tRNA synthetases are established targets for antimicrobial chemotherapy, the foremost example being the bacterial isoleucyl-tRNA synthetase, which is the molecular target of mupirocin, an antibiotic used topically for the treatment of bacterial infections, including Staphylococcus aureus
infections. This precedent indicated to us the potential for developing selective inhibitors of aaRS enzymes for other infectious pathogens such as trypanosomes. The MetRS enzyme was a particularly attractive target for piggyback drug development for neglected tropical diseases (9
) because of the existence of potent MetRS inhibitors under development in Pharma for other indications (22
). The similarities between the bacterial MetRS enzymes and those of T. brucei
and T. cruzi
led to our hypothesis that the existing compounds might bind trypanosomal MetRS enzymes and give rise to antitrypanosomal activity.
Before the activity of compounds was investigated, genetic experiments were conducted to establish that the MetRS enzyme was essential for trypanosome growth. The RNA interference method was used to knock down mRNA levels of the single MetRS gene identified in the T. brucei genome. The observed RNAi results () confirmed our expectation that MetRS of T. brucei is critical for normal growth since protein synthesis is obviously dependent on minimum levels of charged tRNAMet. Cell growth was inhibited by a factor of 106; however, complete killing was not detected, possibly because the RNAi method resulted in incomplete knockdown of the MetRS transcript to only 57 to 77% below normal levels in two clones.
Three compounds (compounds 2, 3, and 7) were synthesized on the basis of published reports of MetRS inhibitors that had antibacterial activity (15
). These compounds were first tested on T. brucei
cultures and observed to have remarkably potent activity, with EC50
s of 8, 30, and 75 nM, respectively (). In order to determine if the compounds bound to the T. brucei
MetRS enzyme, the thermal melt assay using recombinant enzyme was employed. Pronounced shifts in melting temperatures were observed (ΔTm
s, 12.9, 12.2, and 10.1°C, respectively). Generally, temperature shifts exceeding 2°C are considered significant; thus, the data indicated that the compounds were tightly bound by the recombinant MetRS enzyme. With these encouraging results, an additional 18 compounds were synthesized to further explore structure-activity relationships, primarily focusing on the phenyl group on the left side of the molecule (). The compound series resulted in a broad range of activities against T. brucei
cultures from the most active compound, compound 1, with an EC50
of 4 nM, to the least active compound, compound 21, with an EC50
of >5,000 nM. All the compounds were tested in the thermal melt assay, and the correlation between ΔTm
was plotted (, upper line). A very high correlation was observed (R2
= 0.92), meaning that compounds that bind the enzyme with the highest affinity were the most potent at inhibiting T. brucei
cell growth. This was evidence that the compounds act on the parasites through inhibition of the target enzyme and not by another off-target mechanism.
The compounds were also tested against the other major trypanosome pathogen, T. cruzi, and were again observed to have potent growth inhibition activity (). There was a high correlation between the compounds most active against T. brucei and T. cruzi, which is expected due to the 100% sequence identity in the predicted inhibitor binding sites (see Table S4 in the supplemental material). The EC50s against T. cruzi were generally higher (by a factor of 10 to 40) than those observed against T. brucei. Since T. cruzi amastigotes are grown intracellularly in mammalian 3T3 fibroblasts, the higher observed EC50s suggest that the compounds may be partially excluded from the intracellular environment of the host cells. Experiments of binding of the compounds to recombinant T. cruzi MetRS yielded results very similar to those observed with the T. brucei MetRS enzyme. As before, the binding affinity (ΔTm) and cell growth inhibition (EC50) were highly correlated (, bottom line).
In order to establish that binding to the recombinant T. brucei
MetRS was related to inhibition of its enzymatic function, an aminoacylation assay was adopted for the T. brucei
enzyme. The method assesses the complete enzyme reaction by measuring the incorporation of [3
H]methionine into the tRNA substrate. The most potent compound, compound 1, inhibited the enzyme by 99.2% at 50 nM, suggesting that the 50% inhibitory concentration (IC50
) is well below 50 nM, which is consistent with the low EC50
of 4 nM observed on T. brucei
cells. An exact IC50
(possibly subnanomolar) for the highly potent compounds could not be determined because an enzyme concentration of 4 nM was necessary to give a robust signal in the assay. The correlation between enzyme inhibitory activity and the thermal melt results was very tight (R2
= 0.821, P
< 0.0001) and supports the use of the thermal method before an enzyme assay has been developed and optimized (17
The effect of the MetRS inhibitors on growth of the mammalian lymphocytic cell line CRL-8155 was also assessed. Since no inhibition of growth was observed at 20 μM for any of the compounds, this indicated a remarkably high selectivity index for the parasites over mammalian cells. The molecular modeling provides the explanation for why the compounds are unlikely to bind the human cytoplasmic MetRS enzyme (see Table S4 in the supplemental material). However, the differences between the trypanosomatid MetRS and the human mitochondrial MetRS are less pronounced (see Table S4 in the supplemental material) (discussed below). These data establish the potential to selectively inhibit the trypanosomatid MetRS and inhibit trypanosome growth while avoiding toxicity to mammalian cells.
In vitro washout experiments were conducted to determine the time required for compound 2 to lead to irreversible death of the parasites. When bloodstream forms of T. brucei were exposed to compound 2 at 8 times its EC50 for 72 h, the parasites did not recover. Similarly, when they were exposed to compound 2 at 16 times its EC50 for 48 h, they failed to recover. The conditions leading to irreversible growth arrest were similar to those of pentamidine, a drug in clinical use for early-stage HAT. Thus, sustained inhibition of MetRS completely kills T. brucei grown in vitro. This trypanocidal activity will be a necessary feature if the drug is to be used in late-stage HAT, where drugs that completely kill parasites are needed to clear infection from the central nervous system and cerebrospinal fluid.
Compound 1 had a short plasma half-life in mice (~1 h). For the purposes of these studies, we were able to attain adequate plasma concentrations of the compound in mice by administering it via continuous infusion using osmotic minipumps. Plasma concentrations of ~0.5 μM were measured in the mice receiving the minipumps. Using pumps designed to deliver compound for 3 days, profound suppression of parasitemia was observed. However, the parasites were not completely eradicated, as there was delayed recrudescence of parasitemia at about day 7 or 8 and the mice succumbed to the infection. There was also longer survival in the treated group (~9 to 10 days) than the controls (4 days). These data indicate that compound 1 had a profound effect to suppress parasitemia and prolong survival in mice. It also suggests that either higher blood levels or longer exposure to compound 1 are required for cures. Since plasma levels were ~100 times higher than the EC50 (4 nM), one might have expected complete clearance of the parasites on the basis of the results of the washout experiments described above. However, other factors, such as protein binding and access to parasites sequestered in tissues outside the blood compartment, may be reasons that complete cures were not observed.
The analysis of the amino acids that form the predicted binding site of diaryl diamine compounds allowed comparisons between the trypanosomatid and the human MetRS enzymes (see Table S5 in the supplemental material). Substantial differences between the trypanosomatid and human cytosolic MetRS enzymes were observed; specifically, 13 of 25 residues are different, suggesting that the same inhibitor would be unlikely to bind tightly to this pocket in both enzymes. This fits with published data that the diaryl diamine REP8839 () is essentially inactive on the human cytosolic enzyme, with Ki
being >20,000 nM (12
). Fewer differences in the inhibitor binding site were observed between trypanosomatid and human mitochondrial MetRS enzymes, with only 5 of 25 residues being different. This also fits with published data that REP8839 inhibits the human mitochondrial enzyme with a Ki
of 10 nM (12
). Of note, REP8839 inhibits the S. aureus
MetRS with a Ki
of 10 pM (12
), suggesting that extremely tight binding is possible. Unfortunately, we do not know the Ki
s of diaryl diamines against the trypanosomatid enzymes because the methods for accurately measuring the Ki
s of highly active compounds against MetRS (involving an adaptation of an ATP-PPi
exchange assay [12
]) have not been developed for the trypanosomatid enzymes. If the Ki
on trypanosomatid MetRS is very low (
1 nM), then it is possible that the greater activity of the diaryl diamines on trypanosomatid cells than mammalian cells () is due to selective activity on the trypanosomatid enzyme. However, it seems likely that there are other factors contributing to the lack of toxicity of the diaryl diamines against mammalian cells and live animals. One possibility is that the compounds may not penetrate into mammalian cells as well as they do into trypanosomatid cells. The observation that T. cruzi
(grown inside mammalian fibroblasts) has EC50
s as much as 44-fold higher than those for T. brucei
(grown in axenic culture), despite having identical binding pockets in the MetRS enzyme (see Table S4 in the supplemental material), suggests that the compounds do not efficiently penetrate the mammalian cell membrane. It could also be that so little compound passes through the second barrier (the mitochondrial membrane) that the mitochondrial MetRS is not significantly inhibited. As new compounds are made to alter the pharmacological properties, the changes could affect the permeability properties. As a result, it will be important to carefully test for toxicity on mammalian cells that may be mediated through inhibition of the mitochondrial MetRS.
Fig. 6. Structures of MetRS inhibitors in clinical trials for bacterial infections (5, 6, 25).
In summary, the MetRS enzyme has been genetically and chemically validated to be a drug target for T. brucei
and T. cruzi
. Importantly, we provide a proof of concept that the T. brucei
MetRS can be targeted for antitrypanosomal chemotherapy in the mouse model of T. brucei
infection. More work is required to improve the pharmacological properties of the compounds obtained so far, including improving oral bioavailability and penetration through the blood-brain barrier. The pharmacological issues are critical, as the highest priority with respect to African sleeping sickness is to discover new therapeutics (preferably orally administered) that will effectively treat stage 2 disease, i.e., when the parasites have entered the central nervous system (http://www.dndi.org/diseases/hat/target-product-profile.html
). The reported research is an exciting first step toward our goal of developing safe and effective new treatments for HAT and perhaps Chagas' disease.