**Acknowledgements**

One of the strengths of the Maybridge Rule of 3 Fragment Library is the chemical diversity, additionally a range of analogous structures can normally be found within it allowing initial structure activity relationships to be formulated. There are several close analogues of com‐ pound 278 (1-methyl-(1H-indol-6-yl)methanol), which highlight that the N-methyl indole moiety seems necessary to have any *Tb*CK inhibition and the methanol portion of the mole‐ cule can not be replaced by a carboxylic acid. Investigation of the CHEMBL database for similar compounds identified 1-Methyl-1H-pyrrolo[2,3-c]pyridine (CHEMBL594467) which was screened as one of a library of tricyclic and bicyclic analogues of indoloquinoline alka‐ loids against a variety of protozoan parasites. The analogue mentioned here showed weak trypanocidal activity (624 µM) against *Trypanosoma brucei rhodesiense*, but significantly better

Another analogue, 1-Methyl-1H-indole (CHEMBL19912) has been shown to interact with human intracellular adhesion molecules and highlights the importance of selectivity [53]. 1H-indol-5-yl-methanol (CHEMBL1650258) has previously been screened against *Leishmania*

For the relatively simple compound 346 (3-pyridin-3-ylaniline), there are several analo‐ gous structures in the library, including compound 262 (2-(1H-imidazo-1-yl)aniline) which shows ~55% *Tb*CK enzyme inhibition and is trypanocidal. Compound 347 (4-pyri‐ din-3-ylaniline) is a structural isomer of 346 but shows no *Tb*CK enzyme inhibition and is not trypanocidal. The only related structure in the CHEMBL database was 3-(pyri‐ din-3-yl)benzenaminium (CHEMBL1778131) which was shown to be a weakinhibitor of

In this study, screening of a comparatively small fragment library by two different screening methods has allowed identification of several compounds that interact with and inhibit *Tb*CK, a genetically validated drug target against African sleeping sickness. Some of the in‐ hibitory fragments were also selectively trypanocidal, considering these are relatively sim‐ ple molecules with no optimization, finding low µΜ inhibitors is very encouraging. Moreover some of the morphological phenotypes of these trypanocidal compounds include cell-cycle arrests similar to those observed for the *Tb*CK conditional knockout grown under

This study highlights that if faced with a drug target that is problematic to screen, prior ther‐ mal shift analysis could significantly triage the number of compounds to be screened, there‐ by significantly increasing the potential to identify lead compounds. This approach obviously has the limitation that potential inhibitors could be missed if they do not signifi‐

Future follow up work with *Tb*CK will include expanding the structure activity relationship of our most promising hits identified by this study. Their trypanocidal mode of action will

as a potential PTR1 inhibitor but was shown to be inactive at 500 µM [54]

(37 µM) against *Plasmodium falciparum* [52].

metallo-β−lactamase IMP-1 [55].

**4. Conclusions**

426 Drug Discovery

permissive conditions.

cantly alter the Tm of the protein.

This work was supported in part by a Wellcome Trust Senior Research Fellowship (067441) and Wellcome Trust project grants 086658 and 093228. We thank the late and sorely missed Dr Rupert Russell (St Andrews), supported by SUSLA, for access to the May Bridge Rule of 3 Fragment Library.
