Using a non-image-based medium-throughput assay for screening compounds targeting N-myristoylation in intracellular Leishmania amastigotes.

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 N-myristoyl 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.

Synthesis and evaluation of a focused library of pyridine dicarbonitriles against prion disease.

We report the preparation and screening of a set of 55 pyridine dicarbonitriles as potential prion disease therapeutics. Use of microwave irradiation in an attempt to improve the synthesis typically led to only small enhancement in yields but gave cleaner reactions facilitating product isolation. The library was analysed for binding to human prion protein (huPrPC) by surface plasmon resonance and for inhibition of the formation of its partially protease resistant isoform PrPSc in mouse brain cells (SMB). A total of 26 compounds were found to bind to huPrPC whilst 12 showed discernable inhibition of PrPSc formation, five displaying EC(50)s in the range 2.5-9microwo compounds were found to reduce PrPSc levels to below 30% relative to an untreated control at 50nM.