications to treat Chagas disease are still limited to two drugs, nifurtimox and benznidazole, which were developed more than 40 years ago and present important efficacy and safety limitations. Drug repositioning (i.e. finding second or further therapeutic indications for known drugs) has raised considerable interest within the international drug development community. There are many explanations to the current interest on drug repositioning including the possibility to partially circumvent clinical trials and the consequent saving in time and resources. It has been suggested as a particular attractive approach for the development of novel therapeutics for neglected diseases, which are usually driven by public or non-profit organizations. Here we review current computer-guided approaches to drug repositioning and reports on drug repositioning stories oriented to Chagas disease, with a focus on computer-guided drug repositioning campaigns.

Neglected Tropical Protozoan Diseases: Drug Repositioning as a Rational Option.

Cruzipain (Cz) is the major cysteine protease of the protozoan Trypanosoma cruzi, etiological agent of Chagas disease. A conformation-independent classifier capable of identifying Cz inhibitors was derived from a 163-compound dataset and later applied in a virtual screening campaign on the DrugBank database, which compiles FDA-approved and investigational drugs. 54 approved drugs were selected as candidates, 3 of which were acquired and tested on Cz and T. cruzi epimastigotes proliferation. Among them, levothyroxine, traditionally used in hormone replacement therapy in patients with hypothyroidism, showed dose-dependent inhibition of Cz and antiproliferative activity on the parasite.

Discovery of novel polyamine analogs with anti-protozoal activity by computer guided drug repositioning

Neglected tropical diseases represent a major sanitary problem and a huge economic burden to endemic countries, and are currently expanding to non-endemic countries owing to migration currents. Though long abandoned in the past, recent research on novel therapeutics has already started to show results. Drug repositioning is one of the prominent, more successful strategies to approach the development of new treatments for these diseases. Here we present an overview on the limitations of the current available medications to treat African trypanosomiasis, Chagas disease and Leishmaniasis, along with a review on drug candidates presently undergoing clinical trials and drug candidates identified through drug repositioning initiatives.

The regulation of autophagy differentially affects Trypanosoma cruzi metacyclogenesis

Chagas disease is a parasitic infection caused by the protozoa Trypanosoma cruzi that affects about 6 million people in Latin America. Despite its sanitary importance, there are currently only two drugs available for treatment: benznidazole and nifurtimox, both exhibiting serious adverse effects and limited efficacy in the chronic stage of the disease. Polyamines are ubiquitous to all living organisms where they participate in multiple basic functions such as biosynthesis of nucleic acids and proteins, proliferation and cell differentiation. T. cruzi is auxotroph for polyamines, which are taken up from the extracellular medium by efficient transporters and, to a large extent, incorporated into trypanothione (bis-glutathionylspermidine), the major redox cosubstrate of trypanosomatids. From a 268-compound database containing polyamine analogs with and without inhibitory effect on T. cruzi we have inferred classificatory models that were later applied in a virtual screening campaign to identify anti-trypanosomal compounds among drugs already used for other therapeutic indications (i.e. computer-guided drug repositioning) compiled in the DrugBank and Sweetlead databases. Five of the candidates identified with this strategy were evaluated in cellular models from different pathogenic trypanosomatids (T. cruzi wt, T. cruzi PAT12, T. brucei and Leishmania infantum), and in vitro models of aminoacid/polyamine transport assays and trypanothione synthetase inhibition assay. Triclabendazole, sertaconazole and paroxetine displayed inhibitory effects on the proliferation of T. cruzi (epimastigotes) and the uptake of putrescine by the parasite. They also interfered with the uptake of others aminoacids and the proliferation of infective T. brucei and L. infantum (promastigotes). Trypanothione synthetase was ruled out as molecular target for the anti-parasitic activity of these compounds.