A rapid in vitro bioluminescence-based rate-of-kill (BROK) assay to screen the MMV “Malaria Box” Imran Ullah, Rhiannon Blow and Paul Horrocks Centre for Applied Entomology and Parasitology, Keele University, Staffordshire, ST5 5BG Introduction
Rapid rate-of-kill (ROK) is a key target product profile for future antimalarial drugs [1]. Whilst typically determined in either animal models or during early human clinical trails, ROK determination in vitro offers a valuable opportunity to triage candidate “hits” early in the expensive development process. Current in vitro ROK assays rely on a recrudescence assay, and though offering a “gold standard” assessment, this assay takes 3-4 weeks and is not readily scalable [2]. We describe here work exploiting the immediate dynamic response of luciferase-expressing Plasmodium falciparum exposed to antimalarial drugs [3] in the validation of a rapid and scalable bioluminescence-based ROK assay and our progress in screening the 400 compounds provided in the Medicine for Malaria Venture’s Malaria Box [4]. 1. The BROK assay
Fig. 3B Parasite reduction ratio
P. falciparum genetically modified to express high levels of luciferase at the trophozoite stage [3] are exposed to increasing IC50-fold doses. During drug incubation, samples are removed and the level of luciferase expression, compared to an untreated control, plotted. Fig. 1 illustrates the dose- and time-dependent effect on luciferase expression for “fast ROK” chloroquine (CQ) and “slow ROK” atovaquone (ATOV).
3. BROK screening of the Malaria Box
Fig. 1
Fig. 4 illustrates the application of the BROK assay in determining ROK of MMV Malaria Box candidates against a set of “benchmark” controls (CQ, ATOV and DHA, dihydroartemisinin). Exemplars of estimated “fast ROK” and “slow ROK” are shown along with their structures. Note – this level of data has been developed for 66 of the 400 Malaria Box compounds to date.
Fig. 4 2. Validation
“Fast ROK”
The BROK assay was applied to a range of “benchmark” antimalarial drugs using 3hr (black bars) and 6hr (gray bars) incubations. Fig. 2 illustrates the relative order of the ROK. This order compares well with available in vitro ROK data [2]. Note the correlation of the BROK data (Fig. 3) with available in vitro determined parasite clearance time (A PCT, time to produce 99.9% reduction in parasitaemia) and parasite reduction ratio (B PRR, Log10 fold decrease in parasitaemia achieved over 48 hrs). Based on these observations, the IC50 doses used were revised to 9 to 0.3 fold for screening of the MMV Malaria Box.
“Slow ROK”
Fig. 2
“Benchmarks”
4. Moving forward……
Fig. 3A Parasite clearance time
Our study to date validates the BROK assay and provides experience in the application of the assay. We are now proceeding to screen the remaining drug-like and probe-like compounds. All 200 of the drug-like compounds have been explored in a provisional (6hr, 3x and 9x IC50) screening format – with completion of the full set during the summer 2014. All the probe-like compounds to be similarly screened by end of year 2014. References
Acknowledgements We would like to acknowledge financial support from the BBSRC, Royal Society and Biochemical Society in the initial development of the BROK assay. IU is supported by a Keele University ACORN award and his presentation of this poster at the British Society of Parasitology Spring Meeting 2014 is supported by this society and the Keele Postgraduate Association.
1www.mmv.org 2Sanz
et al., (2012). P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action. PloS One, 7(2), pp. e30949-e30949. 3Hasenkamp et al., (2013). Evaluation of bioluminescence-based assays of anti-malarial drug activity. Malaria Journal, 12, pp. 58. 4Spangenberg et al., (2013). The open access Malaria Box: a drug discovery catalyst for neglected diseases. PloS One, 8(6), pp. e62906-e62906. Dr. Paul Horrocks:
[email protected] Imran Ullah:
[email protected]
