Antitrypanosomal Activities of Proteasome Inhibitors

Seven peptidyl proteasome inhibitors were tested for in vitroactivity against Trypanosoma brucei bloodstream forms. Two compoundsshowed activity in the low nanomolar range. In general, trypanosomeswere more susceptible to the compounds than were human HL-60cells. The data support the potential of proteasome inhibitorsfor rational antitrypanosomal drug development.

INTRODUCTION

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Introduction

Trypanosoma brucei is the causative agent of human sleepingsickness in Africa. Over 60 million persons are at risk of acquiringthe infection, and approximately 50,000 new cases are reportedannually (21). If left untreated, the disease is fatal. Forchemotherapy of sleeping sickness only four drugs, with seriousside effects, are available (5). Therefore, the identificationof novel targets for chemotherapy is urgently required if newtreatments of the disease are to be developed.

The proteasome is a multicatalytic proteinase complex whichplays a critical role in intracellular protein degradation (4).Inhibitors of the proteasome have recently received attentionas a novel class of anticancer drugs (2, 8, 12, 15, 17). However,drugs developed as potential antitumor agents could also beof use against sleeping sickness, as has been shown for theornithine decarboxylase inhibitor D,L- ${alpha}$ -difluoromethylornithine(3). We therefore investigated the trypanocidal activities ofdifferent peptidyl proteasome inhibitors against in vitro-culturedbloodstream form T. brucei.

Proteasome inhibitors.

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Proteasome inhibitors.

N-Acetyl-leucyl-leucyl-norleucinal (Ac-Leu-Leu-Nle-CHO), N-acetyl-leucyl-leucyl-methional(Ac-Leu-Leu-Met-CHO), N-benzyloxycarbonyl-isoleucyl- ${gamma}$ -t-butyl-glutamyl-alanyl-leucinal [Z-Ile-Glu(OtBu)-Ala-Leu-CHO],N-benzyloxycarbonyl-leucyl-leucyl-leucinal (Z-Leu-Leu-Leu-CHO),and N-benzyloxycarbonyl-leucyl-leucyl-tyrosyl ${alpha}$ -keto aldehyde(Z-Leu-Leu-Tyr-COCHO) were purchased from Bachem BiochemicaGmbH (Heidelberg, Germany), and N-benzyloxycarbonyl-leucyl-leucyl-phenylalanal(Z-Leu-Leu-Phe-CHO) and N-benzyloxycarbonyl-leucyl-leucyl-leucylboronic acid [Z-Leu-Leu-Leu-B(OH)₂] were purchased from AlexisDeutschland GmbH (Grünberg, Germany). All compounds havebeen shown previously to be inhibitors of the proteasome (8-10,16, 17).

Assays.

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Assays.

Bloodstream form T. brucei (TC221) and human myeloid leukemiaHL-60 cells were grown axenically as described previously (11).

For toxicity tests, cells were seeded into 24-well plates atappropriate densities (10⁴ trypanosomes/ml; 5 x 10⁴ HL-60 cells/ml)in 1 ml of medium containing various concentrations (10^-4 to10^-12 M) of proteasome inhibitors dissolved in 100% dimethylsulfoxide (DMSO). The controls contained DMSO alone. In allexperiments, the final DMSO concentration was 1%, which hadno effect on the cell growth (11). After 48 h of incubation,living cells were counted with a Neubauer hemocytometer. Thecontrol cell counts were 10⁶ trypanosomes/ml and 5 x 10⁵ HL-60cells/ml. Each experiment was set up in duplicate and repeatedthree times.

For detection of apoptosis, cells were exposed to proteasomeinhibitors at various concentrations for 24 h, harvested bycentrifugation, and fixed overnight with 70% ethanol at -20°C.Then, cells were washed twice with HBSS (Hanks balanced saltsolution) and 5 min with 900 µl of HBSS plus 300 µlof 200 mM Na₂HPO₄-100 mM citrate (pH 7.8) and stained with 200µl of HBSS containing 0.5 mg of RNase per ml and 0.5 µgof propidium iodide per ml. After incubation for 30 min at roomtemperature, the DNA content of propidium iodide-stained cellswas analyzed with a FACScan analytical cytometer using CellQuestSoftware (Becton Dickinson, Heidelberg, Germany). Cells fromthe sub-G₀/G₁ peak were counted as apoptotic cells (14).

Antitrypanosomal activities of proteasome inhibitors.

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Antitrypanosomal activities of...

Peptide aldehydes are the best-characterized inhibitors of theproteasome. These agents are substrate analogues, and cellsare permeable to them. Previous studies have revealed that peptidealdehydes of the scaffold R-Leu-Leu-Xaa-CHO are the most potentinhibitors of the proteasome (1). Therefore, Ac-Leu-Leu-Nle-CHO,Ac-Leu-Leu-Met-CHO, Z-Leu-Leu-Leu-CHO, Z-Leu-Leu-Phe-CHO, andZ-Leu-Leu-Tyr-COCHO were chosen to have their antitrypanosomalactivities tested. Since peptide boronates are much more potentinhibitors of the proteasome (1, 8), the trypanocidal activityof the boronate analogue of Z-Leu-Leu-Leu-CHO, Z-Leu-Leu-Leu-B(OH)₂,was also investigated. Z-Ile-Glu(OtBu)-Ala-Leu-CHO was includedin this study because this compound has been shown to inhibitthe proteasome purified from T. brucei (13). All seven compoundsexhibited antitrypanosomal activities against bloodstream formT. brucei, with 50% effective doses (ED₅₀s) and MICs varying10,000-fold (Table 1). The most trypanocidal proteasome inhibitorswere Z-Ile-Glu(OtBu)-Ala-Leu-CHO and Z-Leu-Leu-Leu-B(OH)₂, withED₅₀s of 0.086 and 0.32 nM and MICs of 40 and 10 nM, respectively.Thus, the antitrypanosomal activities of these two proteasomeinhibitors are comparable to that of pentamidine isethionate,which is used to treat sleeping sickness (ED₅₀ range, 1 to 2.5nM; MIC range, 5.8 to 35 nM [11]).

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TABLE 1. ED₅₀s and MICs of proteasome inhibitors for bloodstream form T. brucei and human HL-60 cells^a

The general cytotoxicities of the proteasome inhibitors wereassayed with HL-60 cells (Table 1). All compounds were alsoactive against the human cells, with ED₅₀s ranging from 1.1nM to 20 µM. Encouragingly, the MICs of six compoundswere $>=$ 100 µM, the highest concentration tested; only Z-Leu-Leu-Leu-B(OH)₂had a MIC of 1 µM. However, the ED₅₀ and MIC ratios ofcytotoxic to trypanocidal activities were found to be in a modestrange for nearly all proteasome inhibitors (Table 2). Only Z-Ile-Glu(OtBu)-Ala-Leu-CHOgave substantial ED₅₀ and MIC ratios, with values of 400 and2,500, respectively. For comparison, anti-sleeping sicknessdrugs generally have higher ED₅₀ and MIC ratios (11).

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TABLE 2. ED₅₀ and MIC ratios of cytotoxic to trypanocidal activities of proteasome inhibitors^a

Proteasome inhibitors have been shown to induce apoptosis inmany oncogenic cell types (17), and trypanosomes have been reportedto be able to undergo apoptosis (18-20). This prompted us toinvestigate whether the growth-inhibitory effects of Z-Leu-Leu-Leu-B(OH)₂and Z-Ile-Glu(OtBu)-Ala-Leu-CHO, the two most toxic proteasomeinhibitors studied (Table 1), can be attributed to inductionof apoptosis. Based on hypodiploid DNA content, only 45% oftrypanosomes appeared as apoptotic cells compared to 90% ofHL-60 cells after exposure to Z-Leu-Leu-Leu-B(OH)₂ or Z-Ile-Glu(OtBu)-Ala-Leu-CHOfor 24 h (Fig. 1). The latter finding is in agreement with previousobservations that treatment of HL-60 cells with proteasome inhibitorsresults in induction of apoptosis (6). Since only half of thetrypanosomes appeared as apoptotic cells after treatment withboth compounds, it is questionable whether the trypanocidaleffect of proteasome inhibitors can be actually ascribed toinduction of programmed cell death in bloodstream form T. brucei.Targets other than, or in addition to, the proteasome may beaffected (8, 17).

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FIG. 1. Effects of Z-Leu-Leu-Leu-B(OH)₂ and Z-Ile-Glu(OtBu)-Ala-Leu-CHO in inducing apoptosis in T. brucei and human cells. TC221 bloodstream forms (•) and HL-60 cells ( ${square}$ ) were incubated with various concentrations of the proteasome inhibitors. After 24 h of culture, cells were stained with propidium iodide and analyzed with a fluorescence-activated cell sorter. Cells from the sub-G₀/G₁ peak were counted as apoptotic cells. The percentages of apoptotic cells were calculated in relation to the respective controls in the absence of proteasome inhibitor. Data points represent mean values from three independent experiments. The standard errors were typically less than 10%. 1.E-11 to 1.E-04, 10^-11 to 10^-4.

Although the compounds investigated in this study are not suitablefor clinical use because of their cytotoxic effects, the resultsprovide evidence that inhibition of the proteasome representsa new approach for the development of antitrypanosomal drugs.This finding may also be exploited in the future by utilizingthe wealth of information currently being generated on proteasomeinhibitors as anticancer agents (8, 12, 15, 17). For example,the relatively high trypanocidal activity of Z-Leu-Leu-Leu-B(OH)₂suggests that novel boronic acid proteasome inhibitors currentlyunder clinical evaluation (2) may provide a new class of anti-sleepingsickness drugs in the future. Since trypanosome and mammalianproteasomes differ in terms of their substrate specificities(7), specific and nontoxic proteasome inhibitors are the rationalchoice for future antitrypanosomal drug development.

ACKNOWLEDGMENTS

This work was supported in part by the Deutsche Forschungsgemeinschaft(SFB 544/Kontrolle Tropischer Infektionskrankheiten and GraduiertenKolleg 388/Biotechnologie).

We thank Kathy Andrews for critical reading of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: School of Biological Sciences, University of Bristol, Woodland Rd., Bristol BS8 1UG, United Kingdom. Phone: 44-117-9288254. Fax: 44-117-9257374. E-mail: dsteverding{at}hotmail.com.

REFERENCES

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