powers erythrocyte invasion (6), while PfCDPK5 was shown to be critical for egress of P. falciparum merozoites from erythrocytes (7). In this report, we describe bumped kinase inhibitors (BKIs) that block infection of mosquitoes by malaria parasites. These compounds selectively and potently inhibit CDPK4, which is required for exflagellation of Plasmodium berghei microgametes (8) and has recently been shown to be connected with induction of exflagellation in P. falciparum microgametes (9), before fusion with the macrogamete, to form a zygote. The zygote undergoes transitional ookinete and oocyst stages to mature into infective sporozoites that are injected into a mammalian host during the female mosquito blood meal. Blocking exflagellation through the selective inhibition of Plasmodium CDPK4 would be expected to interrupt malaria transmission without being toxic to humans (10). Results and DiscussionWe have previously demonstrated that the ATP-binding pockets of Toxoplasma gondii and Cryptosporidium parvum CDPK1 can be selectively targeted by BKIs with large aromatic moieties displayed from the 3 position of the pyrazolopyrimidine scaffold due to the anomalously small gatekeeper residues (glycine) present in these kinases. Selective inhibition of Tg/CpCDPK1 with BKIs leads to blockage of mammalian-host cell invasion (11,12). PfCDPK4 has a serine at the gatekeeper position (Figure 1), smaller than the gatekeeper in almost all mammalian kinases, and an overall binding pocket that is very similar to those of TgCDPK1 and CpCDPK1. A number of compounds in our Tg/CpCDPK1 BKI library were found to inhibit recombinant PfCDPK4 (rPfCDPK4), the most potent being BKI-1 with an IC 50 (concentration to inhibit 50% of enzyme activity) of 4 nM (Table 1). However, not all compounds that are potent inhibitors of Tg/CpCDPK1 have comparable activity against rPfCDPK4. Despite the overall structural similarities in the ATP-binding pockets of CDPKs, small differences in the size of the gatekeeper residue may have a large effect on inhibitor potency.Effective control and eradication of malaria will require new tools to prevent transmission. Current antimalarial therapies targeting the asexual stage of Plasmodium do not prevent transmission of circulating gametocytes from infected humans to mosquitoes. Here, we describe a new class of transmission-blocking compounds, bumped kinase inhibitors (BKIs), which inhibit microgametocyte exflagellation. Oocyst formation and sporozoite production, necessary for transmission to mammals, were inhibited in mosquitoes fed on either BKI-1-treated human blood or mice treated with BKI-1. BKIs are hypothesized to act via inhibition of Plasmodium calcium-dependent protein kinase 4 and predicted to have little activity against mammalian kinases. Our data show that BKIs do not inhibit proliferation of mammalian cell lines and are well tolerated in mice. Used in combination with drugs active against asexual stages of Plasmodium, BKIs could prove an important tool for malaria control and eradication.

Malaria parasites are transmitted by mosquitoes, and blocking parasite transmission is critical in reducing or eliminating malaria in endemic regions. Here, we report the pharmacological characterization of a new class of malaria transmission-blocking compounds that acts via the inhibition of Plasmodia CDPK4 enzyme. We demonstrate that these compounds achieved selectivity over mammalian kinases by capitalizing on a small serine gatekeeper residue in the active site of the Plasmodium CDPK4 enzyme. To directly confirm the mechanism of action of these compounds, we generated P. falciparum parasites that express a drug-resistant methionine gatekeeper (S147 M) CDPK4 mutant. Mutant parasites showed a shift in exflagellation EC50 relative to the wild-type strains in the presence of compound 1294, providing chemical-genetic evidence that CDPK4 is the target of the compound. Pharmacokinetic analyses suggest that coformulation of this transmission-blocking agent with asexual stage antimalarials such as artemisinin combination therapy (ACT) is a promising option for drug delivery that may reduce transmission of malaria including drug-resistant strains. Ongoing studies include refining the compounds to improve efficacy and toxicological properties for efficient blocking of malaria transmission.

Toxoplasmosis is a disease of prominent health concern that is caused by the protozoan parasite, Toxoplasma gondii. Proliferation of T. gondii is dependent on its ability to invade host cells, which is mediated, in part, by calcium-dependent protein kinase 1 (CDPK1). We have developed ATP competitive inhibitors of TgCDPK1 that block invasion of parasites into host cells, preventing their proliferation. The presence of a unique glycine gatekeeper residue in TgCDPK1 permits selective inhibition of the parasite enzyme over human kinases. These potent TgCDPK1 inhibitors do not inhibit the growth of human cell lines and represent promising candidates as toxoplasmosis therapeutics.

The fight against malaria is increasingly threatened by failures in vector control due to growing insecticide resistance. This review examines the recent primary research that addresses the putative relationship between agricultural insecticide use and trends in insecticide resistance. To do so, descriptive evidence offered by the new research was categorized, and additional factors that impact the relationship between agricultural insecticide use and observed insecticide resistance in malaria vectors were identified. In 23 of the 25 relevant recent publications from across Africa, higher resistance in mosquito populations was associated with agricultural insecticide use. This association appears to be affected by crop type, farm pest management strategy and urban development.