Activity of anti-cancer protein kinase inhibitors against Leishmania spp.

The protozoan parasite Leishmania donovani is the causative agent of visceral leishmaniasis, a disease potentially fatal if not treated. Current available treatments have major limitations, and new and safer drugs are urgently needed. In recent years, advances in high-throughput screening technologies have enabled the screening of millions of compounds to identify new antileishmanial agents. However, most of the compounds identified in vitro did not translate their activities when tested in in vivo models, highlighting the need to develop more predictive in vitro assays. In the present work, we describe the development of a robust replicative, high-content, in vitro intracellular L. donovani assay. Horse serum was included in the assay media to replace standard fetal bovine serum, to completely eliminate the extracellular parasites derived from the infection process. A novel phenotypic in vitro infection model has been developed, complemented with the identification of the proliferation of intracellular amastigotes measured by EdU incorporation. In vitro and in vivo results for miltefosine, amphotericin B, and the selected compound 1 have been included to validate the assay.T he leishmaniases are a complex of diseases, with visceral and cutaneous manifestations caused by protozoan parasites of the genus Leishmania. Visceral leishmaniasis (VL) has been the main focus for drug research and development over the past 2 decades, due to the large disease burden in East Africa and South Asia (1) and potential patient death if not treated. For VL, there has been progress in treatment over the past decade, with clinical evidence for efficacy of and registration for use of oral miltefosine, paromomycin, and the liposomal formulation of amphotericin B (AmBisome, Gilead, USA) in South Asia (2), as well as combinations of these standard drugs (3). The need for new drugs to treat VL remains, as (i) miltefosine is the only approved oral treatment but requires 28 days of treatment and potential teratogenicity limits its use (4), (ii) paromomycin requires 21 days of treatment and intramuscular administration (http://www.dndi.org/diseases-projects/diseases /vl/current-treatment/current-treatment-vl.html), and (iii) liposomal amphotericin B formulations, which have successful cure rates with a single dose (5), require intravenous (i.v.) infusion, have a high cost if not donated, and have a requirement for cold storage, limiting use in countries where the disease is endemic (6). As part of the drive to find new treatments, there has been a refocus on the assays and models used to identify and develop new molecules as antileishmanial drugs. For in vitro screens and assays, this has ranged from the need to develop methods that (i) are adaptable to and enable high-throughput screens against the replicative intracellular-macrophage amastigote stage of Leishmania donovani, one of the causative species of VL (7); and (ii) include high-throughput technologies that enable the collection of more information compared to the traditionally use...

Section: Assay Developmentsupporting

confidence: 92%

A Replicative In Vitro Assay for Drug Discovery against Leishmania donovani

Tegazzini

Díaz

Aguilar

et al. 2016

“…In association with drug delivery systems, drug repositioning has been an important tool for neglected parasitic diseases. From the old antimony salts to the newest oral drug miltefosine, all drugs used to clinically treat visceral leishmaniasis were introduced by the repositioning approach (19)(20)(21). Buparvaquone (BPQ), a veterinary drug used to treat bovine theileriosis, demonstrated antileishmanial activity; Croft et al demonstrated not only a potent in vitro activity but also a limited efficacy to reduce the parasite load in an L. donovani-BALB/c mouse model (7).…”

Section: Discussionmentioning

confidence: 99%

Nanoliposomal Buparvaquone Immunomodulates Leishmania infantum-Infected Macrophages and Is Highly Effective in a Murine Model

Costa-Silva

Galisteo

Lindoso

et al. 2017

Visceral leishmaniasis is a fatal parasitic neglected disease affecting 1.5 million people worldwide. Based on a drug repositioning approach, the aim of this work was to investigate the in vitro immunomodulatory potential of buparvaquone (BPQ) and to establish a safe regimen to evaluate the in vivo efficacy of BPQ entrapped by negatively charged nanoliposomes (BPQ-LP) in Leishmania infantum-infected hamsters. Small-angle X-ray scattering, dynamic light scattering, and the -potential were applied in order to study the influence of BPQ on the liposome structure. Our data revealed that BPQ was located in the polar-apolar interface, snorkeling the polar region, and protected against aggregation inside the lipophilic region. The presence of BPQ also decreased the Z-average hydrodynamic diameter and increased the surface charge. Compared to intravenous and intramuscular administration, a subcutaneous route was a more effective route for BPQ-LP; at 0.4 mg/kg, BPQ-LP reduced infection in the spleen and liver by 98 and 96%, respectively. Treatment for 5 days resulted in limited efficacy, but 10 days of treatment resulted in an efficacy similar to that of a 15-day regimen. The nanoliposomal drug was highly effective, with a mean 50% effective dose of 0.25 mg/kg, reducing the parasite load in bone marrow by 80%, as detected using quantitative PCR analysis. In addition, flow cytometry studies showed that BPQ upregulated cytokines as tumor necrosis factor, monocyte chemoattractant protein 1, interleukin-10 (IL-10), and IL-6 in Leishmania-infected macrophages, eliminating the parasites via a nitric oxide-independent mechanism. This new formulation proved to be a safe and effective treatment for murine leishmaniasis that could be a useful candidate against visceral leishmaniasis.KEYWORDS Leishmania, drug delivery, liposomes, therapy T he worldwide prevalence and severity of leishmaniasis has led the World Health Organization to consider it a priority infectious disease (1). Due to the complexity of host-parasite interaction, as well as contradictory results from vaccination models, the control of infection is mainly dependent on chemotherapeutic intervention (2). There are now a number of therapies for various forms of leishmaniasis; the preferences for first-line and second-line treatment vary based on the type of disease and are often guided by regional practice. Pentavalent antimonials, such as Pentostam and Glucantime, have been used to treat leishmaniasis for the last 70 years (3). However, these drugs have multiple adverse effects and decreasing effectiveness due to the development of parasite resistance, limiting their application (4). In Brazil, pentavalent antimony is still effective for treating most infections, despite its high toxicity. Until now, Citation da Costa-Silva TA, Galisteo AJ, Jr, Lindoso JAL, Barbosa LRS, Tempone AG. 2017. Nanoliposomal buparvaquone immunomodulates Leishmania infantuminfected macrophages and is highly effective in a murine model. Antimicrob Agents

“…PP2 is an inhibitor for which no antileishmanial activity against intracellular parasites in THP-1 cells in vitro has been previously described but appears to be efficient in an animal model (53). It has an EC 50 on intracellular parasites of ϳ1 M but SIs of ϳ10 for murine macrophages and Ͼ25 for human cell lines.…”

Section: Discussionmentioning

confidence: 99%

From Drug Screening to Target Deconvolution: a Target-Based Drug Discovery Pipeline Using Leishmania Casein Kinase 1 Isoform 2 To Identify Compounds with Antileishmanial Activity

Durieu

Prina

Olivier

et al. 2016

h Existing therapies for leishmaniases present significant limitations, such as toxic side effects, and are rendered inefficient by parasite resistance. It is of utmost importance to develop novel drugs targeting Leishmania that take these two limitations into consideration. We thus chose a target-based approach using an exoprotein kinase, Leishmania casein kinase 1.2 (LmCK1.2) that was recently shown to be essential for intracellular parasite survival and infectivity. We developed a four-step pipeline to identify novel selective antileishmanial compounds. In step 1, we screened 5,018 compounds from kinase-biased libraries with Leishmania and mammalian CK1 in order to identify hit compounds and assess their specificity. For step 2, we selected 88 compounds among those with the lowest 50% inhibitory concentration to test their biological activity on host-free parasites using a resazurin reduction assay and on intramacrophagic amastigotes using a high content phenotypic assay. Only 75 compounds showed antileishmanial activity and were retained for step 3 to evaluate their toxicity against mouse macrophages and human cell lines. The four compounds that displayed a selectivity index above 10 were then assessed for their affinity to LmCK1.2 using a target deconvolution strategy in step 4. Finally, we retained two compounds, PP2 and compound 42, for which LmCK1.2 seems to be the primary target. Using this four-step pipeline, we identify from several thousand molecules, two lead compounds with a selective antileishmanial activity.