Evaluation of the Anti-Trypanosomal Activity of Tyropeptin A

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

The natural compound tyropeptin A, a new peptidyl aldehyde proteasome inhibitor, was tested for its trypanocidal activity in vitro using culture-adapted bloodstream forms of Trypanosoma brucei. The concentrations of tyropeptin A required to reduce the growth rate by 50 % and to kill all cells were 10 and 100 times lower for bloodstream-form trypanosomes than for human leukaemia HL-60 cells, respectively. Enzymatic analysis showed that the trypsin-like activity of the trypanosome proteasome and the chymotrypsin-like activity of the mammalian proteasome are particularly sensitive to inhibition by tyropeptin A. The results suggest that natural compounds targeting the trypsin-like activity of the proteasome may serve as leads for rational drug development of novel anti-trypanosomal agents.

Trypanosoma brucei is the pathogen responsible for human African trypanosomiasis or sleeping sickness. Over 60 million people living in 36 sub-Saharan countries are at risk of contracting sleeping sickness and the estimated number of cases is thought to be between 300,000 and 500,000 [1]. Sleeping sickness is a serious disease and without treatment patients will ultimately die. The four drugs currently available for chemotherapy of human African trypanosomiasis have major drawbacks, including undesirable toxicity, poor efficacy and emerging drug resistance [2], [3]. Therefore, new drugs for the treatment of sleeping sickness are urgently required.

Natural compounds including alkaloids, phenolic derivatives, quinines and terpenes have been shown to inhibit the growth of African trypanosomes in vitro (reviewed in [4]). Of particular interest are inhibitors of the proteasome, as this enzyme complex has been demonstrated to be a valid target for the development of new drugs to treat sleeping sickness [5]. In fact, one of the most trypanocidal proteasome inhibitors is the natural compound epoxomicin [6]. In this paper, we studied the effect of tyropeptin A, a peptidyl aldehyde compound (Fig. [1]) which is produced by Kitasatospora sp. MK993-dF2 [7], on T. brucei bloodstream forms and on purified trypanosome proteasomes. For comparison, tyropeptin A was also tested against human cells and purified mammalian proteasomes.

The trypanocidal and cytotoxic activities of tyropeptin A were determined with T. brucei bloodstream forms 427 - 221a and leukaemia HL-60 cells, respectively, using the growth inhibition assay described previously [8]. Tyropeptin A showed a dose-dependent effect on the growth of both cell types with 50 % growth inhibition (GI₅₀) values of 3.0 × 10 ^{- 7} M for trypanosomes and 3.4 × 10 ^{- 6} M for human cells (Fig. [2]). The minimal inhibitory concentration (MIC) value was 1 × 10 ^{- 6} M for T. brucei bloodstream forms and > 10 ^{- 4} M for HL-60 cells (Fig. [2]). As a result, the GI₅₀ and MIC ratios of cytotoxic/trypanocidal activities (selectivity indices) were 10 and > 100, respectively.

The inhibition of the proteasomal peptidase activities by tyropeptin A was studied with enzyme complexes purified from bloodstream-form trypanosomes and rat liver. Tyropeptin A inactivated the chymotrypsin-like activity of the mammalian proteasome more strongly than that of the trypanosome proteasome (Table [1]). On the other hand, the compound was more potent in inhibiting the trypsin-like activity of the trypanosome proteasome than that of the mammalian proteasome (Table [1]). The epoxyketone epoxomicin, the most selective proteasome inhibitor known, was used as positive control to demonstrate the functionality of the peptidase inactivation test (Table [1]) [6].

It has been shown that tyropeptin A also inhibits cathepsin L [7]. However, bloodstream forms of T. brucei contain a lysosomal cathepsin L-like cysteine proteinase termed brucipain that is vital for their survival [9]. To determine whether the trypanocidal effect of tyropeptin A was due to inhibition of brucipain, T. brucei bloodstream forms were incubated with fluorescein-labelled transferrin in the presence of 10 μM tyropeptin A. It has been previously shown that inhibition of brucipain leads to accumulation of fluorescein-labelled transferrin in the lysosome of bloodstream-form trypanosomes [9], [10], [11]. However, no accumulation of fluorescein-labelled transferrin was observed in the lysosome of bloodstream-form trypanosomes in the presence of 10 μM tyropeptin A (data not shown). It should be noted that at 10 μM all parasites were killed in the toxicity assay (see Fig. [2]). This result shows that tyropeptin A does not inhibit brucipain activity and that, therefore, its toxic effect on T. brucei bloodstream forms can solely be ascribed to blocking proteasomal activity.

The findings reported in this study are consistent with previous observations that, in contrast to mammalian cells, bloodstream-form trypanosomes are particularly sensitive to inhibition of the trypsin-like activity of the proteasome [6], [12]. This may be due to the high trypsin-like activity and low chymotrypsin-like activity of the trypanosomal proteasome while the exact opposite applies to the mammalian proteasome [13], [14]. The discrepancy between cytotoxic activities (10-fold and 100-fold) and inhibitory activities against the trypsin-like activity of the trypanosome and mammalian proteasome (3-fold) may be explained by substantial inactivation of the cellular proteasome during the 48 h incubation time of the growth inhibition assay.

In conclusion, proteasome inhibitors specifically targeting the trypsin-like activity of the proteasome are the rational choice for future anti-trypanosomal drug development. Such agents would display low cytotoxicities, as the host cells rely less on the trypsin-like activity of the proteasome. Although tyropeptin A is not suitable for immediate clinical use because of its less favourable selectivity indices compared with the commercially available drugs [15], the results of this study showed that natural compounds may serve as leads for the development of novel drugs to treat human African trypanosomiasis.

Materials and Methods

Reagents: Tyropeptin A (Isovaleryl-L-tyrosyl-L-valyl-DL-tyrosinal) was obtained from Merck Bioscience (Nottingham, UK). Suc-LLVY-AMC (N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amido-4-methylcoumarin) and Boc-LSTR-AMC (N-tert-butoxycarbonyl-leucyl-seryl-threonyl-arginyl-7-amido-4-methylcoumarin) were purchased from the Peptide Institute (Osaka, Japan).

Cell culture assays: Bloodstream forms of T. brucei clone 427 - 221a [16] and human myeloid leukaemia HL-60 cells [17] were grown in Baltz medium [18] and RPMI medium, respectively. Both media were supplemented with 16.7 % heat-inactivated foetal calf serum. Trypanosomes and human cells were incubated at 37 °C in a humidified atmosphere containing 5 % CO₂.

For toxicity assays, cells were seeded at an appropriate density (10⁴ trypanosome/mL; 10⁵ HL-60 cells/mL) into 24-well tissue culture plates in 1 mL medium containing various concentrations (10 ^{- 4} to 10 ^{- 9} M) of tyropeptin A and 1 % DMSO as solvent. Wells containing cells, medium and 1 % DMSO served as controls. After 48 h incubation, live cells were counted using a haemocytometer. Each test was repeated three times. The 50 % growth inhibition value (GI₅₀), i. e., the inhibitor concentration necessary to reduce the growth rate of the cells by 50 % of that of controls, was determined by linear interpolation [19]. The minimum inhibitory concentration (MIC), i. e., the lowest concentration of the inhibitor at which all cells were killed, was determined microscopically.

Assay of proteasome activity: Trypanosome and rat liver proteasomes were purified as described previously [6], [20]. Peptidase activities of proteasomes were determined in 50 mM Hepes buffer, pH 5.0 with 40 μM Suc-LLVY-AMC (chymotrypsin-like activity) or Boc-LSTR-AMC (trysin-like activity). Each test was set up in duplicate and repeated three times. The 50 % inhibitory concentration (IC₅₀) was determined by a regression method based on the S-shaped concentration-inhibition relationship [6].

Acknowledgements

The authors thank Robert J. Genn and Emily Smith for technical assistance.

References

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  PubMedSearch in Google Scholar

Priv.-Doz. Dr. Dietmar Steverding

School of Medicine, Health Policy and Practice

University of East Anglia

Norwich NR4 7TJ

United Kingdom

Fax: +44-1603-59-3752

Email: dsteverding@hotmail.com

References

• 1 Barrett M P, Burchmore R J, Stich A, Lazzari J O, Frasch A C, Cazzulo J J. et al . The trypanosomaises.  Lancet. 2003;  362 1469-80
  Search in Google Scholar
• 2 Fairlamb A H. Chemotherapy of human African trypanosomiasis: current and future prospects.  Trends Parasitol. 2003;  19 488-94
  CrossrefPubMedSearch in Google Scholar
• 3 Matovu E, Seebeck T, Enyaru J CK, Kaminsky R. Drug resistance in Trypanosoma brucei spp., the causative agents of sleeping sickness in man and nagana in cattle.  Microbes Infect. 2001;  3 763-70
  CrossrefPubMedSearch in Google Scholar
• 4 Hoet S, Opperdoes F, Brun R, Quetin-Leclercq J. Natural products active against African trypanosomes: a step towards new drugs.  Nat Prod Rep. 2004;  21 353-64
  CrossrefPubMedSearch in Google Scholar
• 5 Nkemgu-Njinkeng J, Rosenkranz V, Wink M, Steverding D. Antitrypanosomal activities of proteasome inhibitors.  Antimicrob Agents Chemother. 2002;  46 2038-40
  CrossrefPubMedSearch in Google Scholar
• 6 Glenn R J, Pemberton A J, Royle H J, Spackman R W, Smith E, Rivett A J. et al . Trypanocidal effect of α′,β′-epoxyketones indicates that trypanosomes are particularly sensitive to inhibitors of proteasome trypsin-like activity.  Int J Antimicrob Agents. 2004;  24 286-9
  PubMedSearch in Google Scholar
• 7 Momose I, Sekizawa R, Hashizume H, Kinoshita N, Homma Y, Hamada M. et al . Tyropeptins A and B, new proteasome inhibitors produced by Kitasatospora sp. MK933-dF2. 1. Taxonomy, isolation, physio-chemical properties and biological activities.  J Antibiot (Tokyo). 2001;  54 997-1003
  PubMedSearch in Google Scholar
• 8 Scory S, Steverding D. Differential toxicity of ricin and diphtheria toxin for bloodstream forms of Trypanosoma brucei .  Mol Biochem Parasitol. 1997;  90 289-95
  CrossrefPubMedSearch in Google Scholar
• 9 Scory S, Caffrey C R, Stierhof Y D, Ruppel A, Steverding D. Trypanosoma brucei: killing of bloodstream forms in vitro and in vivo by the cysteine proteinase inhibitor Z-Phe-Ala-CHN₂ .  Exp Parasitol. 1999;  91 327-33
  CrossrefPubMedSearch in Google Scholar
• 10 Steverding D, Stierhof Y D, Fuchs H, Tauber R, Overath P. Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei .  J Cell Biol. 1995;  131 1173-82
  CrossrefPubMedSearch in Google Scholar
• 11 Kabiri M, Steverding D. Trypanosoma evansi: demonstration of a transferrin receptor derived from expression site-associated genes 6 and 7.  J Parasitol. 2001;  87 1189-91
  CrossrefPubMedSearch in Google Scholar
• 12 Steverding D, Spackman R W, Royle H J, Glenn R J. Trypanocidal activities of trileucine methyl vinyl sulfone proteasome inhibitors.  Parasitol Res. 2005;  95 73-6
  CrossrefPubMedSearch in Google Scholar
• 13 Hua S B, To W Y, Nguyen T T, Wong M L, Wang C C. Purification and characterization of proteasomes from Trypanosoma brucei .  Mol Biochem Parasitol. 1996;  78 33-46
  CrossrefPubMedSearch in Google Scholar
• 14 Wang C C, Bozdech Z, Liu C L, Shipway A, Backes B J, Harris J L. et al . Biochemical analysis of the 20S proteasome of Trypanosoma brucei .  J Biol Chem. 2002;  278 15 800-8
  PubMedSearch in Google Scholar
• 15 Merschjohann K, Sporer F, Steverding D, Wink M. In vitro effect of alkaloids on bloodstream forms of Trypanosoma brucei and T. congolense .  Planta Med. 2001;  67 623-7
  Thieme ConnectPubMedSearch in Google Scholar
• 16 Hirumi H, Hirumi K, Doyle J J, Cross G AM. In vitro cloning of animal-infective bloodstream forms of Trypanosoma brucei .  Parasitology. 1980;  80 371-82
  PubMedSearch in Google Scholar
• 17 Collins S R, Gallo R C, Gallagher R E. Continuous growth and differentiation of human myeloid leukaemic cells in suspension cultures.  Nature. 1997;  270 347-9
  PubMedSearch in Google Scholar
• 18 Baltz T, Baltz D, Giroud C, Crockett J. Cultivation in a semi-defined medium of animal infective forms of Trypanosoma brucei, T. equiperdum, T. evansi, T. rhodesiense and T. gambiense .  EMBO J. 1985;  4 1273-7
  PubMedSearch in Google Scholar
• 19 Huber W, Koella J C. A comparison of three methods of estimating EC₅₀ in studies of drug resistance of malaria parasites.  Acta Trop. 1993;  55 257-61
  CrossrefPubMedSearch in Google Scholar
• 20 Rivett A J, Savory P J, Djaballah H. Multicatalytic endopeptidase complex: proteasome.  Methods Enzymol. 1994;  244 331-50
  PubMedSearch in Google Scholar

Priv.-Doz. Dr. Dietmar Steverding

School of Medicine, Health Policy and Practice

University of East Anglia

Norwich NR4 7TJ

United Kingdom

Fax: +44-1603-59-3752

Email: dsteverding@hotmail.com