ABSTRACT
Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
Subject(s)
Cryptosporidiosis , Cryptosporidium parvum/enzymology , Enzyme Inhibitors/pharmacology , Lysine-tRNA Ligase/antagonists & inhibitors , Malaria, Falciparum , Plasmodium falciparum/enzymology , Protozoan Proteins/antagonists & inhibitors , Animals , Cryptosporidiosis/drug therapy , Cryptosporidiosis/enzymology , Disease Models, Animal , Enzyme Inhibitors/chemistry , Humans , Lysine-tRNA Ligase/metabolism , Malaria, Falciparum/drug therapy , Malaria, Falciparum/enzymology , Mice, SCID , Protozoan Proteins/metabolismABSTRACT
Three structures of the histidine triad family protein from Entamoeba histolytica, the causative agent of amoebic dysentery, were solved at high resolution within the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The structures have sulfate (PDB entry 3oj7), AMP (PDB entry 3omf) or GMP (PDB entry 3oxk) bound in the active site, with sulfate occupying the same space as the α-phosphate of the two nucleotides. The C(α) backbones of the three structures are nearly superimposable, with pairwise r.m.s.d.s ranging from 0.06 to 0.13 Å.
Subject(s)
Adenosine Monophosphate/metabolism , Entamoeba histolytica/chemistry , Guanosine Monophosphate/metabolism , Hydrolases/chemistry , Sulfates/metabolism , Amino Acid Sequence , Crystallization , Entamoeba histolytica/genetics , Entamoeba histolytica/metabolism , Hydrolases/genetics , Hydrolases/metabolism , Molecular Sequence Data , Protein Binding/physiology , Protein Structure, SecondaryABSTRACT
The crystal structures of prostaglandin F synthase (PGF) from both Leishmania major and Trypanosoma cruzi with and without their cofactor NADP have been determined to resolutions of 2.6 Å for T. cruzi PGF, 1.25 Å for T. cruzi PGF with NADP, 1.6 Å for L. major PGF and 1.8 Å for L. major PGF with NADP. These structures were determined by molecular replacement to a final R factor of less than 18.6% (Rfree of less than 22.9%). PGF in the infectious protozoa L. major and T. cruzi is a potential therapeutic target.
Subject(s)
Hydroxyprostaglandin Dehydrogenases/chemistry , Leishmania major/chemistry , NADP/chemistry , Trypanosoma cruzi/chemistry , Amino Acid Sequence , Crystallization , Humans , Hydroxyprostaglandin Dehydrogenases/genetics , Leishmania major/genetics , Molecular Sequence Data , NADP/genetics , Protein Structure, Secondary , Trypanosoma cruzi/geneticsABSTRACT
The great power of protein crystallography to reveal biological structure is often limited by the tremendous effort required to produce suitable crystals. A hybrid crystal growth predictive model is presented that combines both experimental and sequence-derived data from target proteins, including novel variables derived from physico-chemical characterization such as R(30), the ratio between a protein's DSF intensity at 30°C and at T(m). This hybrid model is shown to be more powerful than sequence-based prediction alone - and more likely to be useful for prioritizing and directing the efforts of structural genomics and individual structural biology laboratories.