Combining tubercidin and cordycepin scaffolds results in highly active candidates to treat late-stage sleeping sickness

African trypanosomiasis is a disease caused by Trypanosoma brucei parasites with limited treatment options. Trypanosoma is unable to synthesize purines de novo and relies solely on their uptake and interconversion from the host, constituting purine nucleoside analogues a potential source of antitrypanosomal agents. Here we combine structural elements from known trypanocidal nucleoside analogues to develop a series of 3’-deoxy-7-deazaadenosine nucleosides, and investigate their effects against African trypanosomes. 3’-Deoxytubercidin is a highly potent trypanocide in vitro and displays curative activity in animal models of acute and CNS-stage disease, even at low doses and oral administration. Whole-genome RNAi screening reveals that the P2 nucleoside transporter and adenosine kinase are involved in the uptake and activation, respectively, of this analogue. This is confirmed by P1 and P2 transporter assays and nucleotide pool analysis. 3’-Deoxytubercidin is a promising lead to treat late-stage sleeping sickness.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.

n/a Confirmed
The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Genome sequence and annotation information was obtained from TritrypDB (http://www.tritrypDB.org).

nature research | reporting summary
October 2018 Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Sample size
Animal experimental studies were with a lower number of animals (n= 3-4/group) than calculated by our standard power analysis, suggesting the use of 6 mice/group (2-sample t-test, power= 80%, alpha=0.05). Ethical considerations, combined with experience with these models and a relative low variability have been the basis of justifying these reduced numbers. Observations were confirmed in an independent repeat experiment.
Data exclusions BLI images of mice that succumbed due to the stage II CNS disease prior to the start of treatment were not included. These data are not relevant to the action of the compound.

Replication
All compound evaluations and in vivo efficacy studies were replicated in at least two independent experiments.
Randomization Allocation of animals to experimental groups was random.

Blinding
Follow-up of parasitemia and BLI imaging were not conducted in a blinded fashion. Drug administration of the reference drug (topical) and the test compound and vehicle control (oral gavage) were different and could therefore not be blinded.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Wild animals
No wild animals were used in this study.

Field-collected samples
No field-collected samples were used in this study.

Ethics oversight
Animal experimental work was approved by the ethical committee of the University of Antwerp (UA-ECD 2015-90).
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation Culture-derived T. brucei NY-SM cells were exposed to different concentrations of compound 9 for 24 hours. Cells were harvested and washed with PBS before staining with Hoechst 33342 at 5 μg/mL for 25 minutes at 37 °C.

Instrument
MACSQuant flow cytometer (Miltenyi Biotec) Software FlowJo X Cell population abundance Analyses were performed on pure T. brucei parasite cultures Gating strategy Parasites were analyzed within an appropriate FCS/SSC gate, followed by a singlet (SSC-A/SSC-W) gating. The gating strategy is documented in Fig. 7.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.