Exploiting malaria parasite invasion of host cells as a drug target.

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1. Summary

There is growing evidence that the parasite causing malaria, one of the biggest causes of childhood mortality in the world today, is becoming resistant to our best and safest antimalarials. Alternative drug therapies with novel targets are urgently needed to replace those with waning efficacy. One novel strategy receiving increasing attention is inhibition of parasite invasion into the human red blood cell, a scenario that would see parasites rapidly cleared before they could infect a red blood cell and cause disease. In a major positive for this drug strategy, I have developed creative methodologies to rapidly screen for drugs that inhibit parasite invasion of red blood cells, opening up new possibilities in the field of antimalarial development.

2. Description

Background

Half of the world’s population is at risk of infection by mosquito-borne malaria parasites such as Plasmodium falciparum, the most virulent of the human malaria’s. Despite the recent effectiveness of artemisinin combination therapies (a combination of fast acting artemisinins with a slowly cleared partner drug) in treating malaria, 584,000 childhood deaths still occur every year. Alarmingly, resistance to these drugs is spreading in South East Asia raising the very real spectre of a doubling in malaria related mortality1,2. The Australian Government recognises that artemisinin-resistant malaria parasites pose a major threat to our region, including to our nearest neighbours (Indonesia, Timor Leste, Papua New Guinea), and has invested significantly in programs to halt the spread of resistance. Funding for development of new drugs to treat malaria and other tropical diseases was at the forefront of the May 2015 federal budget, and such investments are of critical importance if we are to avoid a humanitarian disaster caused by the spread of artemisinin-resistant parasites.

Malaria invasion of host cells as a novel drug target

Malaria has a critical bottleneck in its lifecycle, namely, the invasion of oxygen carrying red blood cells (RBCs) by the small egg shaped merozoite stage of the lifecycle. RBC invasion by the malaria merozoite is an ideal drug target because (i) extracellular parasites are directly exposed to antimalarials in the bloodstream, (ii) many proteins required for invasion are not shared with host cells, and (iii) failure to invade immediately ends the blood stage of the parasite lifecycle, the stage that causes all the debilitating disease symptoms. Inhibition of host cell invasion is a validated strategy for anti-microbial development against bacteria and viruses, and is of increasing interest for antimalarial development3. In a major technological breakthrough, my colleagues and I developed the first reliable method for the purification of viable P. falciparum merozoites, allowing us to rapidly identify inhibitors of merozoite invasion of the host RBC for further drug development4-7.

A safe antibiotic that inhibits invasion and parasite growth

In an exciting discovery, I identified the safe, long lasting antibiotic azithromycin as a rapid inhibitor of P. falciparum invasion of human RBCs7. Azithromycin is known to target the apicoplast (a remnant plastid) of P. falciparum and has been investigated as a potential partner drug for artemisinin combination therapy, due to favourable properties and antimalarial activity. My work has uncovered a secondary mode of action for the drug that is independent of its activity against the apicoplast, lending support for development of azithromycin analogues as antimalarials with dual modalities7. Preliminary studies identified analogues that were even better inhibitors of invasion than azithromycin itself, a first step for optimisation of the drugs invasion inhibitory activity. On the back of these promising results, I am undertaking further studies to retarget azithromycin as a drug with dual modalities that, (i) reduces risk of resistance developing by targeting two independent mechanisms, and (ii) improves parasite clearance by rapidly killing at the point of invasion and inhibiting parasite growth via activity against the apicoplast. This novel drug treatment strategy has clinical and practical advantages for use in combination therapy.

Identification of new candidate inhibitors of merozoite invasion

P. falciparum invasion of the host cell is a complex process that is likely to require the co-ordinated activity of many essential, non-redundant, parasite proteins. My research has applied creative solutions to develop robust merozoite drug screening assays that are the ideal platform for the identification of novel merozoite invasion inhibitory compounds targeting these proteins4,5,7. Taking full advantage of the advancements made to date, I will screen larger, more diverse, drug libraries for novel inhibitors of merozoite invasion. Starting with the Medicines for Malaria Venture ‘Malaria Box’ (400 diverse antimalarials with unknown targets) I will refine methodologies for medium and high-throughput drug screens using purified merozoites. This preliminary screen will enable optimisation of methods such that larger drug libraries can be tested for parasite invasion inhibitors, potentially in collaboration with high-throughput screening facilities. This strategy offers the exciting possibility of uncovering new lead compounds for antimalarial development and provides an avenue to explore malaria invasion biology in detail.

Conclusion

The spread of artemisinin resistance in South East Asia is an alarming reminder that failure to have effective replacements for first-line treatments can lead to significant increases in malaria mortality1,2. There is a critical and ongoing need to develop new drugs that provide a stable of antimalarials to replace those with waning efficacy8. Retargeting azithromycin as a rapid inhibitor of merozoite invasion, whilst maintaining its activity against the apicoplast, is an exciting prospect for a safe partner drug for combination therapies. Dual modality could see azithromycin rapidly clear parasites by inhibiting invasion and then offer long-term protection by targeting the apicoplast. Such a double hit drug treatment strategy has significant potential to increase efficacy while reducing the chances for the development of resistance. The untapped potential of the Malaria Box and larger drug libraries, along with my experience with the required methodologies, places me in a strong position to identify and develop a wider selection of novel invasion inhibitory drugs. The outcomes of this study are highly relevant to the search for new antimalarials and funds from the Lawrence creative prize will assist in the identification and development of readily translatable drug treatments targeting malaria invasion of the RBC.

3. Additional Details

References

  1. Leang R, Barrette A, Bouth DM, et al. Antimicrob Agents Chemother. 2013;57:818-26.
  2. Trape JF, Pison G, Spiegel A, et al. Trends Parasitol. 2002;18:224-30.
  3. Srinivasan P, Yasgar A, Luci DK, et al. Nature communications. 2013;4:2261.
  4. #Boyle MJ, #Wilson DW, Richards JS, et al. Proc Natl Acad Sci U S A. 2010;107:14378-83. # Equal contribution.
  5. Wilson DW, Langer C, Goodman CD, et al. Antimicrob Agents Chemother. 2013;57:1455-67.
  6. Riglar DT, Richard D, Wilson DW, et al. Cell Host Microbe. 2011;9:9-20.
  7. Wilson DW, Goodman CD, Sleebs BE, et al. BMC Biology. 2015;13:52.
  8. Burrows JN, van Huijsduijnen RH, Mohrle JJ, et al. Malar J 2013;12:187.

 

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