Final Report

1. Project objective

The objective of the project was to identify novel antimalarial compounds derived from a Daiichi Sankyo RD Novare library of natural product extracts. The hit compounds identified should meet MMV and GHIT criteria for antimalarial hit compounds and be suitable for progression to the hit to lead phase.

2. Project design

Daiichi Sanyko RD Novare (Tokyo, Japan) supplied collaborators at Griffith University (Melbourne, Australia) with a library of 30,000 natural product extracts for testing in a whole cell assay to measure growth inhibition of the asexual blood stage of the malaria parasite. The extracts were tested in drug sensitive (Pf3D7) and drug resistant (PfDd2) strains of the malaria parasite and for cytotoxicity in HEK293 cells. The extracts that were active and not cytotoxic were fractionated and the active fractions identified. The active fractions were purified, and the active component characterized (chemical structure and parasitological profile).

3. Results, lessons learned

The library of 30,000 natural product extracts was screened at Griffith University and 5 hit strains (A113594, A113830, A114750, F36316 and F82189) were identified with antimalarial activity (3D7) and acceptable cytotoxicity (HEK293). The active microbe strains belong to actinomycetes or fungi. The active strains were re-cultured on a large scale and the cultures were fractionated (EtOAc extraction, ODS column and HPLC). 189 fractions were prepared and tested against Plasmodium falciparum (3D7 strain; drug sensitive) and HEK293 mammalian cells for cytotoxicity. No active fraction was identified from strains A113594, F36316 and F82189. On the other hand, a fraction from strain A114750 showed full parasite inhibition at all dilutions tested. However, the active component of A114750, was determined to be Borrelidin. Borrelidin is a macrolide known to inhibit mammalian, bacterial, and protozoan threonyl-tRNA synthetases (ThrRS) [Otoguro K, et al. J Antibiot (Tokyo) 2003;56(8):727–729.]. However, despite a compelling parasitological profile Borrelidin has suboptimal PK and insufficient safety margins and is unsuitable for further development as a single dose antimalarial drug. Strain A113830 is the last active strain the team are investigating as part of project S2015-112. It was not possible to isolate an active fraction from the extract using standard fractionation protocols. However, potent activity was detected in the pre-fractionated samples (C17 and G19) and the samples were not cytotoxic. The activity of the pre-fractionated samples in an asexual blood stage growth inhibition assay (Pf3D7) was confirmed at two independent test centers (GRIDD, Australia and TCGLS, India). The pre-fractionated samples are currently being tested against (P.berghei) liver stage schizonts (UCSD, USA) and in a functional dual gamete formation assay (ICL; UK). Obtaining the lifecycle data for the pre-fractionated samples is the final activity that will be carried out as part of project S2015-112. Following completion of the parasitological profiling of the pre-fractionated samples of A113830 (liver, blood, and sexual stages) the team will analyze the data and if the data is sufficiently compelling (antimalarial activity in >1 parasite lifecycle stage assays) we will submit a new GHIT HTLP proposal for the characterization and further development of the active components.