The current standard of care for malaria is combination therapy based on artemisinin, which is highly valued as a potent, rapidly active antiparasitic compound. Like artemisinin, synthetic ozonides contain an endoperoxide bond. A first-generation ozonide, arterolane (OZ277), has already been licensed for clinical use in India as a combination therapy with piperaquine. However, concern has arisen that there may be a loss of potency against kelch13 mutant parasites, which are artemisinin resistant ^{30– 33} . Other synthetic ozonides, including artefenomel (OZ439), have been developed ³⁴ . Artefenomel displays activity in transmission-blocking assays in vitro ³⁵ , and clinical studies support its use in a single-exposure combination therapy ³⁶ . Unlike artemisinin’s peroxide bond, artefenomel’s peroxide bond is more stable and has an improved half-life in plasma: 23 hours compared with 0.5 hours ³⁴ . Promisingly, artemisinin-resistant mutants do not appear to be cross-resistant to artefenomel ^{30, 31} , although some mutations in kelch13 may lead to partial cross-resistance ^{30– 33} . Together, these properties support the continued efforts toward development and licensure of artefenomel for clinical use.

Ferroquine is a third-generation 4-aminoquinoline and a derivative of the antimalarial chloroquine. Although chloroquine resistance has spread to nearly all areas of the world, ferroquine efficacy is not impeded by chloroquine resistance mechanisms and resistance selection has not been observed in the laboratory ³⁷ . Ferroquine also retains activity against parasites resistant to chloroquine, mefloquine, quinine, and piperaquine ^{38, 39} . An initial clinical study with ferroquine in combination with artesunate showed a high malaria cure rate, and treatment with ferroquine also displayed post-treatment prophylaxis activity for at least 2 months ⁴⁰ . Recent phase 2 trials replaced artesunate with the more effective artefenomel ^{40– 42} , and a combination of artefenomel and ferroquine therapy is in the patient-exploratory stage of the MMV portfolio.

Plasmodium spp. depend on de novo pyrimidine synthesis because they lack pyrimidine salvage enzymes. An essential enzyme in the pyrimidine biosynthesis pathway is dihydroorotate dehydrogenase (DHODH). Large high-throughput screens were conducted to identify PfDHODH inhibitors ^{43, 44} . These screens identified several classes of molecules that target DHODH, such as triazolopyrimidines, phenylbenzamides, ureas, and naphthamides ⁴⁴ . One triazolopyrimidine that has potent activity against asexual and liver-stage parasites is DSM265 ⁴⁵ . DSM265 selectively inhibits Plasmodium spp. DHODH enzymes over human orthologues ²⁴ . DSM265 is currently in the patient-exploratory stage of the MMV pipeline and exhibits promising single-dose efficacy. Interestingly, DSM265 is predicted to remain at therapeutic concentrations in humans for more than a week after a single dose because of its favorable pharmacokinetic properties ⁴⁵ . DSM265 also has promising prophylactic activity, providing some protection against infection with a single dose up to 7 days before parasite challenge ^{46, 47} . The single-dose efficacy of DSM265 makes it exceptionally promising for both prophylaxis and treatment of disease. Furthermore, the potency of DSM265 supports future development of compounds that target Plasmodium DHODH.

Cipargamin (KAE609), a spiroindolone, also has potent activity against blood-stage malaria parasites ^{23, 48} . A recent phase 2 study used once-daily dosing of cipargamin for 3 days on 21 adults with either uncomplicated P. vivax or P. falciparum malaria ⁴⁹ . Parasite clearance rates in this clinical study and in vitro are among the fastest of any antimalarial yet characterized ^{23, 49} . Cipargamin likely targets the P-type Na ⁺ ATPase, PfATP4, because resistance mutations have emerged ²³ . PfATP4 appears to regulate parasite ion homeostasis, which is essential for survival, through active Na ⁺ export ⁵⁰ . Inhibition of PfATP4, through cipargamin treatment, perturbs ion homeostasis in the parasite and increases host cell membrane rigidity, resulting in blocked blood-stage development and transmission to mosquitoes ^{28, 29, 50– 52} . Cipargamin is currently in the patient-exploratory stage of the MMV portfolio; however, it is not the only PfATP4 inhibitor currently in development. A diverse range of compounds, including spiroindolones ^{23, 51, 53} , pyrazoleamides ²⁹ , aminopyrazoles ⁵³ , dihydroisoquinolones ²⁸ , and other compounds ⁵⁴ , have been shown to target PfATP4. A dihydroisoquinolone that likely targets PfATP4, SJ733, is in the human volunteer stage of the MMV portfolio. Resistance selection with SJ733, like cipargamin, has generated point mutations, some unique to SJ733 and not induced by cipargamin, in the pfatp4 gene ^{28, 29} . The antimalarial properties of PfATP4 inhibitors, such as cipargamin and SJ733, are exceptionally promising and support future development of compounds with this mechanism of action.

A novel class of antimalarials, imidazolopiperazines, has recently emerged and been found to have potent asexual blood-stage and liver-stage activity ^{55, 56} . One such imidazolopiperazine, KAF156, is currently in the patient-exploratory stage of the MMV portfolio in combination with lumefantrine. Lumefantrine is a clinically approved partner agent; however, it has been modified to a new once-daily formulation for use with KAF156. In addition to displaying asexual blood- and liver-stage activity, KAF156 also inhibits the growth of sexual blood-stage parasites, including mature gametocytes ⁵⁷ . Therefore, KAF156 may be effective in preventing parasite transmission from humans to mosquitoes. The antimalarial mechanism of KAF156 is still unclear because resistance in vitro is thought to be indirectly mediated through mutation of pfcarl, pfact, and pfugt, which encode a conserved protein of unknown function, an acetyl-CoA transporter, and a UDP-galactose transporter, respectively ^{25, 26, 55} . Future studies may illuminate the parasiticidal mechanism of imidazolopiperazines, but this class of drugs has great potential in the treatment of acute disease and reduction of parasite transmission.

Another novel chemical class of antimalarials, 2-aminopyridines, was identified to have potent single-dose activity against in vitro P. falciparum and in vivo P. berghei ⁵⁸ . From this initial screen of 2-aminopyridines, compound 15 (now known as MMV048) was identified with robust antimalarial activity. A follow-up study with MMV048 replicated the potent in vitro and in vivo activity of asexual blood-stage malaria parasites and also confirmed transmission-blocking and liver-stage activity ²⁷ . Genomic and chemoproteomic approaches identified Plasmodium phosphatidylinositol 4-kinase (PI4K) as the likely target of MMV048 ²⁷ . PI4K functions in membrane trafficking and membrane assembly during asexual blood-stages ⁵⁹ . PfPI4K is likely essential during the asexual blood-stage because attempts to insert an early stop codon were unsuccessful ⁵⁹ . The multi-stage antimalarial activity, prolonged half-life, and single-dose efficacy of MMV048 make it a promising new antimalarial in the patient-exploratory stage of the MMV pipeline.

Although infection with P. falciparum represents the largest burden of malaria deaths, there is also a need to develop medicines that prevent the relapse of P. vivax and P. ovale. Unlike P. falciparum, both P. vivax and P. ovale have a dormant liver-stage form called a hypnozoite. Hypnozoites can reactivate without warning, leading to the onset of malarial symptoms. This dormant stage remains both a challenge to treat and a potent barrier to malaria elimination. Tafenoquine, an 8-aminoquinoline, is currently under development for the prevention of P. vivax relapse. Tafenoquine has high activity as a single-dose treatment and has promising anti-hypnozoite activity in humans ⁶⁰ . However, tafenoquine has therapeutic restrictions similar to those of the current radical cure standard for P. vivax and P. ovale, primaquine, which might limit its therapeutic impact. Both primaquine and tafenoquine cause dose-dependent acute hemolytic anemia in individuals with glucose-6-phosphate dehydrogenase deficiency ^{61, 62} . Because of its longer half-life, tafenoquine requires a higher glucose-6-phosphate dehydrogenase activity threshold than primaquine; thus, a greater proportion of individuals will be ineligible for tafenoquine treatment and primaquine will still be needed ⁶³ . In July 2018, the US Food and Drug Administration approved tafenoquine under the trade name Krintafel. Tafenoquine is the first new antimalarial in 60 years to prevent relapse of P. vivax. However, its limitations highlight the need for development of additional compounds that target relapsing malaria.

Two recent studies highlighted the importance of plasmepsins IX (PMIX) and X (PMX) for parasite development ^{71, 72} . Plasmepsins comprise a family of 10 aspartic proteases in the P. falciparum genome. A number of studies have focused on the digestive vacuole plasmepsins I to IV (PMI to PMIV), including development of chemical inhibitors ^{73– 76} . Subsequent functional genetic studies of PMI–PMIV revealed that they are not essential for parasite survival ⁷⁷ . PMIX and PMX are expressed in asexual blood-stage parasites ⁷⁸ . Conditional knockdown of PMIX in P. falciparum revealed that it is essential for red blood cell invasion ^{71, 72} . PMX knockdown similarly interrupted red blood cell invasion but also revealed an additional requirement of PMX in red blood cell egress ⁷² . A recent study employed a combination of in vitro and in vivo rodent experiments to find that hydroxyl-ethyl-amine-based scaffold compound 49c (referred to as 49c) inhibits both PMIX and PMX ⁷¹ . 49c is an effective inhibitor against P. falciparum in vitro and the rodent parasite Plasmodium berghei in vivo ^{79, 80} . Treatment with 49c inhibits asexual, sexual, and liver-stage development, indicating that 49c or other PMIX or PMX inhibitors may have value to both treat symptomatic malaria and block transmission ⁷¹ . Together, these observations provide compelling evidence that PMIX and PMX are promising targets for antimalarial development.

In the recent P. berghei functional genetic screen, one of the cellular pathways most enriched with essential genes is that of isoprenoid biosynthesis ⁶⁵ . A number of studies have previously highlighted the requirement of isoprenoid biosynthesis for P. falciparum asexual replication ^{81– 83} . Isoprenoids are necessary for protein prenylation, the post-translational lipid modification of proteins. Because chemical inhibition of protein prenylation in the malaria parasite disrupts asexual parasite growth ^{84– 90} , prenylated malarial proteins are potential antimalarial targets. Recent chemical labeling approaches have revealed that only 15 to 19 proteins are prenylated in blood-stage malaria and a majority of these proteins are Rab GTPases ^{91, 92} . Rab GTPases function in docking vesicles to membranes and their prenylation aids in association with target membranes ⁹³ . Rab11a, a Rab GTPase, is expressed and prenylated in asexual blood-stage malaria parasites ^{91, 92, 94} and is essential for asexual parasite replication ⁹⁵ . In the parasite, Rab11a functions as a mediator of PI4K signaling and is a binding partner of PI4K, the target of imidazopyrazines and MMV048 ^{27, 59, 96} . Mutation of Rab11a confers resistance to the imidazopyrazine, KAI715 ⁵⁹ . Interestingly, Rab11a has very low genetic diversity when sequenced in 2,000 Plasmodium clinical isolates, and only one non-synonymous mutation has been identified ⁹⁷ . These features suggest that Rab11a may represent a promising target because of both its prenylation and interactions with essential signaling pathways within the parasite. Additional studies are needed to evaluate the biological roles of the remaining prenylated proteins in blood-stage malaria.

During asexual replication, the malaria parasite must exit the red blood cell before invading a new cell. This process, called egress, requires the rupture of both the parasitophorous vacuole membrane (PVM), which surrounds the parasite, and the red blood cell membrane (RBCM). Egress is protease dependent ⁹⁹ , and recently two proteases—SUB1 and SERA6—were identified as mediators of PVM and RBCM rupture ⁹⁸ . SUB1, a serine protease, moves to the parasitophorous vacuole before egress ^{100– 104} and cleaves multiple substrates ^{100, 103, 105– 107} . One substrate cleaved by SUB1 is SERA6 ^{108– 110} , a putative cysteine protease, which requires proteolytic processing by SUB1 to function ⁹⁸ . P. falciparum parasites that lack SUB1 fail to rupture the PVM, thus stalling parasite development ⁹⁸ . Interestingly, parasites that lack SERA6 can rupture the PVM, but RBCM rupture does not occur ⁹⁸ . Therefore, SUB1 and SERA6 have distinct roles in parasite egress. Because SUB1 and SERA6 are essential for asexual blood-stage growth and orthologues are found in other Plasmodium species ⁹⁸ , compounds that inhibit these proteins may be useful in treating multiple types of malarial disease.