Paape, D, Bell, AS, Heal, WP, Hutton, JA, Leatherbarrow, RJ, Tate, EW and Smith, DF (2014) Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes. PLoS Neglected Tropical Diseases, 8 (12). ISSN 1935-2727

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Abstract

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC50 values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania Nmyristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.

Item Type:	Article
Uncontrolled Keywords:	06 Biological Sciences, 11 Medical And Health Sciences
Subjects:	Q Science > QD Chemistry Q Science > QH Natural history > QH301 Biology Q Science > QR Microbiology
Divisions:	Vice-Chancellor's Office
Publisher:	Public Library of Science
Related URLs:	Author
Date Deposited:	05 Jun 2018 09:12
Last Modified:	04 Sep 2021 02:39
DOI or ID number:	10.1371/journal.pntd.0003363
URI:	https://researchonline.ljmu.ac.uk/id/eprint/8769

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