Visceral leishmaniasis treatment: What do we have, what do we need and how to deliver it?

AbstractLeishmaniasis is one of the most neglected tropical disease in terms of drug discovery and development. Most antileishmanial drugs are highly toxic, present resistance issues or require hospitalization, being therefore not adequate to the field. Recently improvements have been achieved by combination therapy, reducing the time and cost of treatment. Nonetheless, new drugs are still urgently needed.In this review, we describe the current visceral leishmaniasis (VL) treatments and their limitations. We also discuss the new strategies in the drug discovery field including the development and implementation of high-throughput screening (HTS) assays and the joint efforts of international teams to deliver clinical candidates

Nitroheterocyclic compounds are more efficacious than CYP51 inhibitors against Trypanosoma cruzi: implications for Chagas disease drug discovery and development

Advocacy for better drugs and access to treatment has boosted the interest in drug discovery and development for Chagas disease, a chronic infection caused by the genetically heterogeneous parasite, Trypanosoma cruzi. in this work new in vitro assays were used to gain a better understanding of the antitrypanosomal properties of the most advanced antichagasic lead and clinical compounds, the nitroheterocyclics benznidazole, nifurtimox and fexinidazole sulfone, the oxaborole AN4169, and four ergosterol biosynthesis inhibitors -posaconazole, ravuconazole, EPL-BS967 and EPL-BS1246. Two types of assays were developed: one for evaluation of potency and efficacy in dose-response against a panel of T. cruzi stocks representing all current discrete typing units (DTUs), and a time-kill assay. Although less potent, the nitroheterocyclics and the oxaborole showed broad efficacy against all T. cruzi tested and were rapidly trypanocidal, whilst ergosterol biosynthesis inhibitors showed variable activity that was both compoundand strain-specific, and were unable to eradicate intracellular infection even after 7 days of continuous compound exposure at most efficacious concentrations. These findings contest previous reports of variable responses to nitroderivatives among different T. cruzi strains and further challenge the introduction of ergosterol biosynthesis inhibitors as new single chemotherapeutic agents for the treatment of Chagas disease.DNDiInstitut Pasteur Korea (IPK)Reconstruction Credit Institution-Federal Ministry of Education and Research (KfW-BMBF)/GermanyMedecins Sans Frontieres (Doctors without Borders)/InternationalKorean government (MSIP), Gyeonggi-doKISTIInst Pasteur Korea, Ctr Neglected Dis Drug Discovery CND3, Songnam, South KoreaCtr Nacl Pesquisa Energia & Mat, Lab Nacl Biociencias LNBio, Campinas, SP, BrazilUniversidade Federal de São Paulo UNIFESP, Depto Microbiol Imunol & Parasitol, São Paulo, BrazilDrugs Neglected Dis Initiat DNDi, Geneva, SwitzerlandUniversidade Federal de São Paulo UNIFESP, Depto Microbiol Imunol & Parasitol, São Paulo, BrazilKorean government (MSIP), Gyeonggi-do: 2007-00559Web of Scienc

Current and future chemotherapy for Chagas disease

Luís Gaspar is thankful to FCT for funding (scholarship reference: SFRH/BD/81604/2011). The research leading to these results has received funding from the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED) and No. 603240 (Project NMTrypI).American trypanosomiasis, commonly called Chagas disease, is one of the most neglected illnesses in the world and remains one of the most prevalent chronic infectious diseases of Latin America with thousands of new cases every year. The only treatments available have been introduced five decades ago. They have serious, undesirable side effects and disputed benefits in the chronic stage of the disease – a characteristic and debilitating cardiomyopathy and/or megavisceras. Several laboratories have therefore focused their efforts in finding better drugs. Although recent years have brought new clinical trials, these are few and lack diversity in terms of drug mechanism of action, thus resulting in a weak drug discovery pipeline. This fragility has been recently exposed by the failure of two candidates, posaconazole and E1224, to sterilely cure patients in phase 2 clinical trials. Such setbacks highlight the need for continuous, novel and high quality drug discovery and development efforts to discover better and safer treatments. In this article we will review past and current findings on drug discovery for Trypanosoma cruzi made by academic research groups, industry and other research organizations over the last half century. We will also analyze the current research landscape that is now better placed than ever to deliver alternative treatments for Chagas disease in the near futurePostprintPeer reviewe

Telomeric Heterochromatin Propagation and Histone Acetylation Control Mutually Exclusive Expression of Antigenic Variation Genes in Malaria Parasites

SummaryMalaria parasites use antigenic variation to avoid immune clearance and increase the duration of infection in the human host. Variation at the surface of P. falciparum-infected erythrocytes is mediated by the differential control of a family of surface antigens encoded by var genes. Switching of var gene expression occurs in situ, mostly from telomere-associated loci, without detectable DNA alterations, suggesting that it is controlled by chromatin structure. We have identified chromatin modifications at telomeres that spread far into telomere-proximal regions, including var gene loci (>50 kb). One type of modification is mediated by a protein homologous to yeast Sir2 called PfSir2, which forms a chromosomal gradient of heterochromatin structure and histone hypoacetylation. Upon activation of a specific telomere-associated var gene, PfSir2 is removed from the promoter region and acetylation of histone occurs. Our data demonstrate that mutually exclusive transcription of var genes is linked to the dynamic remodeling of chromatin

Amazonian plant natural products:perspectives for discovery of new antimalarial drug leads

Plasmodium falciparum and P. vivax malaria parasites are now resistant, or showing signs of resistance, to most drugs used in therapy. Novel chemical entities that exhibit new mechanisms of antiplasmodial action are needed. New antimalarials that block transmission of Plasmodium spp. from humans to Anopheles mosquito vectors are key to malaria eradication efforts. Although P. vivax causes a considerable number of malaria cases, its importance has for long been neglected. Vivax malaria can cause severe manifestations and death; hence there is a need for P. vivax-directed research. Plants used in traditional medicine, namely Artemisia annua and Cinchona spp. are the sources of the antimalarial natural products artemisinin and quinine, respectively. Based on these compounds, semi-synthetic artemisinin-derivatives and synthetic quinoline antimalarials have been developed and are the most important drugs in the current therapeutic arsenal for combating malaria. In the Amazon region, where P. vivax predominates, there is a local tradition of using plant-derived preparations to treat malaria. Here, we review the current P. falciparum and P. vivax drug-sensitivity assays, focusing on challenges and perspectives of drug discovery for P. vivax, including tests against hypnozoites. We also present the latest findings of our group and others on the antiplasmodial and antimalarial chemical components from Amazonian plants that may be potential drug leads against malaria

An Essential Nuclear Protein in Trypanosomes Is a Component of mRNA Transcription/Export Pathway

In eukaryotic cells, different RNA species are exported from the nucleus via specialized pathways. The mRNA export machinery is highly integrated with mRNA processing, and includes a different set of nuclear transport adaptors as well as other mRNA binding proteins, RNA helicases, and NPC-associated proteins. The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease, a widespread and neglected human disease which is endemic to Latin America. Gene expression in Trypanosoma has unique characteristics, such as constitutive polycistronic transcription of protein-encoding genes and mRNA processing by trans-splicing. In general, post-transcriptional events are the major points for regulation of gene expression in these parasites. However, the export pathway of mRNA from the nucleus is poorly understood. The present study investigated the function of TcSub2, which is a highly conserved protein ortholog to Sub2/ UAP56, a component of the Transcription/Export (TREX) multiprotein complex connecting transcription with mRNA export in yeast/human. Similar to its orthologs, TcSub2 is a nuclear protein, localized in dispersed foci all over the nuclei —except the fibrillar center of nucleolus— and at the interface between dense and non-dense chromatin areas, proposing the association of TcSub2 with transcription/processing sites. These findings were analyzed further by BrUTP incorporation assays and confirmed that TcSub2 is physically associated with active RNA polymerase II (RNA pol II), but not RNA polymerase I (RNA pol I) or Spliced Leader (SL) transcription, demonstrating participation particularly in nuclear mRNA metabolism in T. cruzi. The double knockout of the TcSub2 gene is lethal in T. cruzi, suggesting it has an essential function. Alternatively, RNA interference assays were performed in Trypanosoma brucei. It allowed demonstrating that besides being an essential protein, its knockdown causes mRNA accumulation in the nucleus and decrease of translation levels, reinforcing that Trypanosoma-Sub2 (Tryp-Sub2) is a component of mRNA transcription/export pathway in trypanosomes

Chroman-4-One Derivatives Targeting Pteridine Reductase 1 and Showing Anti-Parasitic Activity

Flavonoids have previously been identified as antiparasitic agents and pteridine reductase 1 (PTR1) inhibitors. Herein, we focus our attention on the chroman-4-one scaffold. Three chroman-4-one analogues (1-3) of previously published chromen-4-one derivatives were synthesized and biologically evaluated against parasitic enzymes (Trypanosoma brucei PTR1-TbPTR1 and Leishmania major-LmPTR1) and parasites (Trypanosoma brucei and Leishmania infantum). A crystal structure of TbPTR1 in complex with compound 1 and the first crystal structures of LmPTR1-flavanone complexes (compounds 1 and 3) were solved. The inhibitory activity of the chroman-4-one and chromen-4-one derivatives was explained by comparison of observed and predicted binding modes of the compounds. Compound 1 showed activity both against the targeted enzymes and the parasites with a selectivity index greater than 7 and a low toxicity. Our results provide a basis for further scaffold optimization and structure-based drug design aimed at the identification of potent anti-trypanosomatidic compounds targeting multiple PTR1 variants

Automated Nuclear Analysis of Leishmania major Telomeric Clusters Reveals Changes in Their Organization during the Parasite's Life Cycle

Parasite virulence genes are usually associated with telomeres. The clustering of the telomeres, together with their particular spatial distribution in the nucleus of human parasites such as Plasmodium falciparum and Trypanosoma brucei, has been suggested to play a role in facilitating ectopic recombination and in the emergence of new antigenic variants. Leishmania parasites, as well as other trypanosomes, have unusual gene expression characteristics, such as polycistronic and constitutive transcription of protein-coding genes. Leishmania subtelomeric regions are even more unique because unlike these regions in other trypanosomes they are devoid of virulence genes. Given these peculiarities of Leishmania, we sought to investigate how telomeres are organized in the nucleus of Leishmania major parasites at both the human and insect stages of their life cycle. We developed a new automated and precise method for identifying telomere position in the three-dimensional space of the nucleus, and we found that the telomeres are organized in clusters present in similar numbers in both the human and insect stages. While the number of clusters remained the same, their distribution differed between the two stages. The telomeric clusters were found more concentrated near the center of the nucleus in the human stage than in the insect stage suggesting reorganization during the parasite's differentiation process between the two hosts. These data provide the first 3D analysis of Leishmania telomere organization. The possible biological implications of these findings are discussed

Exploiting the 2-Amino-1,3,4-thiadiazole Scaffold To Inhibit <i>Trypanosoma brucei </i>Pteridine Reductase in Support of Early-Stage Drug Discovery

Pteridine reductase-1 (PTR1) is a promising drug target for the treatment of trypanosomiasis. We investigated the potential of a previously identified class of thiadiazole inhibitors of Leishmania major PTR1 for activity against Trypanosoma brucei (Tb). We solved crystal structures of several TbPTR1-inhibitor complexes to guide the structure-based design of new thiadiazole derivatives. Subsequent synthesis and enzyme- and cell-based assays confirm new, mid-micromolar inhibitors of TbPTR1 with low toxicity. In particular, compound 4m, a biphenyl-thiadiazole-2,5-diamine with IC50 = 16 μM, was able to potentiate the antitrypanosomal activity of the dihydrofolate reductase inhibitor methotrexate (MTX) with a 4.1-fold decrease of the EC50 value. In addition, the antiparasitic activity of the combination of 4m and MTX was reversed by addition of folic acid. By adopting an efficient hit discovery platform, we demonstrate, using the 2-amino-1,3,4-thiadiazole scaffold, how a promising tool for the development of anti-T. brucei agents can be obtained

Visual Genome-Wide RNAi Screening to Identify Human Host Factors Required for Trypanosoma cruzi Infection

The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy