Evaluation of trypanocidal activity of combinations of anti-sleeping sickness drugs with cysteine protease inhibitors

Dietmar Steverding*

BioMedical Research Centre, Norwich Medical School, University of East Anglia, Norwich,

United Kingdom

* Corresponding authors at: BioMedical Research Centre, Norwich Medical School,

University of East Anglia, Norwich NR4 7TJ, United Kingdom. Tel.: +44-1603-591291; fax:

+44-1603-591750.

E-mail address: dsteverding@hotmail.com (D. Steverding).

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A B S T R A C T

Chemotherapy of human African trypanosomiasis (HAT) is unsatisfactory because only a

few drugs, with serious side effects and poor efficacy, are available. As drug combination

regimes often achieve greater therapeutic efficacy than monotherapies, here the trypanocidal

activity of the cysteine protease inhibitor K11777 in combination with current anti-HAT

drugs using bloodstream forms of Trypanosoma brucei was investigated. Isobolographic

analysis was used to determine the interaction between cysteine protease inhibitors (K11777,

CA-074Me and CAA0225) and anti-HAT drugs (suramin, pentamidine, melarsoprol and

eflornithine). Bloodstream forms of T. brucei were incubated in culture medium containing

cysteine protease inhibitors or anti-HAT drugs alone or in combination at a 1:1 fixed-dose

ratio. After 48 h incubation, live cells were counted, the 50% growth inhibition values

determined and combination indices calculated. The general cytotoxicity of drug

combinations was evaluated with human leukemia HL-60 cells. Combinations of K11777

with suramin, pentamidine and melarsoprol showed antagonistic effects while with

eflornithine a synergistic effect was observed. Whereas eflornithine antagonises with CA-

074Me, an inhibitor inactivating the targeted TbCATL only under reducing conditions, it

synergises with CAA0255, an inhibitor structurally related to CA-074Me which inactivates

TbCATL independently of thiols. These findings indicate an essential role of thiols for the

synergistc interaction between K11777 and eflornithine. Encouragingly, the

K11777/eflornithine combination displayed higher trypanocidal than cytotoxic activity. The

results of this study suggest that the combination of the cysteine protease inhibitor K11777

and eflornithine display promising synergistic trypanocidal activity that warrants further

investigation of the drug combination as possible alternative treatment of HAT.

Keywords:

Trypanosoma brucei

Cysteine protease inhibitors

Anti-sleeping sickness drugs

Drug combination

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1. Introduction

Human African trypanosomiasis (HAT) or sleeping sickness is one of the most neglected

tropical diseases. HAT affects mainly poor people living in 250 rural foci scattered over 36

sub-Saharan African countries where it is a major cause for morbidity and mortality (WHO,

2014). The disease is caused by the flagellated protozoan Trypanosoma brucei and

transmitted by the bite of infected tsetse flies. The extracellular parasites live first in the

blood and lymphatic system and later enter the central nervous system causing the typical

symptoms of HAT which are confusion, disturbed sleep pattern, sensory disturbances,

extreme lethargy, poor condition and coma (WHO, 2014). If left untreated, HAT patients will

ultimately die within months or years (WHO, 2014). Sadly, only a few drugs (suramin,

pentamidine, melarsoprol and eflornithine) and one drug combination

(nifurtimox/eflornithine) are currently available for chemotherapy of HAT (Steverding,

2010). All these drugs are outdated and have major drawbacks including poor efficacy,

significant toxicity and the requirement for parental administration (Fairlamb, 2003;

Steverding, 2010). In addition, the compounds are increasingly subject to drug resistance

(Matovu et al., 2001; Delespaux and de Koning, 2007). Hence, effective and better tolerated

chemotherapies are urgently needed for treatment of HAT.

It is well established that drug combination regimes often achieve greater therapeutic

efficacy than monotherapies (e.g. in the treatment of cancer, AIDS and tuberculosis). Drug

combinations may be less toxic than monotherapy regimes and have the additional advantage

of reducing the risk for development of drug resistance. This is because drug combinations

may interact synergistically, i.e. their combined effect may be greater than the sum of their

separate effects (Chou, 2006). However, additive effects are also desirable as they permit

decreased dosage while maintaining efficacy (Chou, 2006). The recently introduced

nifurtimox/eflornithine combination therapy as first line treatment of second stage HAT,

however, failed to show synergistic trypanocidal activity in vitro. Biochemical analysis

revealed that the metabolomic changes associated with the combination are the sum of those

found in each monotherapy with no indication of additional effects (Vincent et al., 2012). In

addition, nifurtimox as a nitro-heterocyclic drug has mutagenic, toxic and carcinogenic side

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effects. Therefore, other drug combinations should be investigated to find combination

therapies with less toxic adverse effects.

Proteases play a key role in the life cycle of T. brucei and in the pathogenicity of HAT

(Steverding, 2013). A chemically validated drug target is the T. brucei cathepsin L-like

protease, TbCATL (aka brucipain, rhodesain or trypanopain-Tb) (Steverding et al., 2012), a

cysteine protease belonging to the C1 family of papain-like enzymes (Caffrey and

Steverding, 2009). TbCATL is found in the lysosome of bloodstream forms of T. brucei

where it is involved in the degradation of endocytosed host proteins (Scory et al., 1999).

Targeting TbCATL with small-molecule inhibitors is trypanocidal both in vitro and in vivo

(Scory et al., 1999; Troeberg et al., 1999; Caffrey et al., 2000). One promising inhibitor that

has emerged over the last 15 years is the peptidomimetic vinyl sulphone K11777 (Fig. 1)

(Caffrey and Steverding, 2008). This inhibitor has been shown to reduce the parasitaemia and

to prolong the survival of T. brucei-infected mice (Caffrey et al., 2000). Considering that

K11777 has been approved by the FDA to enter a phase I safety trial in the United State

(Rodenko and de Koning, 2013), the main aim of this study was to evaluate the trypanocidal

efficacy of K11777 and anti-HAT drug combinations on bloodstream forms of T. brucei in

vitro.

2. Materials and methods

2.1. Drugs

Eflonithine (DL-α-difluoromethylornithine), suramin, pentamidine and CA-074Me ((L-3-

trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester) were

purchased from Sigma-Aldrich (Gillingham, UK). Z-FR-AMC (benzyloxycarbonyl-

phenylalanyl-arginyl-7-amido-4-methyl coumarin) was from Enzo Life Sciences Ltd. (Exeter,

UK). Melarsoprol was a gift from Sanofi-Aventis Deutschland GmbH (Frankfurt, Germany).

K11777 ((N-methyl-piperazinyl-phenylalanyl-homophenylalanyl-vinylsulfone-phenyl)) was

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kindly provided by Dr Conor R. Caffrey (University of California San Francisco, USA).

CAA0225 ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-

ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) was synthesised as previously

described (Steverding et al., 2012).

2.2. Cell Culture

Bloodstream forms of T. brucei clone 427-221a (Hirumi et al., 1980) and human

myelocytic leukemia HL-60 cells (Collins et al., 1977) were grown in Baltz medium (Baltz et

al., 1985) and RPMI 1640 medium (Moore et al., 1967), respectively. Both media were

supplemented with 16.7 % heat-inactivated foetal bovine serum. Trypanosomes and HL-60

cells were cultured at 37°C in a humidified atmosphere containing 5% CO2.

2.3. Isobolographic analysis.

The interaction of drugs was evaluated using the isobolographic method as described

previously (Steverding and Wang, 2009). First, the 50% growth inhibition (GI50) value, i.e.,

the drug concentration necessary to reduce the growth of cells by 50% compared to that of

controls, was determined for each drug. Bloodstream forms of T. brucei were seeded at an

initial density of 104/ml into a 24-well plate in 1 ml medium containing various drug

concentrations. Wells without drugs served as controls. After 48 h of incubation, live

trypanosomes were counted using a haemocytometer. GI50 values were determined by linear

interpolation according to the method described by Huber and Koella (1993). Based on the

GI50 values, trypanosomes were incubated with twofold serially diluted 1:1 ratios of drug

combination. For controls, trypanosomes were cultured with twofold serially diluted

concentrations of each drug alone. After 48 h incubation, live cells were counted and the GI 50

value for each drug in the absence and in the presence of the other co-administered drug was

determined. The combination index (CI) for each drug combination was calculated using the

equation

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GI50(A,com) GI50(B,com)

CI = ————— + —————GI50(A,sin) GI50(B,sin)

where GI50(A,com) and GI50(B,com) are the concentrations of drug A and B used in the combination

to achieve 50% growth inhibition and GI50(A,sin) and GI50(B,sin) are the concentrations for the

single drugs to achieve the same effect. A CI value of <1, =1, and >1 indicates synergism,

additive effect, and antagonism, respectively.6

2.4. TbCATL activity assay

The effect of the cysteine protease inhibitors K11777, CA-074Me and CAA0225 on the

catalytic activity of recombinant TbCATL was determined by pre-incubating the enzyme (6.1

μg/ml) with 100 μM of the compounds in 100 mM citrate, pH 5, 10% DMSO, in the presence

or absence of 1.5 mM DTT for 2 h. Controls were treated with 10% DMSO alone in the

presence or absence of 1.5 mM DTT for the same period of time. Then, 50 μl of the enzyme

solution was added to 1950 μl measuring buffer (100 mM citrate, pH 5, 2 mM DTT)

containing 10 μM Z-FR-AMC. The final enzyme concentration in the assay was 0.305 μg/ml.

After 10 min incubation at room temperature, the reaction was stopped by adding iodoacetic

acid to a final concentration of 5 mM. Release of 7-amino-4-methylcoumarin (AMC) was

measured at excitation and emission wavelengths of 360 and 460 nm in a BIORAD

VersaFlour fluorometer. Data were calculated as specific activity (pmol AMC

released/min/μg protein) using a standard curve constructed with AMC.

2.5. Cytotoxic evaluation of drug combinations

The general toxicity of drug combinations that act synergistically on trypanosomes was

assayed with HL-60 cells. Drugs were tested alone or together at 1, 10 and 100 the

concentration of the drugs in the combination that inhibited the growth of trypanosomes by

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50%. HL-60 cells were seeded at an initial density of 105/ml into a 24-well plate in 1 ml

medium in the presence of the drugs to be tested. After 48 h incubation, live cells were

counted using a haemocytometer in the presence of the vital dye Trypan Blue.

3. Results and discussion

Combinations of K11777 with suramin, pentamidine or melarsoprol showed antagonistic

effects with CIs of 1.67±0.43, 1.46±0.10 and 1.46±0.08, respectively (Fig. 2A-C). Whereas

the GI50 of K11777 dropped from around 3.5 μM to about 2.0 μM, the GI50 of all three anti-

HAT drugs remained almost unchanged (suramin: from 0.10±0.02 to 0.10±0.03 μM;

pentamidine: from 10.3±1.9 to 8.3±0.4 nM; melarsoprol: from 9.6±0.5 to 8.7±0.2 nM). These

results indicate that lysosomal TbCATL activity, the enzyme activity targeted by K11777,

may play a role in the trypanocidal action of the three anti-HAT drugs. At least for suramin

this suggestion seems to be true as TbCATL knock down bloodstream form trypanosomes

display a fourfold lower sensitivity for the drug (Alsford et al., 2012).

In contrast to suramin, pentamidine and melarsoprol, eflornithine in combination with

K11777 showed a synergistic effect with a CI of 0.67±0.22 (Fig. 2D). The GI50 of K11777

dropped from 3.6±0.5 to 0.9±0.2 μM and the GI50 of eflornithine from 41.2±4.0 to 17.1±4.5

μM. As eflornithine inhibits the polymanine biosynthesis leading to a decrease in the

concentration of trypanothione (the glutathione analogue in trypanosomes) (Fairlamb et al.,

1987), and as the activity of TbCATL is dependent on thiols (Troeberg et al., 1996), it is

reasonable to suggest that the synergistic effect of the K11777/eflornithine combination is

associated with a reduced thiol concentration in the lysosome. In this context it should be

mentioned that the intralysosomal milieu has been shown to be a reducing environment

(Pisoni et al., 1990). To prove the suggestion, another cysteine protease inhibitor, CA-074Me

(Fig. 1), which in contrast to K11777 inhibits TbCATL only under reducing conditions

(Table 1) (Steverding et al., 2012), was tested in combination with eflornithine. The CA-

074Me/eflornithine combination showed an antagonistic effect with a CI of 1.33±0.09 (Fig.

3A). Accordingly, the GI50 of both compounds decreased only a little (CA-074Me: from

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8.6±0.6 to 5.1±0.7 μM; eflornithine: from 39.3±3.2 to 28.0±7.9 μM). To rule out the

possibility that the observed difference in the interaction between the two cysteine protease

inhibitor and eflornithine is due to their differences in structure and pharmacophore (K11777

is a vinyl sulphone whereas CA-074 is an oxirane), a third cysteine protease inhibitor

structurally related to CA-074Me, CAA0225 (Fig. 1), which inhibits TbCATL independently

of thiols (Table 1) (Steverding et al., 2012), was also tested. In contrast to CA-074Me,

CAA0225 showed a synergistic effect in combination with eflornithine with a CI of

0.89±0.17 (Fig. 3B). The drop in GI50 values of CAA0225 (from 1.8±0.6 to 0.6±0.1 μM) and

eflornithine (from 40.7±3.8 to 21.6±2.6 μM) was of similar extent compared to that of

K11777 and eflornithine (see above). These results confirm the suggestion that the

underlying mechanism for synergy of the K11777/eflornithine combination is indeed

facilitated by the reduction in thiol levels. This suggested mechanism is further corroborated

by the observation that eflornithine antagonises with suramin whose trypanocidal action

relies on the activity of the thiol-dependent TbCATL (Alsford et al., 2012).

The general cytotoxicity of the K11777/eflornithine combination which acted

synergistically on trypanosomes was assayed with human myeloid leukaemia HL-60 cells.

The concentration that inhibited the growth of bloodstream forms of T. brucei by 50% (0.9

μM K11777/17.1 μM eflornithine; see Fig. 2D) had no effect on the proliferation of HL-60

cells (Fig. 4). Even a 10-fold higher concentration (9 μM K11777/171 μM eflornithine)

slowed down the growth of HL-60 cells by only 25% (Fig. 4). However, a 100-fold higher

concentration (90 μM K11777/1710 μM eflonithine) was toxic to the human cells whereby

the cytotoxic effect of the combination was almost entirely due to the action of the cysteine

protease inhibitor K11777 (Fig. 4). It should be noted that HL-60 cells compared to non-

malignant cells are generally more sensitive to cytotoxic drugs as, for example, has been

recently shown for the ionophore salinomycin (Steverding and Sexton, 2013). Thus,

compared to normal cells, the cytotoxicity of the K11777/eflornithine combination

determined with HL-60 cells is likely to be overestimated.

This study has shown that the cysteine protease inhibitor K11777 acts synergistically with

the anti-HAT drug eflornithine to inhibit the growth of bloodstream forms of T. brucei and

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that the synergistic interaction between the two drugs was due to a reduction in thiols levels.

In addition, the K11777/eflornithine combination was found to be less toxic to human HL-60

cells than to trypanosomes. These findings suggest that the combination of K11777 and

eflornithine could be developed into an alternative treatment of HAT. However, as this was

an in vitro study, further investigations are required in order to establish the in vivo efficacy

of the K11777/eflornithine combination. In this respect, it is worth mentioning that K11777 is

currently undergoing clinical trials as a potential new drug for the treatment of Chagas

disease caused by Trypanosoma cruzi. As in T. brucei, K11777 also targets a cathepsin L-like

protease in T. cruzi, TcCATL, which is closely related to TbCATL (Caffrey and Steverding,

2009). In addition, both enzymes are found in the lysosome of both parasites (although

TcCATL has been additionally localized to the prelysosomal reservasome, the flagellar

pocket and the plasma membrane) and contribute to nutrient processing (Sajid et al., 2011).

Moreover, both TbCATL and TcCATL prefer basic and bulky, hydrophobic residues in the

S1 subsite and in the S2 subsite, respectively (Dunny et al., 2013). As both enzymes share

common features, it is reasonable to assume that the soon available pharmacokinetic and

pharmacodynamic data for K11777 from the clinical trials will help with the design of in vivo

studies to determine the efficacy of the compound in combination with eflornithine to cure

animals experimentally infected with T. brucei.

Acknowledgements

I thank Dr Darren Sexton for critical reading of the manuscript.

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Tables

Table 1

Effect of dithiothreitol (DTT) on the inhibition of TbCATL by cysteine protease inhibitors

Inhibitor

Activity as pmol/min/μg protein (%)a

‒ DTT + DTT

DMSO 515±74 (100%) 802±112 (100%)

K11777 24±3 (4.7%) 1±1 (0.1%)

CA-074Me 522±67 (101.1%) 7±2 (0.9%)

CAA0225 31±2 (6.0%) 1±1 (0.1%)

Recombinant TbCATL was pre-treated with 100 μM of inhibitor or equivalent volume of

DMSO in the absence or presence of 1.5 mM DTT and 2 h later assayed for hydrolysis of Z-

FR-AMC. Data are means ± SD of three independent experiments.

a Percent activity relative to DMSO controls.

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Figure legends

Fig. 1. Chemical structures of K11777, CA-074Me and CAA0225.

Fig. 2. Isobolographic plot for the interaction between K11777 and suramin (A), pentamidine

(B), melarsorpol (C), and eflornithine (D). Bloodstream forms of T. brucei were incubated

with twofold serial dilutions of drug combinations (1:1) or drugs alone. After 48 h of

incubation, live cells were counted and GI50 values determined. The dotted line that connects

the GI50 points for the single drug treatments (filled squares) is the theoretical additive line.

The GI50 of the combinations is indicated by open circles. Each point represents the mean ±

SD of three independent experiments.

Fig. 3. Isobolographic plot for the interaction between eflornithine and CA-074Me (A), and

CAA0225 (B). For experimental details see Fig. 2.

Fig. 4. Effect of eflornithine and K11777 on the growth of HL-60 cells. Cells were incubated

with eflornithine alone (dark grey columns), eflornithine/K11777 combined (medium grey

columns) or K11777 alone (light grey columns) at 1, 10 or 100 of the drug combination

that inhibited the growth of trypanosomes by 50% (17.1 μM and 0.9 μM, respectively). Mean

values ± SD of three independent experiments are shown.

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333

334

335

336

337

338

339

340

341

342

343

344

345346

Figure 1

O

NH

NH

SO O

O

N

NCH 3

K11777

OCH 3

O

N

O

NH

O

ONH

O

CA-074Me

NH O

O

NH

OHO

O

NH

CAA0225

15

347

348

349

350

351

352

353

354

355

356

357

358

359360

Figure 2

A

0

1

2

3

4

0 0.03 0.06 0.09 0.12

K11

777

(μM

)

Suramin (μM)

B

0

1

2

3

4

0 2 4 6 8 10 12

K11

777

(μM

)

Pentamidine (nM)

16

361362363

364365

366367

Figure 2 (cont.)

C

0

1

2

3

4

0 2 4 6 8 10

K11

777

(μM

)

Melarsoprol (nM)

D

0

1

2

3

4

0 10 20 30 40

K11

777

(μM

)

Eflornithine (μM)

17

368369370

371372

373374

Figure 3

A

0

2

4

6

8

0 10 20 30 40

CA-

074M

e (μ

M)

Eflornithine (μM)

B

0

0.6

1.2

1.8

2.4

0 10 20 30 40

CAA

0225

(μM

)

Eflornithine (μM)

18

375376377

378379

380381

Figure 4

0

20

40

60

80

100

120

1X 10X 100X

Cel

l Gro

wth

(% o

f Con

trol

)

Concentration of DFMO/K11777 (17.1 μM/0.9 μM)

DFMO DFMO/K11777 K11777

19

382

383

384