Despite considerable progress in combating malaria, it remains one of the world’s most important infectious diseases, with 50% of the world population at risk of developing the disease and a mortality rate of ∼0.5 million annually (1). The lack of an effective vaccine and the relentless ability of the Plasmodium parasite responsible for malaria to develop drug resistance has contributed to the continuing disease burden (2–4). Artemisinin-combination therapies (ACTs) are the mainstay of current treatment regimens, but decreased effectiveness, particularly in Southeast Asia, threatens our ability to control this disease. A global effort to develop new drugs for the treatment and prevention of malaria is under way but not guaranteed to succeed (3, 5, 6). These efforts include a systematic attempt to target all life-cycle stages of the parasite to allow combination therapies to be developed, which are also likely to reduce the development of resistance. High-throughput screens (HTSs) designed to identify small drug-like molecules that prevent growth of blood-stage parasites (7, 8) and target-based approaches have identified new compounds that are currently in preclinical development and/or various stages of human clinical trials for treatment of malaria (3). Missing from these efforts has been a high-throughput technology to find liver stage–specific chemotypes. On page 1129 of this issue, Antonova-Koch et al. (9) report an HTS effort that has filled this gap. They identify a substantial number of new chemical starting points with potent liver-stage antimalarial activity, promising a new capacity to feed compounds through the drug development pipeline for chemoprotection.
As efforts to eliminate malaria increase, the need for chemoprotective agents to protect vulnerable populations will also increase (3). The idea is to find a long-lasting agent to treat infections before they become symptomatic and to develop these into a chemical vaccine (that is, a drug that protects against disease). The best malaria stage of infection to target for this approach is the one in the liver. The malaria life cycle begins when an infected mosquito injects sporozoites into a person, some of which find their way to the liver to establish infection (10) (see the figure). After replication in hepatocytes, malaria parasites burst out and infect erythrocytes, setting up an amplifying intraerythrocytic cycle. From 101 sporozoites that reach the liver, up to 105 merozoites will emerge into the blood, and up to 1012 will then build up in the bloodstream during a severe infection. A drug that blocks parasite replication in the liver works on a much lower parasite burden and thus has a lower chance of encountering and selecting for a rare parasite with a resistance mutation than do blood stage–active compounds. This is particularly so if a compound does not have activity on both stages and therefore does not put selective pressure on a large blood-stage parasite load.
Plasmodium falciparum is responsible for most malaria cases, and it is the most deadly, whereas Plasmodium vivax has the greatest global distribution. Antonova-Koch et al. made a strategic choice to use the rodent malaria parasite Plasmodium berghei for their screen. This conferred many advantages over using a human parasite: ease of production, minimal biohazard risk, more rapid life cycle, and ability to infect hepatoma cell lines that are more facile to use and do not detoxify the compounds being screened. From an initial hit rate of ∼4%, a subset (∼104) were prioritized for evaluation in confirmation assays, leading to the validation of ∼103 compounds with good druglike properties that have potent liver-stage activity and minimal cytotoxicity on host liver cells. Of these, 631 were profiled on additional Plasmodium species and life-cycle stages. Interestingly, two-thirds of these hits are specific for liver-stage parasites, highlighting the previously unknown biology of this stage and promising new cellular insights if compound targets can be determined. This is a goal that will require innovative approaches. The subset of compounds that were also active against blood-stage P. falciparum parasites contained a high proportion of mitochondrial inhibitors (43%) across diverse scaffolds. The mitochondrion in malaria parasites is critical for pyrimidine biosynthesis, a pathway that is essential for cell replication to generate the mature schizont in both blood and liver infections (see the figure). Demand for pyrimidine nucleotide bases is even greater in the liver stage, in which one sporozoite is replicated to generate 20,000 merozoites (10). Drugs that target enzymes required for pyrimidine nucleotide biosynthesis are effective for both malaria treatment and chemoprevention, including the cytochrome bc1 inhibitor atovaquone, which is an approved antimalarial agent used mainly for chemoprevention, and DSM265, an inhibitor of dihydroorotate dehydrogenase, currently in clinical development (3, 5, 6). The surprisingly high percentage of dual-acting compounds that hit these targets suggests that this pathway is one of the most vulnerable pathways shared between the blood and liver stages.
Not all hits from the P. berghei HTS worked on liver-stage P. vivax infections; the crossover was only ∼25%. This may be partially explained by assay differences and by compound metabolism in the primary human hepatocytes used for the P. vivax assay. This latter issue could be engineered out of any compound series during lead optimization. It remains to be seen how many of the identified chemotypes will ultimately have liver-stage activity against both P. vivax and the deadly P. falciparum. Extrapolating from experience with compounds on blood stages of the rodent and human parasites, a large majority are likely to be effective against all Plasmodium species.
Now comes the hard work of prioritizing these hit compounds and optimizing them to have the properties of a chemical vaccine for clinical development. Recent work to develop chemical vaccines for HIV (11) and to formulate atovaquone as an injectable for chemoprevention in malaria (12) provide the beginnings of proof of concept for this strategy. The potential advantages of liver stage–specific chemoprotection in terms of simpler field implementation and low resistance propensity must be balanced with a need for high safety when used to protect a whole asymptomatic community (more so than a short-term treatment given to a discrete population of patients). Additionally, compounds must be stable, have a long half-life, and be amenable to slow delivery formulation, such as a long-acting injectable that will also have the benefit of improving compliance. Because of these complexities, there is a need to have a substantial list of candidate compounds. Thanks to the work of Antonova-Koch et al., we have such a list.