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In the Acknowledgments, the following text should be added at the end, as it was missed through oversight in the original submission: "This work is based on research funded in part by the Bill & Melinda Gates Foundation (Investment ID OPP1093639) and with UK aid from the UK Government (Project 300504) through GALVmed. The findings and conclusions contained within are those of the authors and do not necessarily reflect positions or policies of the Bill & Melinda Gates Foundation or the UK Government."

SUMMARY Dr David Livingstone died on May 1st 1873. He was 60 years old and had spent much of the previous 30 years walking across large stretches of Southern Africa, exploring the terrain he hoped could provide new environments in which Europeans and Africans could cohabit on equal terms and bring prosperity to a part of the world he saw ravaged by the slave trade. Just days before he died, he wrote in his journal about the permanent stream of blood that he was emitting related to haemorrhoids and the acute intestinal pain that had left him incapable of walking. What actually killed Livingstone is unknown, yet the years spent exploring sub-Saharan Africa undoubtedly exposed him to a gamut of parasitic and other infectious diseases. Some of these we can be certain of. He wrote prolifically and described his encounters with malaria, relapsing fevers, parasitic helminths and more. His graphic writing allows us to explore his own encounters with tropical diseases and how European visitors to Africa considered them at this time. This paper outlines Livingstone's life and his contributions to understanding parasitic diseases.

The parasitic protozoan Trypanosoma brucei causes Human African Trypanosomiasis and Nagana in other mammals. These diseases present a major socio-economic burden to large areas of sub-Saharan Africa. Current therapies involve complex and toxic regimens, which can lead to fatal side-effects. In addition, there is emerging evidence for drug resistance. AN5568 (SCYX-7158) is a novel benzoxaborole class compound that has been selected as a lead compound for the treatment of HAT, and has demonstrated effective clearance of both early and late stage trypanosomiasis in vivo. The compound is currently awaiting phase III clinical trials and could lead to a novel oral therapeutic for the treatment of HAT. However, the mode of action of AN5568 in T. brucei is unknown. This study aimed to investigate the mode of action of AN5568 against T. brucei, using a combination of molecular and metabolomics-based approaches.Treatment of blood-stage trypanosomes with AN5568 led to significant perturbations in parasite metabolism. In particular, elevated levels of metabolites involved in the metabolism of S-adenosyl-L-methionine, an essential methyl group donor, were found. Further comparative metabolomic analyses using an S-adenosyl-L-methionine-dependent methyltransferase inhibitor, sinefungin, showed the presence of several striking metabolic phenotypes common to both treatments. Furthermore, several metabolic changes in AN5568 treated parasites resemble those invoked in cells treated with a strong reducing agent, dithiothreitol, suggesting redox imbalances could be involved in the killing mechanism., Author summary Human African Trypanosomiasis (HAT) is a neglected tropical disease affecting mostly rural communities of sub-Saharan Africa. New drugs for this parasitic disease have been slow to develop, but one new class of drug, the benzoxaboroles, has proven to be highly effective in preclinical models and holds promise for on-going clinical trials. The lead compound in this class is AN5568, also known as SCYX-7158. Here, we study the effects of AN5568 on intracellular metabolism of the parasite and show changes to pathways involved in S-adenosyl-methionine metabolism. Further work shows similarities between this drug and sinefungin, an inhibitor of methyltransferases and dithiothreitol, an endoplasmic reticulum stress inducer. These phenotypes provide new clues to the mechanism of action of this compound.

Kinetoplastid parasites—trypanosomes and leishmanias—infect millions of humans and cause economically devastating diseases of livestock, and the few existing drugs have serious deficiencies. Benzoxaborole-based compounds are very promising potential novel anti-trypanosomal therapies, with candidates already in human and animal clinical trials. We investigated the mechanism of action of several benzoxaboroles, including AN7973, an early candidate for veterinary trypanosomosis. In all kinetoplastids, transcription is polycistronic. Individual mRNA 5'-ends are created by trans splicing of a short leader sequence, with coupled polyadenylation of the preceding mRNA. Treatment of Trypanosoma brucei with AN7973 inhibited trans splicing within 1h, as judged by loss of the Y-structure splicing intermediate, reduced levels of mRNA, and accumulation of peri-nuclear granules. Methylation of the spliced leader precursor RNA was not affected, but more prolonged AN7973 treatment caused an increase in S-adenosyl methionine and methylated lysine. Together, the results indicate that mRNA processing is a primary target of AN7973. Polyadenylation is required for kinetoplastid trans splicing, and the EC50 for AN7973 in T. brucei was increased three-fold by over-expression of the T. brucei cleavage and polyadenylation factor CPSF3, identifying CPSF3 as a potential molecular target. Molecular modeling results suggested that inhibition of CPSF3 by AN7973 is feasible. Our results thus chemically validate mRNA processing as a viable drug target in trypanosomes. Several other benzoxaboroles showed metabolomic and splicing effects that were similar to those of AN7973, identifying splicing inhibition as a common mode of action and suggesting that it might be linked to subsequent changes in methylated metabolites. Granule formation, splicing inhibition and resistance after CPSF3 expression did not, however, always correlate and prolonged selection of trypanosomes in AN7973 resulted in only 1.5-fold resistance. It is therefore possible that the modes of action of oxaboroles that target trypanosome mRNA processing might extend beyond CPSF3 inhibition.

Animal African trypanosomiasis (or nagana) is a wasting livestock disease found in sub-Saharan Africa and caused by protozoan parasites Trypanosoma congolense, T. vivax and T. brucei. Chemotherapy (mostly with the diamidine diminazene aceturate) and chemoprophylaxis (mostly with the phenanthridine isometamidium chloride) are essential for disease control. However, as current treatments lose efficacy due to increased drug resistance, the need for new veterinary trypanocides becomes a high-priority. Trypanosoma spp. are one of the genera of parasites that are susceptible to DNA minor groove binder drugs synthesised at the University of Strathclyde (S-MGBs). All three of the above African species are susceptible to S-MGBs and in addition, the South American species, T. cruzi has been shown to be similarly susceptible. One of the principal challenges to obtaining compounds useful in the field is to achieve activity across the range of infectious species so that characterisation of the infection is not required at diagnosis. Over 100 S-MGBs have been evaluated at the University of Glasgow and the Swiss Tropical Health Institute, Basel, and compounds with development potential have been identified. As an example, S-MGB 234 has been shown to be curative in in vivo models of trypanosome infection in mice. Importantly S-MGB 234 does not show cross resistance with other antitrypanosomal drugs such as diminazine, isometamidium, or ethidium bromide, which is consistent with a different route into the parasite’s cell. S-MGBs that contain alkene links, such as in S-MGB 234, are the most active sub-class of S-MGB and point the way towards structural optimisation.

LED fluorescence microscopy offers potential benefits to the diagnosis of human African trypanosomiasis, as well as to other aspects of diseases management, such as detection of drug resistant strains. To advance such approaches reliable and specific fluorescent markers to stain parasites in human fluids are needed. Here we report a series of novel green fluorescent diamidines and their suitability as probes to stain trypanosomes.

The treatment of Human African Trypanosomiasis remains a major unmet health need in sub-Saharan Africa. Approaches involving new molecular targets are important and pteridine reductase 1 (PTR1), an enzyme that reduces dihydrobiopterin in Trypanosoma spp. has been identified as a candidate target and it has been shown previously that substituted pyrrolo[2,3-d]pyrimidines are inhibitors of PTR1 from T. brucei (J. Med. Chem. 2010, 53, 221-229). In this study, 61 new pyrrolo[2,3-d]pyrimidines have been prepared, designed with input from new crystal structures of 23 of these compounds complexed with PTR1, and evaluated in screens for enzyme inhibitory activity against PTR1 and in vitro antitrypanosomal activity. 8 compounds were sufficiently active in both screens to take forward to in vivo evaluation. Thus although evidence for trypanocidal activity in a stage I disease model in mice was obtained, the compounds were too toxic to mice for further development.