VU Research Portal Trypanosoma brucei phosphodiesterase B1 as a drug target for Human African Trypanosomiasis Jansen, C.J.W. 2015 document version Publisher's PDF, also known as Version of record Link to publication in VU Research Portal citation for published version (APA) Jansen, C. J. W. (2015). Trypanosoma brucei phosphodiesterase B1 as a drug target for Human African Trypanosomiasis. 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Oct. 2021 An introduction into phosphodiesterases and their potential role as drug targets for neglected diseases Chapter 1 4 CHAPTER 1 1.1 Human African Trypanosomiasis Human African Trypanomiasis (HAT), also known as African sleeping sickness, is a deadly infectious disease caused by the kinetoplastid Trypanosoma brucei (Figure 1A). The spread of HAT is restricted to sub-Saharan Africa by the prevalence of the disease vector, several species of tsetse fly (genus Glossina, Figure 1B).1, 2 As shown in Figure 1C, the tsetse flies are found across 38 central African countries, where they are able to cause localized epidemics following infection by feeding on the blood of infected humans, livestock or wild animals.3 Following eradication efforts and given that not all tsetse flies become infected or are able to become infected, 24 countries have reported recent cases of HAT, leaving an estimated 70 million people at risk of infection.2, 4, 5 While the number of annually reported cases has remained under 10,000 since 2009, the disease has had three major epidemics in the last century.1, 6, 7 Furthermore, efforts to control the spread of the disease are hampered by the remoteness of outbreaks and conflicts in several endemic regions, leading to incomplete reporting of new HAT cases and an increased risk of another major epidemic.8 Figure 1: A) A stylized representation of Trypanosoma brucei infected blood based on scanning and transmission electron micrographs.9 B) A tsetse fly drawing blood with its 5 proboscis inserted in human skin.10 C) A map showing the spread of the distribution of the tsetse fly across Africa overlaid with the cases of HAT reported over the period 2000- 2009.11 The images in A, B and C are reproduced without adaptation from the source referenced. Two different forms of HAT exist and these result from infection with either T.b. gambiense or T.b. rhodesiense. The two strains have different epidemiologies, with T.b. gambiense endemic to central and western Africa accounting for 97% of infections and T.b. rhodesiense endemic to the eastern Africa.5 Disease progression and therapy are also affected by the strain. Infection by T.b. gambiense progresses from the haemolymphatic phase (stage 1) to the meningoencephalitis phase (stage 2) after 2-4 years. In a few cases patients have cleared the parasites during stage 1 without treatment, however once a patient passes to stage 2 the disease is invariably fatal without treatment.12 Infection by T.b. rhodesiense is more aggressive with progression from stage 1 to stage 2 usually occurring within 8 weeks and no cases of parasitological clearance without treatment have been reported.13 The treatment options for HAT have improved in recent years. However significant issues remain.14 For HAT caused by T.b. gambiense the first-line treatment for stage 1 is pentamidine and the second-line treatment is suramin, for stage 2 the first-line treatment is NECT (nifurtimox and eflornithine) and the second-line treatment is melarsoprol (Figure 2). For HAT caused by T.b. rhodesiense the first-line treatment for stage 1 is suramin and the second line treatment is pentamadine, for stage 2 the first-line treatment is melarsoprol and there is no second-line treatment.15 Each of these treatments are regarded as essential medicines by the WHO and they are now available free of charge to endemic countries. The introduction of eflornithine and later NECT to replace melarsoprol as the treatment for stage 2 HAT caused by T.b. gambiense, has improved treatment outcomes significantly, with deaths following treatment dropping from about 6% to 1%.16, 17 The situation for stage 2 HAT caused by T.b. rhodesiense has not seen such an improvement as eflornithine and NECT are not effective against this parasite strain. Given the similarities between the two subspecies, this is an indication that resistance to drug treatments may be able to arise in HAT caused by T.b. gambiense. Resistance to treatments is an ongoing threat for parasitic 6 diseases and in the case of HAT, eradication of the disease is unlikely given the reservoir CHAPTER 1 of trypanosomes found in livestock and wild life.18 This necessitates the discovery of novel drugs to prepare for the eventuality of resistance to current treatments. Given the areas to which HAT is endemic, a further complication in treating HAT is that each of these treatments requires either intravenous or intramuscular administration. Therefore, a key feature sought in novel treatments is that they be orally available. Figure 2: The chemical structures of compounds that form part of the WHO recommended essential medicines for the treatment of HAT, suramin, pentamidine, merlasoprol, and the two active components of NECT treatment, nifurtimox and eflornithine. Two compounds currently in clinical trials for the treatment of HAT, SCYX-7158 and fexinidazole. 7 There are several promising drug candidates undergoing clinical trials for the treatment of HAT. A promising compound is SCYX-7158, a benzoxaborole currently in phase 1 clinical trials and showing the potency and pharmacokinetics consistent with a single dose oral treatment for stage 2 HAT (Figure 2).19 This compound was discovered and developed using phenotypic screening and the mode of action remains unknown. To find out if the mode of action might be related to phosphodiesterase (PDE) inhibition, SCYX-7158 was synthesized in our lab and found to inhibit human PDE4B with an IC50 of 80 µM and TbrPDEB1 with an IC50 of >100 µM, ruling out PDE inhibition as a mode of action (unpublished). Another compound in development for the treatment of HAT is fexinidazole which is currently undergoing phase II/III clinical trials.20 This too would be an orally available drug effective against both strains of trypanosomes causing HAT. While such advances are needed, even with a new drug reaching the market the need for novel treatments would remain, since resistance can be expected to arise. A further benefit to the discovery of multiple novel drugs is that the process of resistance can be slowed significantly by the use of multiple drugs with orthogonal modes of action. For these reasons, developing new drugs with novel targets for the treatment of HAT remains a high priority for institutes tackling neglected diseases.21 The drug target that was explored in the work presented here is phosphodiesterases, specifically TbrPDEB1. 1.2 Phosphodiesterases Phosphodiesterases (PDEs) play a key role in signaling cascades that involve the second messenger molecules cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Through the hydrolysis of cAMP and cGMP to AMP and GMP respectively, PDEs regulate the concentrations of the cyclic nucleotides and thereby signal transmission. Cyclic nucleotide signaling in parasites shows potential as a drug target due to the important role PDEs play in the life cycles of many parasites.22-24 The Trypanosoma brucei cyclic nucleotide signaling pathway begins with the activation of membrane bound adenylyl cyclases (ACs).25 The ACs are bound to the cellular surface with specific distribution densities determined by localization (for example in flagellar ACs) and life cycle stage.26 The ACs are activated by an extracellular receptor domain that passes a signal 8 24, 27 via a transmembrane domain to an intracellular catalytic domain. The activated catalytic CHAPTER 1 domain converts ATP to cAMP passing the signal into cell and amplifying it though the creation of many second messenger molecules. The ACs act as sensors, but also play a role in inhibiting the innate immune response of the host.28 Downstream effectors of cAMP signaling are still poorly understood, however cAMP dependent protein kinase As (PKAs) have been identified.29 The concentration of cAMP determines signaling to downstream effectors and signaling is regulated by the activity of PDEs that hydrolyze the cAMP into AMP, reducing the cAMP concentration. The cyclic nucleotide signaling pathway has been found to play important roles in motility and cytokinesis of Trypanosoma brucei.30, 31. There are 5 trypanosomal PDE genes, A, B1, B2, C and D and the same classification of genes is seen in leishmanial parasites. Plasmodial parasites also encode 5 PDE genes, however they are classed, αA,