Genetic and chemical validation of Trypanosoma brucei adenosine kinase, the intracellular target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine

(1)

Thesis

Reference

Genetic and chemical validation of Trypanosoma brucei adenosine kinase, the intracellular target of

4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine

GRAVEN, Patricia

Abstract

Human African Trypanosomiasis (HAT) is caused by African trypanosomes, a fatal disease if untreated. New safe and potent drugs are needed to treat HAT as the few available drugs are still unsatisfactory. The aim of the thesis is to understand the antiproliferative effect of compound 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine (1) which has recently been identified to hyperactivate Trypanosoma brucei adenosine kinase (TbAK). To this end, an ion-pair HPLC/UV method was developed and used to analyse changes in adenine purine levels of trypanosomes during different growth phases in absence or presence of 1. Several strains of Trypanosoma brucei were tested, e.g. i) the wild type strain, ii) a tetracycline inducible ak overexpression strain (hypothesizing that the presence of 1 is similar to overexpression of TbAK), and iii) a tetracycline inducible null mutant ak overexpression strain to provide insight into the effect of non-physiological high levels of TbAK on the parasite nucleoside/nucleotide metabolism.

GRAVEN, Patricia. Genetic and chemical validation of Trypanosoma brucei adenosine kinase, the intracellular target of

4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine. Thèse de doctorat : Univ. Genève, 2013, no. Sc. 4595

URN : urn:nbn:ch:unige-400462

DOI : 10.13097/archive-ouverte/unige:40046

Available at:

http://archive-ouverte.unige.ch/unige:40046

Disclaimer: layout of this document may differ from the published version.

1 / 1

(2)

Section des Sciences Pharmaceutiques Professeur Leonardo Scapozza

Biochimie Pharmaceutique Docteur Remo Perozzo

Genetic and chemical validation of Trypanosoma brucei adenosine kinase, the intracellular target of

4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine

THÈSE

présentée à la Faculté des sciences de l’Université de Genève

pour obtenir le grade de Docteur ès sciences, mention sciences pharmaceutiques

par

Patricia Graven

de Zermatt (VS)

Thèse Nº 4595

GENÈVE

Atelier d’impression ReproMail 2013

(3)

(4)

To my family

«Dans la vie, rien n’est à craindre, tout est à comprendre.»

Marie Curie (1867-1934)

(5)

(6)

Abbreviations

Ade adenine

AAT African Animal Trypanosomiasis

ADA adenosine deaminase

ADK adenylate kinase

ADP adenosine 5'-diphosphate

AgAK Anopheles gambiae adenosine kinase

AMP adenosine 5'-monophosphate

apoL1 apolipoprotein L1

ATP adenosine 5'-triphosphate blastR blasticidin resistance cassette

BSF bloodstream form

CAC citric acid cycle

CATT card agglutination test for trypanosomiasis cAMP cyclic adenosine monophosphate

CD circular dichroism

CPDD Consortium for Parasitic Drug Development dATP deoxyadenosine triphosphate

DDT dichloro-diphenyl-trichloroethane

DHFR-TS dihydrofolate reductase-thymidylate synthase

DNA deoxyribonucleic acid

DNDi Drug for Neglected Diseases Initiative DRC Democratic Republic of Congo

EC energy charge

EDTA ethylenediamine tetraacetic acid E. coli Escherichia coli

ELO pathway microsomal fatty acid elongase pathway

EtOH ethanol

FA fatty acids

(11)

FAO Food and Agriculture Organization of the United States

FCS Fetal Calf Serum

GMP guanosine monophosphate

GDP guanosine diphosphate

GTP guanosine triphosphate

Gua guanine

Guo guanosine

G418 geneticin

HAT Human African Trypanosomiasis HMI-9 Hirumi’s modified Iscove’s medium 9 HpHbR haptoglobin-hemoglobin receptor

HPLC high performance liquid chromatography HsaAK Homo sapiens adenosine kinase

hygroR hygromycin resistance cassette

Hypo hypoxanthine

IAG-NH inosine-adenosine-guanosine-nucleoside hydrolase IC50 half maximal inhibitory concentration

IMP inosine monophosphate

Ino inosine

IPA ion-pairing agent

IPTG isopropyl-β-D-thiogalactopyranoside ITC isothermal titration calorimetry

KM Michaelis constant

mRNA messenger RNA

MMOA molecular mechanism of action MTA molecular target assessment

MtuAK Mycobacterium tuberculosis adenosine kinase NECT nifurtimox-eflornithine combination therapy NGO non-government organization

NY T. b. brucei New York single marker cell line NY-ak^-(A1) NY TbAK single knock-out strain A1

NY-ak^-(A1C1) NY TbAK double knock-out strain A1C1 NY-ak^-(A1C1B4) NY TbAK triple knock-out strain A1C1B4 NY-ak⁺ NY TbAK overexpression strain

(12)

NY-akD299V⁺ NY dead kinase mutant TbAK overexpression strain D299V NY-at1^- NY TbAT1 double knock-out cell line

NY-mpra⁺ NY TbMRPA overexpression cell line

NY-at1−mrpa⁺ NY double mutant TbAT1 knock-out and TbMRPA overexpression cell line

PAGE polyacrylamide gel electrophoresis

PAS polyadenylation sites

PCF procyclic form

PCR polymerase chain reaction

PfkB phosphofructokinase B

phleoR phleomycin resistance cassette PMSF phenylmethylsulfonyl fluoride PPP public-private partnership PTR1 pteridine reductase

puroR puromycin resistance cassette

RNA ribonucleic acid

RNAi RNA interference

RP reverse phase

RT room temperature

Rt retention time

SAS splice acceptor sites

SD standard deviation

SDS sodium dodecyl sulfate

SRA serum associated gene

tet tetracycline

tetR tetracycline repressor

TRIS tris(hydroxymethyl)aminomethane TbAK T. brucei adenosine kinase

TbAKD299V T. brucei dead mutant adenosine kinase TbAT1 T. brucei P2 purine transporter

TbHK1 T. brucei Hexokinase

TbMRPA T. brucei efflux pump (multi drug resistance protein) T. brucei Trypanosoma brucei

T. b. brucei Trypanosoma brucei brucei

(13)

T. b. gambiense Trypanosoma brucei gambiense T. b. rhodesiense Trypanosoma brucei rhodesiense

TgoAK Toxoplasma gondii adenosine kinase TLF 1/2 trypanolytic factor 1 or 2

TPP target-product profile UTR untranslated region

Vmax maximal velocity of a reaction VSG variant surface glycoprotein

WBC white blood cell

WHO World Health Organisation

Xant xanthine

Xanto xanthosine

XMP xanthosine monophosphate

The one-letter code is used for amino acids.

(14)

Summary

The protozoan parasites Trypanosoma brucei rhodesiense (T. b. rhodesiense) and Trypanosoma brucei gambiense (T. b. gambiense) are the causative agents of Human African Trypanosomiasis (HAT), also known as sleeping sickness. The parasites are transmitted to humans by the bite of an infected tse-tse fly (typus Glossina). Currently 70 million people in sub-Saharan Africa are at risk. HAT evolves into two stages; the first stage (haemolymphatic stage) leads to mild symptoms such as headache, fever and enlarged lymph nodes, whereas in the second stage (neurological stage) the parasites invade the central nervous system and cause mental deterioration and other neural symptoms. If untreated, depending on the parasite involved, the infection will lead to coma and death within months or years.

Available treatments for this disease still remain unsatisfactory, needing parental administration, are toxic, or parasites have acquired resistance. Consequently safe and potent new drugs are needed to treat sleeping sickness.

Trypanosoma brucei adenosine kinase (TbAK), was identified by chemical proteomics to be the intracellular target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]- morpholine (compound 1). This molecule exhibits very good antitrypanosomal activity with an IC50 of 1 µM, and biochemical analysis revealed it to bind specifically to TbAK and to abolish intrinsic substrate-inhibition. In addition, compound 1 was shown to activate TbAK by 2.5 fold. TbAK is involved in the purine salvage pathway, which is distinct from the human one, as unlike mammalians cells, trypanosomes lack de novo purine biosynthesis and are therefore totally dependent on purine salvage from their hosts.

Many drugs fail in further drug development steps (clinical trials) because the biological relevance of the putative target was not sufficiently enough evaluated.

Huge amounts of money and precious time are wasted; therefore target validation is a crucial step in drug development. The aim of the current thesis was the chemical and genetical validation of TbAK as the intracellular target of compound 1 and the elucidation of its mechanism of action.

(15)

To address these questions, a chemical genetics approach, including genetic and chemical validation was undertaken. Genetic perturbation of the system was achieved by means of a functional and non-functional TbAK overexpression strain and by engineering of a knock-out strain in combination with the chemical perturbation in presence of compound 1. The effects of the perturbation have been investigated at the phenotype as well as at the metabolic level by monitoring the purine pool and energy charge variations using an ion-pair RP-HPLC/UV method optimized for the quantification of adenine and guanine pools.

First, a robust and sensitive ion-pair RP-HPLC/UV method was developed to investigate quantitatively the metabolomics of adenine and guanine derivatives and energy charge of trypanosomes during logarithmic phase, stationary phase and senescent phase to provide insights into the metabolic state and health of parasites.

The analysis of adenine and guanine purine levels during the different growth phases revealed that all of the adenine purine levels are in the low mM range. ATP levels only slightly increased during senescent phase, whereas adenosine, AMP, ADP, GDP and GTP levels increased considerably during stationary and senescent phases. However, much more importance was attributed to adenine and guanine purine ratios as their physiological relevance in regulation of biochemical processes is high. The energy charge, a measure of energy that is available within the cells in form of high phosphate bounds of adenine nucleotides, was found to decrease from logarithmic to static and to senescent phase. On the contrary, AMP/ATP, ADP/ATP and GDP/GTP ratios increased in the same order. In addition, adenylate kinase, a key enzyme in the interconversion of AMP, ADP and ATP was found to maintain the reaction of the interconversion at equilibrium, as the AMP/ATP ratio varied as the square of the ADP/ATP ratio.

In order to investigate properties of compound 1, the molecule was tested against drug resistant strains. The results showed that compound 1 is neither a substrate of P2 purine transporter TbAT1 nor of the active efflux pump TbMRPA and therefore its pharmacological effect is not susceptible to these resistance mechanisms. To further study the mechanism of action of compound 1, growing curves and morphological analysis were performed. Compound 1 lead to constant growth reduction and interfered with cell cycle as shown by DAPI staining analysis, leading to multinuclear

(16)

cells. With the intention of testing the sensitivity of trypanosomes expressing less TbAK towards compound 1 a knock-out strain was engineered. Although the triple knock-out strain of TbAK, harboring only one of the four TbAK alleles, expressed less than 10 % of the physiological quantity of TbAK (a full knock-out of TbAK could not be achieved), the same sensitivity towards compound 1 as the wild type strain.

Nevertheless, it was found that the triple knock-out strain of TbAK was the better genetic toll compared to the existing RNAi strain against TbAK, as it expresses less TbAK compared to the RNAi strain.

Having mapped the adenine and guanine metabolites under normal conditions during different growth phases in trypanosomes, it was possible to assess the effect of compound 1 on the metabolites level. Two hypotheses were postulated for the putative mechanism of action of compound 1: (1) ATP burnout or (2) imbalance of GTP/ATP ratio. Results from purine pool analysis of trypanosomes incubated with compound 1 indicated, that neither hypothesis (1) nor (2) explained its activity.

However it could be demonstrated that compound 1 interacts with TbAK in trypanosomes, leading to alteration in adenine purine pool via increase of AMP/adenosine and AMP/ATP ratios and to a decrease of energy charge. However, the observed alterations could only partially explain compound 1 mechanism of action and toxicity.

As hyperactivation of TbAK by compound 1 can be theoretically compared to overexpression of TbAK, a TbAK overexpression strain was constructed.

Furthermore, a dead kinase TbAK overexpression strain (TbAKD299V) was engineered giving more information about the presence of non-physiological high levels of TbAK. Western Blot analysis showed that overexpression of functional and non-functional TbAK lead to about 3 times more protein compared to the wild type strain. Interestingly both overexpression strains showed a growth phenotype, where growth was reduced in the induced strain compared to the non-induced strain.

However growth rates went back to normal after 3 days of induction. Additionally, both overexpression strains interfered with cell cycle status leading to multinuclear cells as demonstrated by DAPI staining. Furthermore, purine pool analysis showed that both overexpression strains interfered with adenine purine pools and energy charge, as both overexpression strains increased AMP/adenosine, AMP/ATP and

(17)

ADP/ATP ratios reduced energy charge. For the overexpression strains growth reduction correlated well with decrease of energy charge.

Surprisingly, the analysis performed with overexpression of functional TbAK in presence of compound 1 showed a constant growth reduction and the development of multinuclear cells. Both observations were less pronounced compared to overexpression of functional TbAK alone. Moreover, purine pool analysis with overexpression of functional TbAK in presence of compound 1, showed that compound 1 abolishes the effect induced by TbAK overexpression alone, indicating that compound 1 is interfering with TbAK activity. Therefore no synergistic effect was observed for overexpression of TbAK in presence of compound 1. This observation was confirmed by IC50 measurement for compound 1, as no significant decrease of the IC50 value was observed when overexpression of TbAK was induced.

Taken together, new insights into the role of TbAK and adenine and purine pools of trypanosomes could be gained with this study. It could be demonstrated that compound 1 is interfering with TbAK activity although the hyperactivation of TbAK conferred by compound 1 only could partially explain the compound toxicity.

However, overexpression of TbAK seems to have more impact on adenine purine pool alteration compared to compound 1. Nevertheless decrease in energy charge could be correlated to decrease in cell growth and AMP rather than ADP is the key energy sensor molecule in trypanosomes. Surprisingly TbAK seems to have an additional role besides converting adenosine to AMP, as overexpression of non- functional TbAKD299V led to a very similar phenotype and showed very similar adenine purine alterations compared to overexpression of functional TbAK. Finally, evidence for essentiality of TbAK could be gained but further experiments need to be undertaken to prove this assumption.

(18)

Résumé

La trypanosomiase humaine africaine (THA) aussi connue sous le nom de maladie du sommeil, est causée par les parasites protozoaires Trypanosoma brucei rhodeseinse (T. b. rhodesiense) et Trypanosoma brucei gambiense (T. b.

gambiense). Les parasites sont transmis à l’homme par une piqure d’une mouche tsé-tsé (du genre Glossina). Actuellement, 70 millions de personnes dans les pays d’Afrique subsaharienne vivent dans des zones à risque. La THA évolue dans 2 stades : le 1^er stade (phase lymphatico-sanguine) se présente sous forme de symptômes légers, comme des céphalées, de la fièvre et des ganglions lymphatiques enflés, tandis que le 2^ème stade de la maladie se caractérise par l’invasion du système nerveux central par les parasites (phase neurologique) qui cause des détériorations mentales et d’autres symptômes neurologiques. Si le patient reste sans traitement, la maladie va évoluer vers le coma et la mort.

Les traitements actuels ne sont pas satisfaisants, car ils nécessitent une administration parentérale, sont toxiques, ou car les parasites ont acquis une résistance. Ainsi de nouveaux médicaments sûrs et efficaces sont requis pour le traitement de la maladie du sommeil.

L’adénosine kinase de Trypanosoma brucei (TbAK) a été identifiée par une approche de protéomique chimique comme cible intracellulaire de 4-[5-(4-phenoxyphenyl)-2H- pyrazol-3-yl]-morpholine (composé 1). Ce composé possède une activité antitrypanosomiale avec une concentration d’inhibition IC50 de 1 µM. Des analyses biochimiques ont révélé que le composé 1 se lie fortement et spécifiquement à TbAK et qu’il augmente son activité enzymatique par un facteur de 2.5. Il a aussi été démontré que le composé 1 supprime l’inhibition par le substrat. La TbAK est une enzyme clé dans le métabolisme des purines, qui est différent de celui de l’homme car les parasites ne sont pas capables de synthétiser des purines par eux-mêmes.

Ainsi, ils dépendent entièrement de sources de purines présentes dans leur hôte.

Dans le processus de développement d'un médicament, beaucoup de candidats échouent pendant les phases cliniques car la pertinence biologique de la cible

(19)

supposée du médicament n’a pas été suffisamment évaluée auparavant. De ce fait, beaucoup d’argent et de temps précieux sont gaspillés ; de ce fait la validation de la cible est une étape cruciale dans le développement d’un médicament. L’objectif de cette thèse était la validation génétique et chimique de TbAK comme cible intracellulaire du composé 1 et l’investigation de son mécanisme d’action.

Pour aborder ces questions, une approche « chemical genetics », comprenant la validation génétique et chimique, a été employé. La perturbation génétique du système a été réalisée par la construction de plusieurs souches de parasites génétiquement modifiées: une surexprimant l’adénosine kinase fonctionnelle (TbAK), une surexprimant l’adénosine kinase non-fonctionnelle (TbAKD299V) et une possédant une invalidation génique (knock-out) de l’adénosine kinase. Ces perturbations génétiques ont été examinées en combinaison avec le composé 1.

L’effet de ces manipulations génétiques ainsi que l’influence du composé 1 ont ensuite été examinées au niveau phénotypique ainsi qu’au niveau métabolique par le suivi des niveaux de purines de l’adénine et de la guanine par la chromatographie de pair d’ions avec détection UV (ion-pair RP-HPLC/UV).

Premièrement une méthode ion-pair RP-HPLC/UV robuste et sensible a été développée pour examiner le niveau de purines de l’adénine et de la guanine pendant différentes phase de croissance (logarithmique, statique et sénescente).

Ainsi, l’état métabolique et l’état de santé des parasites sous conditions normales a pu être étudiée. L’étude a montré que toutes les purines mesurées sont présentes à des basses concentrations micromolaires dans les parasités. Le niveau d’ATP n’a augmenté que légèrement pendant la phase sénescente, alors que les niveaux d’adénosine, d’AMP et d’ADP ont fortement augmentés pendant la phase statique et sénescente en comparaison avec la phase logarithmique. Cependant, plus d’importance a été attribué aux rapports de purines d’adénine et de guanine car leur pertinence biologique en termes de régulation de processus biochimiques est élevée.

La charge énergétique a diminué de la phase logarithmique à la phase statique et à la phase sénescente. A l’inverse, les rapports AMP/ATP, ADP/ATP et GDP/GTP ont augmentés de la phase logarithmique à la phase sénescente. En plus, il a été démontré que l’adenylate kinase, une enzyme clé dans l’interconversion de l’AMP, ADP et ATP, maintien la réaction enzymatique à l’équilibre, car le rapport AMP/ATP à varié comme le carré du rapport ADP/ATP.

(20)

Pour examiner les propriétés du composé 1, la molécule a été testée avec des souches de parasites résistantes, l’une possédant un knock-out d’un transporteur de purines P2 TbAT1, l’autre surexprimant une pompe à efflux TbMRPA. Les résultats des mesures d’IC50 avec ces souches et le composé 1 ont montrés que le composé 1 n’est pas un substrat du transporteur de purines P2 TbAT1, ni de la pompe à efflux TbMRPA. En conséquence, le composé 1 n’est pas susceptible à ces mécanismes de résistances. En plus, des analyses phénotypiques et des courbes de croissance en présence du composé 1 ont été effectuées pour mieux étudier son mécanisme d’action. Le composé 1 a provoqué une réduction constante de la croissance parasitaire ainsi que le développement de cellules multinucléaires. Ensuite, une souche parasitaire exprimant moins de TbAK a été construite pour tester sa sensibilité envers le composé 1. Malgré le fait que la souche triple knock-out de TbAK (knock-out de 3 des 4 allèles de TbAK) exprimait moins que 10 % de la quantité physiologique de TbAK (un knock-out total de TbAK n’a pas pu être obtenu), la sensibilité de cette souche envers le composé 1 mesuré par IC50 n’a pas changé en comparaison avec la souche sauvage. Néanmoins, la souche triple knock-out de TbAK est le meilleur outil génétique en comparaison avec la souche ARN interférent existant de TbAK, car le triple knock-out exprime moins de protéine que la souche ARN interférant.

La détermination des métabolites de l’adénine et de la guanine en conditions normales a permis d’évaluer l’effet du composé 1 au niveau de ces métabolites. Les résultats ont rejetés les deux hypothèses initiales essayant d’expliquer le mécanisme d’action du composé 1 : (1) ni un épuisement en ATP et (2) ni un déséquilibre du rapport GTP/ATP expliquent l’activité du composé 1. Cependant il a pu être affirmé que le composé 1 interagit avec TbAK menant à des perturbations des niveaux de purines de l’adénine en augmentant les rapports AMP/adenosine et AMP/ATP et en diminuant la charge énergétique. Toutefois, ces observations ne permettent d’expliquer que partiellement le mécanisme d’action du composé 1.

L’effet de l’hyperactivation de TbAK peut théoriquement être comparé à une surexpression de TbAK, de ce fait une souche parasitaire surexprimant TbAK a été élaborée. En outre, une souche surexprimant une TbAK non-fonctionnelle (TbAKD299V) a été construite pour étudier l’effet de la présence de niveaux élevés et non-physiologiques de TbAK. Des analyses par transfert de protéines (Western

(21)

Blot) ont montrés que quand la surexpression de TbAK et de TbAKD299V a été induite, 3 fois plus d’adénosine kinase totale est présente dans les parasites par rapport à la souche sauvage. Etonnamment, la surexpression des deux protéines, l’

l’adénosine kinase fonctionnelle et non-fonctionnelle ont conduit à une réduction de la croissance par rapport aux à leurs contrôles respectives (souches non-induites).

Cependant, la croissance des parasites est revenue à la normale après 3 jours d’induction dans les deux souches. En plus, la coloration au DAPI a démontré que les parasites surexprimant la TbAK et la TbAKD299V présentaient une interférence avec le cycle cellulaire et développaient des cellules multinucléaires. Les analyses de métabolites de purines ont révélés que la surexpression d’adénosine kinase fonctionnelle et non-fonctionnelle induisait des altérations des niveaux de purines de l’adénine et de la charge énergétique ; les rapports AMP/adénosine, AMP/ATP et ADP/ATP étaient augmentés et la charge énergétiques était diminuée dans les souches ou la surexpression était induite par rapport aux souches non-induites. Pour les deux souches de surexpression, la réduction de la croissance observée pouvait être reliée à la diminution de la charge énergétique.

Curieusement, quand la TbAK était surexprimé en présence du composé 1 la réduction de croissance était constante. En outre, cette condition a aussi conduit au développement de cellules multinucléaires. Pourtant ces deux observations étaient moins prononcées que pour la surexpression de TbAK seule. De plus, l’analyse de purine avec la surexpression de TbAK en présence du composé 1 a montré que le composé 1 a supprimé la diminution constante de la charge énergétique (effet induit par la surexpression de TbAK seule), indiquant que le composé 1 interfère avec l’activité de TbAK. Ainsi, aucun effet synergique ou additif n’a pu être confirmé pour la surexpression de TbAK en présence du composé 1. Cette constatation a été confirmé par la mesure de la IC50 car aucune diminution significative de cette valeur n’a pu être mesuré quand la TbAK a été surexprimé.

En résumé, cette étude a permis l’acquisition de nouvelles connaissances concernant le rôle de la TbAK et des niveaux de purines de l’adénine et de la guanine dans les trypanosomes. Il a pu être démontré que le composé 1 interagit avec la TbAK même si l’hyperactivation de la TbAK par le composé 1 peut seulement partiellement expliquer la toxicité de la molécule. Néanmoins, la surexpression de TbAK semble avoir plus d’effet sur l’altération des niveaux de purines de l’adénine

(22)

que le composé 1. Cependant une corrélation entre la réduction de la croissance et la réduction de la charge énergétique a pu être établi et il semble que l’AMP plutôt que l’ADP est la molécule clef au niveau de la détection du niveau énergétique de la cellule. Etonnamment, la TbAK semble posséder une fonction supplémentaire en plus de convertir l’adénosine en AMP, car la surexpression de la protéine non- fonctionnelle TbAKD299V cause un phénotype similaire et une altération des purines de l’adénine similaire que la surexpression de la protéine fonctionnelle TbAK.

Finalement, les résultats obtenus suggèrent que la TbAK joue un rôle essentielle dans le métabolisme des purines, toutefois des expériences supplémentaires sont nécessaires pour démontrer cette supposition.

(23)

(24)

PART 1

Introduction

(25)

(26)

Trypanosoma brucei

1.1 Classification of Trypanosomes

Trypanosomes are flagellated protozoan parasites that belong to the order of Kinetoplastidia, one of the most ancient eukaryotic lineages. Parasites of this order are responsible for a wide variety of diseases affecting humans, animals and plants [1]. The three major diseases caused by the trypanosomatid parasites are African trypanosomiasis (Sleeping Sickness, caused by Trypanosoma brucei), Chagas disease (American trypanosomiasis, caused by Trypanosoma cruzi) and Leishmaniasis (a set of trypanosomal diseases caused by various species of Leishmania). This group of parasites is often referred to the “Tritryps”. The genus Trypanosoma is divided into two main groups based on their mode of transmission by the insect vector: salivaria and stercoraria. Stercoraria comprises species that develop in the arthropod’s hindgut (intestinal tract) and are therefore found in the insect’s feces (Trypanosome cruzi). Salivaria includes species that are found in the salivary glands of the tsetse fly (Glossina) and are transmitted via inoculation into mammalian skin (Trypanosoma brucei) [2]. In this work, we focus on Trypanosoma brucei, African trypanosomes.

African trypanosomes are divided into three subgeni: Nannomonas (the congolense group), Duttonella (the vivax group) and Trypanozoon. The subgenus Trypanozoon is further divided into 3 species: Trypanosoma brucei (T. brucei), T. evansi and T.

equiperdum. Finally, T. brucei is subdivided into 3 subspecies: T. brucei brucei (T. b.

brucei), T. brucei gambiense (T. b. gambiense) and T. brucei rhodesiense (T. b.

rhodesiense) [3]. The T. brucei subspecies are morphologically indistinguishable and exhibit only minimal biological differences (T. b. brucei and T. b. rhodesiense can differ by as little as the expression of a single gene). Therefore the distinction into subspecies can mostly be explained by pathogenic mechanism, host range variation and habitat [4]. Wild and domestic animals as well as humans can be long term reservoir of these parasites. T. b. brucei is responsible for African Animal

(27)

Trypanosomiasis (AAT, also known as Nagana disease); however humans are resistant to this subgenus because of serum factors trypanosome lytic factor TLF1 and TLF2 [5, 6]. Apolipoprotein-L1 (apoL1) is present in both factors and is responsible for the lysis of the parasite due to the development of pores in the lysosomal membrane [7]. In contrast, T. b. rhodesiense and T. b. gambiense are able to infect humans, causing Human African Trypanosomiasis (HAT, or sleeping disease). The two subspecies have developed different resistance mechanisms to TLF. Interestingly, different antigenic variants of the same cloned line of T. b.

rhodesiense can be human serum sensitive or resistant [8]. The difference between serum sensitive and resistant clones is due to the expression of the serum-resistance associated antigen (SRA) [9, 10]. This gene is located in a variant surface glycoprotein (VSG) expression site and is only expressed when that particular expression site is active [11]. SRA is able to interact with apoL-1 and prevent its effect. In T. b. gambiense several resistance mechanisms to TLF have evolved:

amino acid substitutions on the haptoglobin-hemoglobin receptor (HpHbR) that binds apo-L1 and is responsible for its uptake into the cell, reduced expression of this receptor and resistance to the effects of apoL1 in the lysosome [12]. Table 1.1 shows the characteristics of the T. brucei group.

Table 1.1 Characteristics of subspecies within T. brucei group [4, 13]

Subspecies Distribution Host range Growth in

rodents

HSR

phenotype^a T. b. brucei Tropical

Africa

Wild & domestic mammals, not humans

Fast -

T. b. rhodesiense East Africa Humans, wild &

domestic mammals

Fast +/-

T. b. gambiense Group 1

West and Central Africa

Humans, wild &

domestic mammals

Slow +++

T. b. gambiense Group 2

Ivory Coast Humans, wild &

domestic mammals

Fast +/-

aHSR, resistance to lysis by human serum.+++, resistant; +/-, variable resistance; -, sensitive.

(28)

1.2 Morphology and cell structure

African trypanosomes are highly motile single celled organisms. They have a characteristic S shape and measure 10 - 30 µM in length and have a diameter of 1 - 3 µM. The parasites show all the main features of eukaryotic cells, nevertheless they have several single-copy organelles [14]: a single Golgi apparatus, a single mitochondrion with a single kinetoplast that contains the mitochondrial genome and is the major distinguishing feature of the class of the Kinetoplastida. Other characteristics of trypanosomes are the presence of flagella and the existence of a prominent invagination of the plasma membrane called the flagellar pocket. Through this pocket, the parasite can interact with its environment (endo- and exocytosis) [15, 16].

The bloodstream form of T. brucei has an outer surface that is densely coated with a layer of glycoprotein (variable surface glycoprotein, VSG). Through antigenic variation, the composition of the VSG is changed periodically, expressing only one of 1’000 to 2’000 variable antigens at any given time. This process enables the parasite to escape from the host immune antibody mediated lysis [17]. Two predominant mechanisms are used to switch a VSG gene: the telomeric expression site that is being transcribed can be switched, or the recombination machinery transfers genes from locations on other chromosomes to an active expression site [18]. The main features of trypanosomes are shown in Figure 1.1.

Figure 1.1. Schematic diagram of the major organelles of T. brucei BSF. Picture taken from http://www.ilri.org.

(29)

1.3 Vector and Life cycle

All T. brucei group parasites are transmitted to humans by the bite of a tsetse fly (genus Glossina). Consequently, HAT is restricted to Sub-Saharan African countries, the natural habitat of Glossina which stretches from south of the Sahara to the north of the Kalahari desert [19]. Figure 1.2A and B shows trypanosome protozoa and tsetse fly. The development of the fly to the adult stage requires about 27 days at 25°C at a generation time of 43 days. This relatively slow reproduction rate is largely compensated by the survival rate of the adult fly which typically goes above 97%

[20]. Only certain species of Glossina transmit the disease. The Palpitans group is the main vector of T. b. gambiense in West and central Africa. This group is mainly found in gallery forests, swamps and in water sides with closed canopy [21]. In contrast, the Morsitans group is the predominant vector of T. b. rhodesiense. Their typical habitat is open woodland and woodland savannah [21, 22]. Both forms of HAT occur only in Uganda [20].

African trypanosomes encounter many different environments during the course of life cycle. They respond to these by dramatic changes in cell shape, metabolism and patterns of gene expression [23].

Figure 1.2. African trypanosomes and tsetse fly. (A) Colored scanning electron micro- graph (SEM) of T. brucei protozoa (yellow) amongst red blood cells. (B) Tsetse fly Glossina.

Both pictures are taken from SciencePhotoLibrary, www.sciencephoto.com.

(30)

During a blood meal of the tsetse fly metacyclic trypanomastigotes are injected from the salivary gland into the blood where they transform into bloodstream trypanomastigotes which are carried to other sites. The bloodstream form (BSF) parasites pass through various body fluids (lymph, blood, spinal fluid) where they replicate by binary fission causing clinical symptoms (stage 1). After an interval of several weeks to many months the trypanosomes ultimately reach the central nervous system (CNS) and cause CNS disorders (stage 2). BSF parasites exist as long-slender forms (proliferative), short-stumpy forms (non-proliferative) or as intermediate between these two extremes (see Figure 1.3). The slender form parasite undergoes rapid multiplication and is adapted to use the glucose rich milieu of the blood [24]. Slender BSF differentiate into short-stumpy forms (which have a limited life span in the host) at peak of a parasitemia [25].

Figure 1.3. Morphological forms of Trypanosoma brucei bloodstream form. (A) long- slender form, (B) intermediate form, (C) short-stumpy form. Adapted from [26].

Short-stumpy form has all of the components required for a fully active mitochondrial respiratory chain. Once taken up by the tsetse during blood meal, the stumpy forms can therefore more easily adapt to the loss of glucose in the tsetse fly than the slender form [24]. In the tsetse fly midgut, the trypanomastigote transform into procyclic trypanomastigotes and multiply by binary fission. Subsequently, procyclic forms (PCF) leave the midgut, transform into epimastigotes and multiply in the salivary gland. To complete its life cycle, T. brucei generates metacyclic forms in the salivary glands that are infectious for mammals [27].

It is estimated that the whole cycle is probably only successfully accomplished in about one of 10 tsetse flies and about 2-10 % of all flies in an endemic area are infected with trypanosomes. A fly remains infective during all of its lifetime, thus man/fly exposure time is a crucial component of the disease [15]. A scheme of the life cycle of T. brucei is shown in Figure 1.4.

(31)

Figure 1.4. Life cycle of African trypanosomiasis. (a) Tsetse fly takes a blood meal and injects metacyclic trypanomastigotes. (b) Injected metacyclic trypanomastigotes transform into bloodstream trypanomastigotes, which are carried to other sites. (c) Trypanomastigotes multiply by binary fission in various body fluids (blood, lymph, and CSF). (d) Trypanomastigotes in blood. (e) Tsetse fly takes a blood meal from human host, and bloodstream trypanomastigotes are ingested. (f) Bloodstream trypanomastigotes transform into procyclic trypanomastigotes in tsetse flies midgut. Procyclic trypanomastigotes multiply by binary fission. (g) Procyclic trypanomastigotes leave the midgut and transform into epimastigotes. (h) Epimastigotes multiply in salivary gland of tsetse fly, and are transformed into metacyclic trypanomastigotes. Picture adapted from http://jpkc.sysu.edu.cn.

(32)

1.4 Genome

Trypanosomes consist of two genome units: a nuclear genome (85% of the total cellular DNA) and a mitochondrial genome (kinetoplast, 15% of the total cellular DNA). The complete genome sequence was published in 2005 [28].

The nuclear genome is divided into three different groups of chromosomes which are distinguished based on their size: 11 large chromosomes (1-6 Mb), 6 intermediate chromosomes (300-600 Kb), and 100 minichromosomes (50-100 Kb) [28]. In contrast, the kinetoplast is made out of two genetic features: the maxicircles and the minicircles [29]. The parasite has a complex life cycle that alternates between insect and mammalian hosts [30]. To adapt to the different environments, modifications in gene expression are required. Indeed, several hundred mRNAs show at least twofold developmental regulation [31]. As shown in Figure 1.5 the open reading frames of kinetoplastid DNA are arranged in long arrays which are transcribed into polycistronic RNA precursors and further processed into individual mRNAs by trans splicing and polyadenylation [32-34].

Figure 1.5. Polycistronic transcription and processing of mRNAs in trypanosomes.

Trans splicing of spliced leader RNA to the 5′ end of the 5′ UTR of protein coding genes leads to the dissection of polycistronic transcription units. Mature mRNA contains a 5′

conserved spliced leader sequence and a 3′ poly(A) tail. Sequence tags ‘bridging’ the spliced leader sequence and the 5′ end of a gene or the poly(A) tail and the 3′ end of a gene can be used to map splice acceptor sites (SAS) and polyadenylation sites (PAS), respectively.

Figure taken from [35].

(33)

Regulation of mRNA and protein abundance occurs post-transcriptionally at the level of trans splicing and polyadenylation, RNA export, RNA stability, protein translation and protein stability [36, 37]. In several genes in T. brucei, more recent findings show that stabilization and degradation of DNA are the main post-transcriptional mechanisms responsible for the variation in protein expression levels [38, 39].

1.5 Energy metabolism

To maintain viability, trypanoses depend on the carbon sources present in their hosts [40]. The slender BSF of T. brucei has the simplest energy metabolism, which is only based on glycolysis of glucose taken up in the blood of the host (Figure 1.6A) [40].

Pyruvate is excreted as the sole metabolic end product. In trypanosomes, glycolysis is compartmentalized: the glycosomes (peroxisome-like organelles) house the first seven enzymes of glycolysis, whereas last three reactions take place in the cytosol [41]. Hence, glucose degradation via the glycolysis pathway is irreversible. The process starts with 2 molecules ATP and yields 4 molecules of ATP at the end. As hexokinase (HK) and phosphofructokinase (PFK) are not feedback regulated in trypanosomes there is a risk of ‘‘turbo-explosion’’, an autocatalytic acceleration of the pathway accompanied by the accumulation of sugar phosphates [30, 42]. However,

‘‘turbo-explosion’’ in trypanosomes is prevented because of the compartmentalization of glycolysis in glycosomes. Net ATP synthesis coupled to glycolysis only occurs in the cytosolic portion of the pathway. Therefore this ATP is not available to HK or PFK because the glycosomal membrane is not permeable to small and charged molecules. The situation in insect-stage PCF trypanosomes is more complex (Figure 1.6B). Pyruvate, the endproduct of glycolysis, is not excreted but is further metabolized inside the mitochondrion in which it is mainly degraded to acetate which generates extra ATP [43]. In addition to carbohydrate degradation, PCF derive most of their energy by amino acid catabolism (with a preference for L–proline) carried out by mitochondrial enzymes. Although PCF have all citric acid cycle (CAC) enzymes (BSF lack cytochromes and several key citric acid cycle enzymes), they dedicate parts of the CAC for other tasks than for catabolism of mitochondrial substrates (i.e.

transport of acetyl-CoA units from the mitochondrion to the cytosol for biosynthesis of fatty acids, degradation of proline to glutamate to succinate and generation of malate that can be used for gluconeogenesis) [44]. However, recent findings have shown

(34)

that the procyclic form prefers D-glucose over L-proline when grown in medium rich in sugar. The adaptation of L-proline metabolism to D-glucose metabolism (and vice versa) is a fast and stepwise process [45, 46]. This highlights the capacity of PCF to adjust their metabolism to changing conditions in the environment.

Figure 1.6. The energy metabolism of T. brucei (A) BSF and (B) PCF. Enzymes: 1, hexokinase; 2, glucose-6-phosphate isomerase; 3, phosphofructokinase; 4, aldolase; 5, triosephosphate isomerase; 6, glyceraldehyde-3-phosphate dehydrogenase;7, phosphoglycerate kinase; 8, glycerol-3-phosphate dehydrogenase; 9, glycerol kinase; 10, phosphoglyceratemutase; 11, enolase; 12, pyruvate kinase; 13, glycerol-3-phosphate oxidase; 14, phosphoenolpyruvate carboxykinase; 15, Lmalatedehydrogenase; 16, fumarase; 17, fumarate reductase; 18, pyruvate phosphate dikinase; 19, pyruvate dehydrogenasecomplex; 20, acetate:succinate CoA transferase; 21, proline oxidase; 22, ∆'-pyrroline-5-carboxylate reductase; 23, glutamatesemialdehyde dehydrogenase;

24, glutamate dehydrogenase; 25, α-ketoglutarate dehydrogenase; 26, succinyl CoA synthetase;27, FAD-dependent glycerol-3-phosphate dehydrogenase. Abbreviations: AA, amino acid; AcCoA, acetyl- CoA; 1,3-BPGA, 1,3-bisphosphoglycerate; c, cytochrome c; Citr, citrate; DHAP, dihydroxyacetone phosphate; Fum, fumarate; G-3-P, glyceraldehyde3-phosphate; Glu, glutamate; Gly-3-P, glycerol 3- phosphate; KG, α-ketoglutarate; Mal, malate; OA, 2-oxoacid; Oxac,oxaloacetate; PEP, phosphoenolpyruvate; 3-PGA, 3-phosphoglycerate; Succ, succinate; Succ-CoA, succinyl-CoA; UQ, ubiquinone. Substrates and secreted end-products are indicated in green and red, respectively, and boxed. Enzymes involved in reactions represented by dashed lines are present, but experiments indicated that no significant fluxes occurred through these steps. Figure taken from [47].

(35)

Human African Trypanosomiasis

1.6 Epidemiology and current situation

The description of the past situation is reported in [48, 49] and recent information about HAT can be found in [50-53].

In 1373 the historian Ibn Khaldun described that King Diata II (sultan of Mali) suffered from lethargy and died from “... a disease that frequently befalls the inhabitants...".

With the esthablishement of trading routes in the 15th century by Portuguese, later on by French, British and Dutch trade-companies, slave traders were conscious of the menacing symptoms of swollen cervical lymph glands. Thus they used to inspect the slaves necks before purchasing them. The first publications on sleeping sickness in the medical literature date back to the 18^th century and are written by ship doctors or medical officers. In 1857, David Livingstone reported the harmful effects of tsetse bites and described the flies as "poisonous insects to ox, horse and dog". Finally, the link between tsetse fly, T. brucei and sleeping sickness was made in the very beginning of the 20^th century. Émile Brumpt noticed that the geographic distribution of tsetse flies corresponds with that of sleeping sickness, whereas David Bruce revealed that NAT is caused by T. brucei and Robert Michael Forde observed for the first time trypanosomes in human blood.

The African continent has experienced three major HAT epidemics during the last century. The first one took place in 1896 and lasted for 10 years. At the time, colonial administrations charged scientists to study HAT and find medicines to treat the disease. This lead to the discovery of the first drugs to treat trypanosome infections, i.e. atoxyl and trypan red. In 1916 the pharmaceutical company Bayer discovered suramin (a drug that is still in use) and tryparsamide discovered by Michael Heidelberger. Nevertheless, a second epidemic was recorded in 1920 in a number of African countries. Both drugs in combination with the introduction of mobile teams for systematic case detection and vector control (bush clearing, tsetse traps), could significantly reduce the prevalence of HAT until the late 1940s. From 1949 on,

(36)

dichloro-diphenyl-trichloroethane (DDT) insecticide was distributed in large parts of endemic areas and the discovery of new effective drugs for humans (melarsoprol) and animals, made it possible to reach very low levels of new infections in the 1960s.

For diagnosis, gland palpation and gland puncture were performed and each positive case was medicated with the objective to eliminate all possible parasite reservoirs.

Through the relaxation of surveillance, conflicts and the banning of DTT because of its environmental effects, HAT has tremendously reappeared in Africa during the second part of the last century (third epidemic in 1970). In 1998 around 40’000 cases were reported while 300’000 cases were estimated to remain undiagnosed and therefore untreated. During epidemic periods HAT was the first or second greatest cause of mortality in some communities of the Democratic Republic of Congo (DRC), Angola and Southern Sudan, even ahead of HIV/AIDS.

In the last 10 years, over 70% of reported cases occurred in the DRC. Due to strengthened surveillance, the number of reported cases has dropped below 10’000 in 2012. 30’000 cases are the estimated number of current HAT infections. In 2000/

2001 WHO established a public-private partnership with Aventis Pharma (now Sanofi Aventis) and Bayer Health Care which enabled the creation of a WHO surveillance team, providing support to endemic countries in their control activities and the supply of drugs free of charge for the treatment of patients. In 2006 and in 2011 this partnership was renewed. Nevertheless, HAT remains a daily danger to more than 60 million people in 37 countries of sub-Saharan Africa, 22 of which beiing among the least developed countries. Many people live in isolated zones with limited access to adequate health services, which hinders the surveillance and the diagnosis of HAT. In Figure 1.7 the actual situation of HAT in the world is shown. Not only human but also AAT represents a constraint for socioeconomic development of Africa. The food and agriculture Organization of the United Nations (FAO) estimates that AAT is a major cause contributing to rural poverty. Cattle affected by AAT cannot be used for farming work and are useless for milk and meat production.

(37)

HAT and AAT are considered a major hurdle in the creation of a prosperous agriculture providing food security and to the development lead to sustainable economic progress. In addition, conflict is an important factor of sleeping sickness outbreaks and has contributed to disease reappearance (epidemic in Uganda in 1979-86). By the degradation of health systems and services, dislocation of populations, economic effects and insecurity, the spreading of HAT is greatly affected by conflicts [52].

Figure 1.7. HAT in endemic and nonendemic African countries. T. b. gambiense infection is represented in red and T. b. rhodesiense infection is shown in yellow. Figure taken from WHO: Sleeping sickness national control programmes, annual country reports 2010.

(38)

1.7 Pathology and clinical features

Depending on the parasite involved, HAT occurs as a chronic or acute infection. In both forms, HAT evolves through two characteristic stages: the early stage (hemolymphatic stage) and the late stage (encephalitic stage). Table 1.2 summarizes the main differences between the chronic and the acute form of HAT. However, the evolution from the early to the late stage is not always distinct in T. b. rhodesiense infections [54].

First stage of disease, also known as the early stage or haemolymphatic stage

The parasites multiply at the site of inoculation which leads to a trypanosomal chancre (inflammatory nodule, ulcer) in approximately 50% of T. b. rhodesiense but seldom in T. b. gambiense infection [19]. Parasites proliferate in the bloodstream and are transported to the lymph nodes and other organs (spleen, liver, heart and endocrine system). Systemic symptoms of the early stage are often non-specific, e.g.

fever, malaise, facial edema, anemia, lymphadenopathy and arthralgia. Frequently, patients are misdiagnosed to suffer from malaria because of febrile episodes [52],[55]. Later features reflect the involvement of specific organ systems [56].

Second stage of disease, also known as the late stage or encephalic stage

In the second stage of the disease, parasites invade internal organs, cross the blood- brain barrier and infect the Central Nervous System (CNS) [56]. As the disease evolves, headaches become severe and the sleep/wake cycle is disrupted. The patient has nocturnal insomnia and often sleeps during the day [57]. This sleep disorders give the disease its common name. If untreated, or unsuccessfully treated, the state of health of the patient will progressively deteriorate with increasing sleep disturbances, cerebral edema, incontinence, mental deterioration, seizures and lastly death [58]. The progression of the disease is usually much slower and longer in T. b.

gambiense compared with T. b. rhodesiense infection. While the latter may lead to death within a few weeks, the former may last many months or years [56]. Besides the transmission over the infected tsetse fly, the trypanosoma can cross the placenta and infect the fetus (mother-to-child infection).

(39)

Table 1.2. Characteristics of the two forms of HAT: chronic infection and acute infection [51].

Chronic infection Acute infection

Parasite T. b. gambiense T. b. rhodesiense

Reported cases 95 % 5%

Geographical distribution West and central Africa Eastern and southern Africa

Primary reservoir Humans Wild & domestic mammals

First signs and symptoms After months to years Less than 9 months

Development of disease Slowly (late CNS^a disease) Rapidly (early CNS^a disease)

Parasitemia Low High

Lymphadenopathy^b Prominent Minimal

aCNS Central nervous system, ^bLymphadenopathy is a condition where the lymph nodes are enlarged.

(40)

Fight against Sleeping sickness

1.8 Current Treatments for Sleeping sickness

1.8.1 Chemotherapy

Treatment of HAT depends on the stage of the disease and on the species that causes the infection. Drugs used in the first stage of HAT are less toxic and easier to administer compared to drugs used in the second stage of the disease. The earlier HAT is diagnosed; the better is the prospect of a cure. Follow-up should be proposed for all HAT patients after treatment, e.g. 6-monthly for 2 years. Literature for the treatment for HAT can be found in [59-65]. Figure 1.8 and Table 1.3 give an overview of the current treatments for HAT.

Treatment of the first stage of T. b. gambiense infection is based on pentamidine. It is administered intramuscularly or as an intravenous slow infusion during 7 days and is generally well tolerated. Pain at the injection site, hypoglycaemia and hypotension are the most frequent adverse effects. The efficacy of pentamidine in HAT continues to be excellent despite its extensive use in prophylaxis programmes. The mode of action of pentamidine is not fully understood. It is thought that the drug interferes with nuclear metabolism producing inhibition of the synthesis of DNA, RNA, phospholipids, and proteins.

Melarsoprol (organoarsenic compound) has until very recently been the most widely used drug for treatment of second stage HAT caused by T. b. gambiense.

Hospitalization during treatment with melarsoprol is mandatory because of the high rate of adverse drug reactions which may be severe or life threatening such as an encephalopathic syndrome (5–10% frequency with around 50% case-fatality rate), peripheral neuropathy, hepatic toxicity, skin rash, acute phlebitis and vein sclerosis.

Moreover, high melarsoprol failure rates have been reported from several endemic countries (parasites from relapse cases lack the P2 transporter which is necessary to transport melarsoprol into the parasite). Melarsoprol's mode of action is not clearly understood. The schedule of treatment consists of 1 injection daily for 10 days.

(41)

Eflornithine has progressively replaced melarsoprol as the first-line treatment for the treatment of second stage HAT caused by T. b. gambiense because it has been shown to be safer than melarsoprol. However, it needs huge requirements in logistics and nursing care (56 intravenous infusions of over 30 min over 14 days corresponding to ~30 L of drug per patient). Eflornithine is an irreversible specific inhibitor of ornithine decarboxylase, an enzyme involved in the polyamine pathway.

Inactivation of this pathway retains the trypanosomes in the short metacyclic form which is not capable to divide and modify VSG, therefore making human's immune response more effective. Unpublished data (Enock Matovu, Makerere University) points to a substantial increase in eflornithine treatment failures in Northern Uganda.

So far, in vitro experiments could show that eflornithine resistance can easily be achieved through loss of a putative amino acid transporter, TbAAT6. Since January 2010 Medecins sans Frontières has used a nifurtimox–eflornithine combination therapy (NECT) as first-line treatment in several endemic countries. NECT allows shortening and simplifying eflornithine based therapy. The mechanism of action of the prodrug nifurtimox is only partially understood and has been related to induction of oxidative stress in the target cell and/or to the activation of the drug by a eukaryotic type I nitroreductase that leads to a toxic nitrile compound.

Suramin is used for first-stage T. b. rhodesiense disease with a complex dose regimen that lasts up to 30 days. Nephrotoxicity, peripheral neuropathy and bone marrow toxicity are known adverse effects but are usually mild and reversible. The mechanism of action of the compound has not been pinpointed, although it is most likely the sum of many non-specific interactions with different subcellular systems that are toxic for the cell. Suramin resistant T. evansi parasites were found in Uganda, but it is not known if suramin resistant T. b. gambiense or T. b. rhodesiense strains exist today.

Unfortunately, T. b. rhodesiense is inherently resistant to eflornithine (they have a higher ornithine decarboxylase turnover). Thus, melarsoprol remains the only effective treatment for patients with second-stage disease despite its toxicity. The same treatment schedule as for T.b. gambiense infection is recommended.

(42)

Figure 1.8. Chemical structures, brand name and first year of publication of drugs in use for HAT.

(43)

Table 1.3. Current chemotherapeutic treatment for HAT.

Drug Species Stage Pentamidine

isethionate

T. b.

gambiense

Stage 1 Dosage: 4 mg/kg/day IM or IV injection (7 days).

ADR^a: Hyperglycemia, hypotension, site pain, GI symptoms^b .

Suramin sodium

T. b.

gambiense

Stage 1 (treatment of relapse)

Dosage: test dose of 4-5 mg/kg IV injection (1 day), then 20 mg/kg IV every 3-7 days (5 weeks, maximal

dose/injection 1g).

ADR^a: Nephrotoxicity, peripheral

neuropathy, bone marrow, fatal rash and rare acute hypersensitivity.

T. b.

rhodesiense

Stage 1

Melarsoprol T. b.

gambiense

Stage 2 (treatment of relapse)

Dosage: 2.2 mg/kg/day IV infusion (10 days).

ADR^a: Hospitalization is mandatory (high toxicity, organic-arsenic compound).

Encephalopathic syndrome, peripheral motoric or sensorial neuropathies.

T. b.

rhodesiense

Stage 2

Eflornithine T. b.

gambiense

Stage 2 Dosage: 100 mg/kg/day IV infusion every 6 h (14 days, 56 short infusions).

ADR^a: Convulsions, GI symptoms^b, bone marrow toxicity.

NECT^c T. b.

gambiense

Stage 2 Dosage: Eflornithine: 200 mg/kg/day IV infusion every 12 h (7 days, 14 short infusions), Nifurtimox: 15 mg/kg/day PO (10 days).

ADR^a: Eflornithine: see above. Nifurtimox:

ADR are dose related, GI symptoms^b, convulsions, tremor.

aAdverse Drug Reaction (ADR), ^bGastro àà£$

(NECT)

(44)

1.8.2 Vaccine

The development of a vaccine for HAT was thought to be impossible to achieve because of the high antigenic variation of the surface proteins of BSF T. brucei trypanomastigotes. To overcome this issue, research focused on the identification of invariant (or less variant) components to be used as therapeutic targets. So far, some vaccine prototypes were constructed and testing of these vaccines is currently ongoing. According to recent data, vaccination with a DNA plasmid encoding a bloodstream-stage specific invariant surface glycoprotein (ISG) confers partial protection of mice (40%) [66]. Targets for the anti-trypanosome vaccinations are the flagellar pocket, actin, tubulin, trans-sialidases and cation pumps. Alternative approaches are to develop an anti-disease vaccine by targeting infection-associated pathology rather than the parasite itself. The main targets used for this approach are glycosyl-inositol-phosphate anchor (GIP, is responsible for the induction of TNFα production) and cysteine protease (CP, elicits a high IgG response in trypanotolerant cattle) [67]. To date not one single experimental anti-trypanosome vaccination protocol showed efficacy in a field setting. One of the problems might be the use of general murine models for basic trypanosomiasis research that may not reflect the immunological situation of the natural hosts (B-cell function and memory maintenance suffer from trypanosomiasis-associated defects in mice) [67]. Possibly, the use of other, more relevant models for infection could lead to more successful outcomes in vaccine trials.

1.8.3 Screening and Diagnostics

Disease management is made in three steps: screening for potential infection (i), diagnosing if the parasite is present (ii) and stage determination (iii) [51].

A serological card agglutination test for trypanosomiasis (CATT) is used for mass screening of gambiense HAT. The test is based on the detection of antibodys against VSG [68, 69]. Unfortunately false negative can arise because of cross-reactions with non-specific epitopes and T. b. rhodesiense is not sensitive to the test (no shared VSGs have been found). For the moment, East African trypanosomiasis lacks an adapted screening test, but search for diagnostic antigens is ongoing [68]. Presence of the parasite is confirmed by the microscopic examination of blood, cervical lymph node fluid or trypanosomal chancre [60]. Because of the different treatment of the

Genetic and chemical validation of Trypanosoma brucei adenosine kinase, the intracellular target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine

Thesis

Reference

Genetic and chemical validation of Trypanosoma brucei adenosine kinase, the intracellular target of

4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine

Genetic and chemical validation of Trypanosoma brucei adenosine kinase, the intracellular target of

4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine

Patricia Graven

To my family

Table of Contents

Abbreviations

Summary

Résumé

PART 1

Introduction

Trypanosoma brucei

1.1 Classification of Trypanosomes

1.2 Morphology and cell structure

1.3 Vector and Life cycle

1.4 Genome

1.5 Energy metabolism

Human African Trypanosomiasis

1.6 Epidemiology and current situation

1.7 Pathology and clinical features

Fight against Sleeping sickness

1.8 Current Treatments for Sleeping sickness