Malaria is still a major global health problem with significant cases of human mortality and morbidity threatening the rural tropical and subtropical regions in South America and South Asia despite recent progress in global malaria control measures and a decline in the frequency of Plasmodium falciparum (P. falciparum) malaria cases. Presently, two of five species of Plasmodium parasite are major causes of transmissible human malaria by Anopheles mosquito bite; Plasmodium falciparum and Plasmodium vivax.  In 2015, despite progress in reduced malaria cases and death, 95 countries and territories remain malaria-affected and about 3 billion people – nearly half of the world’s population – are still vulnerable to malaria. Moreover, it was estimated that the neglected Plasmodium vivax affects 2.5 billion people yet received 3 % of research effort as compared to its notorious cousin Plasmodium falciparum (Plos Negl Trop Dis 2012 6(9):e1814). Malaria is a rapidly changing disease and recent artemisinin-resistant parasites confirmed in five neighbouring countries in the Greater Mekong subregion: Cambodia, Lao People’s Democratic Republic, Thailand, Myanmar and Vietnam, and increasing global reports of severe P. vivax malaria mostly affecting South-East Asian countries (74%) are new concerns amidst ongoing P. falciparum resistance (WHO Global Malaria Program. World Malaria Report. 2015. http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/). The emergence of a multidrug-resistance parasite and rising P. vivax malaria cases in 2015 are driving the world health organization (WHO) call for P. vivax malaria control and health concerns worldwide due to very limited effective antimalarial medicine, insensitive diagnosis kits for dormant hypnozoites and non-falciparum P. vivax, and patchy knowledge to even successfully combat the malaria parasite anywhere today (http://www.searo.who.int/mediacentre/releases/2015/1607/en/). Under these circumstances, new molecular targets are needed to fight malaria.

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The recommended WHO first-line treatment for malaria is artemisinin combination therapy (ACT) for P. falciparum and for chloroquine-resistant P. vivax parasites (Guideline for the treatment of malaria. 2015 http://www.who.int/malaria/publications/atoz/9789241549127/en/). There is currently no alternative antimalarial drug as effective as artemisinin and its derivatives. Artemisinin is a class of antimalarial compounds with an endoperoxide bridge, derived from the sweet wormwood Artemisia annua and its first emergency malaria use in China’s Project 523 in 1967, after reports of global chloroquine-resistant P. falciparum outbreak in the late 1950s. Its isolation was credited to Youyou Tu, who was recently awarded the Nobel Prize in Medicine in 2015. Artemisinin monotherapy has a short half-life, and thus a combination of artemisinin and an additional long-lasting drug is prescribed, so as to minimize residual parasitic resistance and notably in P. vivax for dormant hypnozoite clearance in liver to prevent future relapse. A study on artemisinin and its derivatives has highlighted at least 124 protein targets and haem-activated artemisinin in latter stage of the P. falciparum life cycle and possibly in P. vivax (Nat Commun 2015 6:10111). Thus parasite resistance is likely to develop before the ring stage, to block the multi-targeting nature of artemisinin and enhance ring-stage parasite growth. Hence the discovery of novel effective antimalarials involves addressing drug resistance developed before the ring stage.

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The life cycle of the malaria parasite is complex, involving both the female Anopheles mosquito and human. When the Anopheles mosquito bites a human host, it releases haploid sporozoites to form exoerythrocytic schizonts in the liver, which produce haploid merozoites in 5-16 days via mitosis or dormant hypnozoites that can cause late relapse even months later. These merozoites exit the liver and invade the red blood cells (RBCs) to undergo either asexual or sexual cycle replication. In the 2-day asexual cycle, the merozoites begin the uninuclear ring and trophozoite stages to become intraerythroytic schizonts which rupture and release fresh merozoites in the ring stage, and repeat the RBC invasion. This asexual cycle involving schizont maturation and merozoite release occurs in synchrony for 1-3 days, and clinical symptoms develop e.g. periodic fever, headaches, anaemia, renal failure and cerebral malaria. Also, some merozoites may instead begin the 10-15 day intraerythroytic sexual cycle and form female and male gametocytes which re-enter a mosquito during blood meal. The gametocytes mature into sexual gametes that fuse into diploid zygotes in meiosis and develop into oocysts within the mosquito midgut. Each oocyst then develops thousands of active sporozoites thus repeating the life cycle of the parasite in subsequent blood meals.

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Structure-based drug design resumes to the forefront of modern preclinical drug discovery due to the lack of successful “hits” in high-throughput screening in the past decade (Computational and Structural Biotechnology Journal 2013 5(6):1). Thus non-artemisinin classes of non-toxic antimalarials that act effectively against novel targets in the parasite have to be developed with protein structure visualization. Unlike high-throughput screening, protein structure visualization methods by nuclear magnetic resonance, X-ray crystallization and recent cryo-electron microscopy advances provide the direct interactions between the protein target and preclinical candidates, needed for detailed development into drugs. Among potential new protein targets for antimalarial are the ubiquitin proteasome system and the Plasmodium proteases, notably the haemoglobin-degrading cysteine proteases (or falcipains). Cysteine proteases of the food vacuole, falcipain-2 and falcipain-3 in P. falciparum and vivaparin-2, vivapain-3 and vivapain-4 in P. vivax, are major enzymes involved in haemoglobin degradation into haem and amino acids for erythrocytic parasite growth (J. Trop. Med. 2012 Article ID 345195, PLoS ONE 2013 8(9): e73530, J. Parasitic Diseases 2011 35:94). But despite these advances in NMR spectroscopy, obtaining suitable pure protein remains the most expensive and time-consuming step in the production of a three-dimensional protein structure. Improving expression, solubility and homogeneity remains key to NMR success.

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Other potential new targets include the melatonin signalling (phospholipase C, adenylyl cyclase, protein kinase A, protein kinase 7) and

Mitochondrion and the apicoplast are both considered as the most important organelles in P. falciparum metabolism. The replication of parasite requires a plentiful lipid supply, specifically fatty acids for the membrane biogenesis. The parasites initially lack the ability to synthesis their own fatty acids and thus take lipids from their hosts. FAS II is not critical for P. falciparum blood stage replication and critical for normal liver stage development. The 23-megabase nuclear genomer of P. falciparum encodes fewer enzymes and transporters compared to the genomes of free-living eukaryotic microbes, but a large proportion of genes are devoted to immune evasion and host-parasite interactions.P. falciparum chromosomes vary considerably in length, with most of the variation occurring in the subtelomeric regions.Subtelomeri deletion often extend well into the chromosome, and in some cases alter the cell adhesion ability owing to the loss of genes encoding adhesion molecules (PNAS 1993 90:8292, Nature 1986 322:474). Of the 5,268 predicted proteins, about 60% did not have sufficient similarity to proteins in other organism to justify provision of functional assignment. Thus almost two-thirds of the proteins appear to be unique to this organism. About 670% of the putative apicoplast-targeted proteins are of unknown function. Several metabolic pathways in the organelle are distinct from host pathways and offer potential parasite-specific targets for drug therapy. There are 237 P. falciparum proteins with strong matches to proteins in all completed eukaryotic genomes but no matches to proteins, even at low stringency in any complete prokaryotic proteome.

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Clinically, resistance is defined as a slower rate of parasite clearance in patients treated with artemisinin derivative or an ACT. The slow clearance rates associated with enhanced survival rates of ring-stage parasite.