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.
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.
Leishmania parasites cause two million new cases of leishmaniasis each year with several hundreds of millions people at risk. Due to the paucity and shortcomings of available drugs, we have undertaken the crystal structure determination of a key enzyme from Leishmania major in hopes of creating a platform for the rational design of new therapeutics. Crystals of the catalytic core of methionyl-tRNA synthetase from L. major (LmMetRS) were obtained with the substrates MgATP and methionine present in the crystallization medium. These crystals yielded the 2.0 脜 resolution structure of LmMetRS in complex with two products, methionyladenylate and pyrophosphate, along with a Mg 2+ ion that bridges them. This is the first class I aminoacyl-tRNA synthetase (aaRS) structure with pyrophosphate bound. The residues of the class I aaRS signature sequence motifs, KISKS and HIGH, make numerous contacts with the pyrophosphate. Substantial differences between the LmMetRS structure and previously reported complexes of E. coli MetRS (EcMetRS) with analogs of the methionyladenylate intermediate product are observed, even though one of these analogs only differs by one atom from the intermediate. The source of these structural differences is attributed to the presence of the product pyrophosphate in LmMetRS. Analysis of the LmMetRS structure in light of the Aquifex aeolicus MetRS-tRNA Met complex shows that major rearrangements of multiple structural elements of enzyme and/or tRNA are required to allow the CCA acceptor triplet to reach the methionyladenylate intermediate in the active site. Comparison with sequences of human cytosolic and mitochondrial MetRS reveals interesting differences near the ATP-and methionine-binding regions of LmMetRS, suggesting that it should be possible to obtain compounds that selectively inhibit the parasite enzyme.
Crystal structures of histidyl-tRNA synthetase from the eukaryotic parasites Trypanosoma brucei and Trypanosoma cruzi provide a first structural view of a eukaryotic form of this enzyme, and reveal differences from bacterial homologs. Histidyl-tRNA synthetases in general contain an extra domain inserted between conserved motifs 2 and 3 of the Class II aminoacyl-tRNA synthetase catalytic core. The current structures show that the three dimensional topology of this domain is very different in bacterial and archaeal/eukaryotic forms of the enzyme. Comparison of apo and histidine-bound trypanosomal structures indicates substantial active site rearrangement upon histidine binding, but relatively little subsequent rearrangement after reaction of histidine with ATP to form the enzyme's first reaction product, histidyladenylate. The specific residues involved in forming the binding pocket for the adenine moiety differ substantially both from the previously characterized binding site in bacterial structures and from the homologous residues in human histidyl-tRNA synthetases. The essentiality of the single histidyl-tRNA synthetase gene in T. brucei is shown by a severe depression of parasite growth rate that results from even partial suppression of expression by RNA interference.
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