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Targets and strategies for drug development against human African sleeping sickness
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. (Anders Hofer)
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Trypanosoma brucei is a causative agent of African sleeping sickness. It is an extracellular parasite which circulates in the blood, lymph and eventually invades the central nervous system. There is a great need for new medicines against the disease and specific properties of nucleoside kinases in the pathogen can be exploited as targets for chemotherapy. 

T. brucei contains a gene where two thymidine kinase sequences are fused into a single open reading frame. These types of tandem thymidine kinases were found only in different types of parasites, which made us to believe that it might be beneficial for them. Each thymidine kinase sequence in these tandem enzymes are here referred to as a domain. By cloning and expressing each domain from T. brucei separately, we found that domain 1 was inactive and domain 2 was as active as the full-length enzyme. T. brucei thymidine kinase phosphorylated the pyrimidine nucleosides thymidine and deoxyuridine and to some extent purine nucleosides like deoxyinosine and deoxyguanosine. Human thymidine kinase increases the affinity to its substrates when it forms oligomers. Similarly, the T. brucei two thymidine kinase sequences, which can be viewed as a pseudodimer, had a higher affinity to its substrates than domain 2 alone. 

T. brucei lacks de novo purine biosynthesis and it is therefore dependent on salvaging the required purine nucleotides for RNA and DNA synthesis from the host. Purine salvage is considered as a target for drug development. It has been shown that in the presence of deoxyadenosine in the growth medium, the parasites accumulate high levels of dATP and the extensive phosphorylation of deoxyadenosine leads to depleted ATP pools. Initially, we wondered if deoxyadenosine could be used as a drug against T. brucei. However, we found that T. brucei is partially protected against deoxyadenosine because it was cleaved by the enzyme methylthioadenosine phosphorylase (MTAP) to adenine and ribose-1-phosphate. At higher concentration of deoxyadenosine, 3 the formed adenine was not efficiently salvaged into ATP and started to inhibit MTAP instead. The deoxyadenosine was then instead phosphorylated by adenosine kinase leading to accumulation of dATP. The MTAP reaction makes deoxyadenosine itself useless as a drug and instead we focused on finding analogues of deoxyadenosine or adenosine that were cleavage-resistant and at the same time good substrates of T. brucei adenosine kinase. Our best hit was then 9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine (FANA-A). An additional advantage of FANA-A as a drug was that it was taken up by the P1 nucleoside transporter family, which makes it useful also against multidrug resistant parasites that often have lost the P2 transporter function and take up their purines solely by the P1 transporter. In parallel with our study of nucleoside metabolism in T. brucei, we also have a collaboration project where we screen essential oils from plants which are used in traditional medicine. If the essential oils are active against the trypanosomes, we further analyze the different components in the oils to identify new drugs against African sleeping sickness. One such compound identified from the plant Smyrnium olusatrum is isofuranodiene, which inhibited T. brucei proliferation with an IC50 value of 3 μM.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2017. , 36 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1884
Keyword [en]
T. brucei thymidine kinase, T. brucei methylthioadenosine phosphorylase, FANA-A purine nucleoside analogues, essential oils
National Category
Biochemistry and Molecular Biology Pharmacology and Toxicology
Identifiers
URN: urn:nbn:se:umu:diva-131074ISBN: 978-91-7601-675-6 (print)OAI: oai:DiVA.org:umu-131074DiVA: diva2:1071945
Public defence
2017-03-03, KB.E3.01, KBC-Huset, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2017-02-10 Created: 2017-02-05 Last updated: 2017-03-23Bibliographically approved
List of papers
1. Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding
Open this publication in new window or tab >>Trypanosoma brucei thymidine kinase is tandem protein consisting of two homologous parts, which together enable efficient substrate binding
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2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 21, 17628-17636 p.Article in journal (Refereed) Published
Abstract [en]

Trypanosoma brucei causes African sleeping sickness, a disease for which existing chemotherapies are limited by their toxicity or lack of efficacy. We have found that four parasites, including T. brucei, contain genes where two or four thymidine kinase (TK) sequences are fused into a single open reading frame. The T. brucei full-length enzyme as well as its two constituent parts, domain 1 and domain 2, were separately expressed and characterized. Of potential interest for nucleoside analog development, T. brucei TK was less discriminative against purines than human TK1 with the following order of catalytic efficiencies: thymidine > deoxyuridine ≫ deoxyinosine > deoxyguanosine. Proteins from the TK1 family are generally dimers or tetramers, and the quaternary structure is linked to substrate affinity. T. brucei TK was primarily monomeric but can be considered a two-domain pseudodimer. Independent kinetic analysis of the two domains showed that only domain 2 was active. It had a similar turnover number (k(cat)) as the full-length enzyme but could not self-dimerize efficiently and had a 5-fold reduced thymidine/deoxyuridine affinity. Domain 1, which lacks three conserved active site residues, can therefore be considered a covalently attached structural partner that enhances substrate binding to domain 2. A consequence of the non-catalytic role of domain 1 is that its active site residues are released from evolutionary pressure, which can be advantageous for developing new catalytic functions. In addition, nearly identical 89-bp sequences present in both domains suggest that the exchange of genetic material between them can further promote evolution.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-58608 (URN)10.1074/jbc.M112.340059 (DOI)000306373000061 ()22442154 (PubMedID)
Available from: 2012-09-04 Created: 2012-09-04 Last updated: 2017-02-09Bibliographically approved
2. Trypanosoma brucei Methylthioadenosine Phosphorylase Protects the Parasite from the Antitrypanosomal Effect of Deoxyadenosine: IMPLICATIONS FOR THE PHARMACOLOGY OF ADENOSINE ANTIMETABOLITES
Open this publication in new window or tab >>Trypanosoma brucei Methylthioadenosine Phosphorylase Protects the Parasite from the Antitrypanosomal Effect of Deoxyadenosine: IMPLICATIONS FOR THE PHARMACOLOGY OF ADENOSINE ANTIMETABOLITES
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2016 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 22, 11717-11726 p.Article in journal (Refereed) Published
Abstract [en]

Trypanosoma brucei causes African sleeping sickness for which no vaccine exists and available treatments are of limited use due to their high toxicity or lack of efficacy. T. brucei cultivated in the presence of deoxyadenosine accumulates high levels of dATP in an adenosine kinase-dependent process and dies within a few hours. Here we show that T. brucei treated with 1 mM deoxyadenosine accumulates higher dATP levels than mammalian cells but that this effect diminishes quickly as the concentration of the deoxynucleoside decreases. Radioactive tracer studies showed that the parasites are partially protected against lower concentrations of deoxyadenosine by the ability to cleave it and use the adenine for ATP synthesis. T. brucei methylthioadenosine phosphorylase (TbMTAP) was found to be responsible for the cleavage as indicated by the phosphate dependence of deoxyadenosine cleavage in T. brucei cell extracts and increased deoxyadenosine sensitivity in TbMTAP knockdown cells. Recombinant TbMTAP exhibited higher turnover number (k(cat)) and K-m values for deoxyadenosine than for the regular substrate, methylthioadenosine. One of the reaction products, adenine, inhibited the enzyme, which might explain why TbMTAP-mediated protection is less efficient at higher deoxyadenosine concentrations. Consequently, T. brucei grown in the presence of adenine demonstrated increased sensitivity to deoxyadenosine. For deoxyadenosine/adenosine analogues to remain intact and be active against the parasite, they need to either be resistant to TbMTAP-mediated cleavage, which is the case with the three known antitrypanosomal agents adenine arabinoside, tubercidin, and cordycepin, or they need to be combined with TbMTAP inhibitors.

National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-123454 (URN)10.1074/jbc.M116.715615 (DOI)000377264800022 ()27036940 (PubMedID)
Available from: 2016-07-18 Created: 2016-07-04 Last updated: 2017-02-06Bibliographically approved
3. 9-(2-Deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine: a potent antitrypanosomal adenosine analogue that circumvents transport-related drug resistance
Open this publication in new window or tab >>9-(2-Deoxy-2-fluoro-ß-D-arabinofuranosyl) adenine: a potent antitrypanosomal adenosine analogue that circumvents transport-related drug resistance
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(English)Manuscript (preprint) (Other academic)
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-131073 (URN)
Available from: 2017-02-05 Created: 2017-02-05 Last updated: 2017-02-09
4. An overlooked horticultural crop, Smyrnium olusatrum, as a potential source of compounds effective against African trypanosomiasis
Open this publication in new window or tab >>An overlooked horticultural crop, Smyrnium olusatrum, as a potential source of compounds effective against African trypanosomiasis
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2017 (English)In: Parasitology international, ISSN 1383-5769, E-ISSN 1873-0329, Vol. 66, no 2, 146-151 p.Article in journal (Refereed) Published
Abstract [en]

Among natural products, sesquiterpenes have shown promising inhibitory effects against bloodstream forms of Trypanosoma brucei, the protozoan parasite causing human African trypanosomiasis (HAT). Smyrnium olusatrum (Apiaceae), also known as Alexanders or wild celery, is a neglected horticultural crop characterized by oxygenated sesquiterpenes containing a furan ring. In the present work we explored the potential of its essential oils obtained from different organs and the main oxygenated sesquiterpenes, namely isofuranodiene, germacrone and β-acetoxyfuranoeudesm-4(15)-ene, as inhibitors of Trypanosoma brucei. All essential oils effectively inhibited the growth of parasite showing IC50 values of 1.9–4.0 μg/ml. Among the main essential oil constituents, isofuranodiene exhibited a significant and selective inhibitory activity against T. brucei (IC50 of 0.6 μg/ml, SI = 30), with β-acetoxyfuranoeudesm-4(15)-ene giving a moderate potentiating effect. These results shed light on the possible application of isofuranodiene as an antiprotozoal agent to be included in combination treatments aimed not only at curing patients but also at preventing the diffusion of HAT.

Keyword
Apiaceae, Essential oil, Isofuranodiene, Trypanosoma brucei, Wild celery
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-131072 (URN)10.1016/j.parint.2017.01.001 (DOI)000394476700023 ()
Available from: 2017-02-05 Created: 2017-02-05 Last updated: 2017-05-23Bibliographically approved

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