title: Identification and Characterization of Novel Anti-leishmanial Compounds.
Creators: name: Zulfiqar, Bilal
Publisher: name: Griffith University
Year: 2017
Subjects: subject: Anti-leishmanial compounds
subject: Trypanosomatid protozoan
subject: Phlebotomus
subject: Lutzomyia
subject: Cytotoxicity assays

description: Leishmaniasis is characterized as a parasitic disease caused by the trypanosomatid protozoan termed Leishmania. Leishmaniasis is endemic in 98 countries around the globe with increased cases of morbidity and mortality emerging each day. The mode of transmission of this disease is via the bite of a sand fly, genus Phlebotomus (Old World) and Lutzomyia (New World). The life cycle of Leishmania parasite exists between the sand fly (promastigote form) and the mammalian host (amastigote form). Leishmaniasis can be characterized as cutaneous, muco-cutaneous or visceral leishmaniasis based on clinical manifestations exhibited in infected individuals. Although leishmaniasis is treatable, it faces challenges largely due to emerging resistance and extensive toxicity for current drugs. Therapeutic efficacy varies depending upon the species, symptoms and geographical regions of the Leishmania parasite. The drug discovery pipeline for neglected trypanosomatid diseases remains sparse. In particular, the field of leishmaniasis drug discovery has had limited success in translating potential drug candidates into viable therapies. Currently there are few compounds that are clinical candidates for leishmaniasis, it is therefore essential that new compounds that are active against Leishmania are identified and evaluated for their potential to progress through the drug discovery pipeline. In order to identify new therapeutics, it is imperative that robust, biologically relevant assays be developed for the screening of anti-leishmanial compounds. The currently available assays have low predictive compatibility and high attrition rate in the identification of the compounds. In an aim to identify compounds with activity against L. donovani (MHOM/IN/80/DD8) promastigotes, a phenotypic, high-throughput, resazurin based 384-well viability based assay was successfully developed to estimate the effect of compound treatment on L. donovani DD8 parasites. Complementary cytotoxicity assays were also established to access the toxicological profile of the compounds. The assays developed are robust and reproducible, using standard statistical parameters to assess assay quality and suitability for use in high throughput screening (HTS) based drug discovery. To identify compounds active against L. donovani DD8 parasites, two compound collections were evaluated, a synthetic scaffold and a natural product (NP) based library. A primary screen of the open scaffold library constituting 5560 structurally diverse synthetic compounds was undertaken. Screening incorporated a promastigote viability assay (extracellular form) and a high-content intracellular amastigote imaging assay. Confirmation of activity was performed together with cytotoxicity studies against both THP-1 (host cell) and HEK-293 cell lines. The second library, Davis open access natural product-based (DOANP) library was tested against kinetoplastids (Leishmania donovani DD8, Trypanosoma brucei brucei and Trypanosoma cruzi) using highthroughput phenotypic assays. This library currently consists of 472 distinct compounds, the majority of which are natural products that have been obtained from Australian natural sources, such as endophytic fungi, plants, macrofungi and marine invertebrates. The confirmed hits were prioritized based on structure activity relationships to identify potential analogues. Then the activities of these analogues were evaluated to access the in vitro effect of these compounds. Based on the anti-leishmanial activity and selectivity, two compounds identified from the synthetic scaffold library were selected to be taken further for characterization and biological profiling. Of these two compounds, the first compound BZ1 exhibited an IC50 value of 0.59 ± 0.13 μM against the intracellular form (amastigote) of the parasite and IC50 value of 2.37 ± 0.85 μM against the extracellular form (promastigote) of L. donovani DD8 (Old World - Indian strain) parasites. The second compound BZ1-I demonstrated to have an IC50 of 0.57 ± 0.17 μM against intracellular amastigotes and an IC50 value of 0.60 ± 1.12 μM against promastigotes. The two compounds were subjected to additional assays which included cidal/static effect, time to kill and pre-incubation studies. The mechanism of action studies were also conducted to assess whether the compounds induce apoptosis in parasites and their effect on mitochondrial morphology. Resistance was generated and confirmed for both the compounds in order to determine the target protein using whole genome sequencing technique. Compounds cytotoxicity was also determined against a cytotoxicity panel constituting of THP-1, HEK-293, HepG2, J774.1, Raw-264.1 cell lines to establish the safety profile. The lead molecules were subsequently progressed to Drug Metabolism and Pharmacokinetics (DMPK) and pH stability studies. In addition to the activity against the L. donovani DD8 (Old World - Indian strain) parasite, compounds also showed activity against other species and strains of the Old World and New World parasites, namely L. infantum (Old World), L. donovani (Old World - Sudanese strain) and L. infantum (New World) amastigotes. The compounds were also shown to have in vitro activity against T. brucei species, T. cruzi and Plasmodium falciparum strains. Screening against the extracellular and intracellular forms of the parasite, as well as across a panel of leishmanial species, provides a unique activity profile for prioritizing the identified hits. These hit compounds provide urgently needed starting points for the development of novel lead series for future anti-leishmanial therapeutics.