The development of chemotherapies against eukaryotic pathogens is especially challenging because of both the evolutionary conservation of drug targets between host and parasite, and the evolution of strain-dependent drug resistance. There is a strong need for new nontoxic drugs with broad-spectrum activity against trypanosome parasites such as Leishmania and Trypanosoma. A relatively untested approach is to target macromolecular interactions in parasites rather than small molecular interactions, under the hypothesis that the features specifying macromolecular interactions diverge more rapidly through coevolution. We computed tRNA Class-Informative Features in humans and independently in eight distinct clades of trypanosomes, identifying parasite-specific informative features, including base pairs and base mis-pairs, that are broadly conserved over approximately 250 million years of trypanosome evolution. Validating these observations, we demonstrated biochemically that tRNA:aminoacyl-tRNA synthetase (aaRS) interactions are a promising target for antitrypanosomal drug discovery. From a marine natural products extract library, we identified several fractions with inhibitory activity toward Leishmania major alanyl-tRNA synthetase (AlaRS) but no activity against the human homolog. These marine natural products extracts showed cross-reactivity towards Trypanosoma cruzi AlaRS indicating the broad-spectrum potential of our network predictions. We also identified Leishmania major threonyl-tRNA synthetase (ThrRS) inhibitors from the same library. We discuss why chemotherapies targeting multiple aaRSs should be less prone to the evolution of resistance than monotherapeutic or synergistic combination chemotherapies targeting only one aaRS.

43The development of chemotherapies against eukaryotic pathogens is especially 44 challenging because of both the evolutionary conservation of drug targets between host 45 and parasite, and the evolution of strain-dependent drug resistance. There is a strong 46 need for new nontoxic drugs with broad-spectrum activity against trypanosome 47 parasites such as Leishmania and Trypanosoma. A relatively untested approach is to 48 target macromolecular interactions in parasites rather than small molecular interactions, 49 under the hypothesis that the features specifying macromolecular interactions diverge 50 more rapidly through coevolution. We computed tRNA Class-Informative Features in 51 humans and eight clades of trypanosomes, identifying parasite-specific informative 52 features (including base-pairs and base mis-pairs) that are broadly conserved over 53 approximately 250 million years of trypanosome evolution. Validating these 54observations, we demonstrated biochemically that tRNA:aminoacyl-tRNA synthetase 55 interactions are a promising target for anti-trypanosomal drug discovery. From a marine 56 natural products extract library, we identified several fractions with inhibitory activity 57 toward Leishmania major alanyl-tRNA synthetase (AlaRS) but no activity against the 58 human homolog. These marine natural products extracts showed cross-reactivity 59 towards Trypanosoma cruzi AlaRS indicating the broad-spectrum potential of our 60 network predictions. These findings support a systems biology model in which 61 combination chemotherapies that target multiple tRNA-synthetase interactions should 62 be comparatively less prone to the emergence of resistance than conventional single 63 drug therapies. 64 65 Author Summary 66Trypanosome parasites pose a significant health risk worldwide. Conventional drug 67 development strategies have proven challenging given the high conservation between 68 humans and pathogens, with off-target toxicity being a common problem. Protein 69 synthesis inhibitors have historically been an attractive target for antimicrobial discovery 70 against bacteria, and more recently for eukaryotic pathogens. Here we propose that 71 exploiting pathogen-specific tRNA-synthetase interactions offers the potential for highly 72 targeted drug discovery. To this end, we improved tRNA gene annotations in 73 trypanosome genomes, identified functionally informative trypanosome-specific tRNA 74 features, and showed that these features are highly conserved over approximately 250 75 million years of trypanosome evolution. Highlighting the species-specific and broad-76 spectrum potential of our approach, we identified natural product inhibitors against the 77 parasite translational machinery that have no effect on the homologous human enzyme. 78 79 nontoxic drugs with broad-spectrum activity against different species of Leishmania and 89 other trypanosomes [6,7]. 90 Given their essential role in protein synthesis, aminoacyl-tRNA synthetases 91 (aaRSs) have been an attractive target for antimicrobial ...

Motivation Structure-conditioned information statistics have proven useful to predict and visualize tRNA Class-Informative Features (CIFs) and their evolutionary divergences. Although permutation P-values can quantify the significance of CIF divergences between two taxa, their naive Monte Carlo approximation is slow and inaccurate. The Peaks-over-Threshold approach of Knijnenburg et al. (2009) promises improvements to both speed and accuracy of permutation P-values, but has no publicly available API. Results We present tRNA Structure–Function Mapper (tSFM) v1.0, an open-source, multi-threaded application that efficiently computes, visualizes and assesses significance of single- and paired-site CIFs and their evolutionary divergences for any RNA, protein, gene or genomic element sequence family. Multiple estimators of permutation P-values for CIF evolutionary divergences are provided along with confidence intervals. tSFM is implemented in Python 3 with compiled C extensions and is freely available through GitHub (https://github.com/tlawrence3/tSFM) and PyPI. Availability and implementation The data underlying this article are available on GitHub at https://github.com/tlawrence3/tSFM. Supplementary information Supplementary data are available at Bioinformatics online.