Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis

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.

Keywords: cryptosporidiosis; malaria; tRNA synthetase.

Fig. 1.

The structure of cladosporin 1, evolution of the lead 5 from the screening hit 2. Enzyme data were obtained with the Kinase-Glo assay. MLM, mouse liver microsomes.

Fig. 2.

Binding modes of ligands bound to PfKRS1 and CpKRS. (A) PfKRS1:Lys:2 showing the binding mode of 2 (C atoms, gold) bound to the ATP site of PfKRS1 (PDB ID code 6AGT) superimposed upon PfKRS1:Lys:cladosporin (cladosporin C atoms, slate; PDB ID code 4PG3). (B) PfKRS1:5 showing binding mode of 5 bound to PfKRS1 (PDB ID code 6HCU). (C) Overlay of CpKRS:Lys:cladosporin (C atoms, gold; PDB ID code 5ELO) compared with PfKRS1:Lys:cladosporin (C atoms, gray; PDB ID code 4PG3). Nonconserved residues within the ligand binding site are labeled. (D) CpKRS:Lys:5 showing binding mode of 5 (C atoms, gold) in complex with CpKRS:Lys (C atoms, gray; PDB ID code 6HCW). H-bonds are shown as dashed lines, and key residues are labeled for clarity.

Fig. 3.

Heatmap showing effects of compounds 1 and 5 on the melting temperature (ΔT_m) of KRS enzymes.

Fig. 4.

In vivo efficacy of compound 5 in mouse models of malaria and cryptosporidiosis infections. (A) The in vivo efficacy data for compound 5 in P. falciparum-infected SCID mice. (B) The levels of compound 5 in blood of the mice during the malaria efficacy experiment, 24 h after the first oral dose and 24 h after the administration of the last dose on day 4. The symbols represent the same individuals depicted in plot A. (C) Determination of daily dose to reduce P. falciparum parasitemia by 90% at day 5 of the experiment. (D) Determination of daily exposure in blood to reduce P. falciparum parasitemia by 90% at day 5 of the experiment. (E) Efficacy of compound 5 in the Nluc-cryptosporidiosis INF-γ–knockout mouse model when dosed orally at a concentration of 20 mg/kg once a day for 7 d. Orange bars show fecal parasite levels for mice treated with compound 5 and blue bars for the vehicle-treated control mice. At day 8, there is a 10,000-fold reduction in parasite levels compared with control. n = 4 mice per group. (F) Efficacy of compound 5 in the cryptosporidiosis NOD SCID gamma mouse model when dosed orally at a concentration of 20 mg/kg once a day for 7 d. Orange bars represent fecal parasite levels for mice treated with compound 5, green bars mice treated with paromomycin (2,000 mg/kg), and blue bars for vehicle-treated control mice. At day 8, there is a 100-fold reduction in parasite levels compared with control.

Fig. 5.

Differential binding mode of compound 5 to PfKRS1, HsKRS, and CpKRS. (A) Sequence alignment of the active sites of PfKRS1, HsKRS, and CpKRS. Two nonconserved residues suggested to be responsible for the selectivity of compound 5 are highlighted in red boxes. (B–D) Multiframe representations of the active site from the MD simulations of the active sites of PfKRS1 (B) (cyan), HsKRS (C) (green), and CpKRS (D) (orange) in the absence (Upper) and presence (Lower) of compound 5. Lysine is shown in magenta, compound 5 is shown in purple, and nonconserved residue labels are highlighted in boxes.