Antitrypanosomal quinazolines targeting lysyl-tRNA synthetase show partial efficacy in a mouse model of acute Chagas disease

Abstract

The protozoan parasite Trypanosoma cruzi causes Chagas disease, which is among the deadliest parasitic infections in Latin America. Current therapies are toxic and lack efficacy against the chronic stage of infection; thus, new drugs are urgently needed. Here, we describe a previously unidentified series of quinazoline compounds with potential against Trypanosoma cruzi and the related trypanosomatid parasites Trypanosoma brucei and Leishmania donovani. We demonstrated partial efficacy of a lead quinazoline compound in a mouse model of acute Chagas disease. Mechanism of action studies using several orthogonal approaches showed that this quinazoline compound series targeted the ATP-binding pocket of T. cruzi lysyl-tRNA synthetase 1 (KRS1). A high-resolution crystal structure of KRS1 bound to the drug indicated binding interactions that led to KRS1 inhibition. Our study identified KRS1 as a druggable target for treating T. cruzi infection in a mouse model. This quinazoline series shows potential for treating Chagas disease but will require further development to become a future treatment for this neglected disease.

Fig. 1. Generation of trifluorinated quinazolines.

(A) Schematic shows the synthesis strategy for generating trifluorinated quinazolines. (B) Shown are the two lead compounds, DMU371 and DMU759, that were further characterized and their mechanism of action investigated. Nu denotes nucleophilic addition.

Fig. 2. Genome-wide overexpression library screen with DMU371.

The library comprises a pooled population of bloodstream form T. brucei transfected with overexpression plasmids containing genomic DNA fragments between 3 and 10 kb in size. The final transfected library provides 10-fold genome coverage with >95% of T. brucei genes represented. The parasite library is grown in the presence of test drug and genomic DNA is harvested from those able to survive selection. Next generation sequencing identifies the genomic fragments harbored by “drug resistant” parasites revealing overexpressed proteins and potential drug targets (19). (A) Growth of the genome-wide overexpression library in the presence or absence of DMU371 selection at a concentration equivalent to twice the established EC₅₀ value. Asterisk indicates sub-culturing of the culture and addition of fresh compound. The arrow indicates when the library was harvested for genomic DNA preparation. (B) Shown is the genomewide map resulting from the overexpression library screen with DMU371. RPKM, reads per kilobase of transcript per million mapped reads. Inset shows the top fragment hit of this library screen containing the full coding sequence of lysyl-tRNA synthetase (Tb927.8.1600). Gene of interest is highlighted in green, other protein-coding regions are indicated in black. Blue and pink peaks represent forward and reverse barcodes (in the sense orientation), respectively. Grey peaks are all other reads (table S3).

Fig. 3. Isothermal TPP for proteome scale identification of quinazoline targets.

Isothermal TPP plots show log₂ fold change in abundance between compound-treated and untreated T. cruzi epimastigote lysates subjected to thermal shock at 48°C across two biological replicates. All proteins identified with >2 unique peptides are shown. “Hits” demonstrating a >1.5-fold shift in abundance (increase or decrease) are shown in blue. TcKRS1 is indicated in red. Hits for experiments with DMU371 and DMU759 are noted in tables S5 and S6, respectively.

Fig. 4. Crystal structure of TcCpKRS1 with DMU371 bound to the active site.

(A) Shown is the crystal structure of TcCpKRS1 with DMU371 (yellow) bound to the active site in the presence of lysine substrate (green). Water molecules are indicated in red and hydrogen bonding interactions formed with water molecules are represented as black dashed lines. Key residues are labeled using CpKRS1 numbering. C. parvum residues mutated to their T. cruzi counterparts are shown in bold italics. (B) Three unoccupied regions of the active site that could be exploited for future quinazoline analog development are highlighted with red, blue and dark grey ovals. The surface of the enzyme is shown in grey and water molecules are shown in red. (C) The most likely conformation of S309L is shown in magenta. Proximity to the cyclopentyl moiety of DMU371 (yellow) and potential for steric clash is evident.

Fig. 5. Assessment of DMU759 efficacy in a murine model of acute Chagas disease.

(A) BALB/c mice infected with bioluminescent T. cruzi CL-Brener strain were treated for 10 days post-infection (dpi) for 5 days by oral gavage with 100 mg/kg benznidazole (BNZ) (n=3) once daily, or with DMU759 50 mg/kg (n=3) twice daily. Ventral images of each mouse are shown at various timepoints following infection. Heat maps are shown on a log₁₀ scale and indicate bioluminescence intensity related to parasite burden (blue, low intensity; red, high intensity). The minimum and maximum radiances for the pseudocolor scale are shown. (B) Graph presents the mean bioluminescence (photons per second, p/s) determined by in vivo imaging of treated and untreated mice infected with T. cruzi. Treatment groups and dosing regimens are indicated. The black horizontal unbroken line indicates background bioluminescence total flux established from uninfected mice (n=3), with the dashed line indicating SD above the average.