Transmission of malaria to mosquitoes blocked by bumped kinase inhibitors

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-1treated 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.