Repurposing auranofin as a lead candidate for treatment of lymphatic filariasis and onchocerciasis.

Two major human diseases caused by filariid nematodes are onchocerciasis, or river blindness, and lymphatic filariasis, which can lead to elephantiasis. The drugs ivermectin, diethylcarbamazine (DEC), and albendazole are used in control programs for these diseases, but are mainly effective against the microfilarial stage and have minimal or no effect on adult worms. Adult Onchocerca volvulus and Brugia malayi worms (macrofilariae) can live for up to 15 years, reproducing and allowing the infection to persist in a population. Therefore, to support control or elimination of these two diseases, effective macrofilaricidal drugs are necessary, in addition to current drugs. In an effort to identify macrofilaricidal drugs, we screened an FDA-approved library with adult worms of Brugia spp. and Onchocerca ochengi, third-stage larvae (L3s) of Onchocerca volvulus, and the microfilariae of both O. ochengi and Loa loa. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective in vitro in killing both Brugia spp. and O. ochengi adult worms and in inhibiting the molting of L3s of O. volvulus with IC50 values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC50 against the microfilariae of L. loa compared with the IC50 for adult female O. ochengi, which may be beneficial if used in areas where Onchocerca and Brugia are co-endemic with L. loa, to prevent severe adverse reactions to the drug-induced death of L. loa microfilariae. Further testing indicated that auranofin is also effective in reducing Brugia adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode.

Introduction

River blindness and lymphatic filariasis (LF) are two major neglected diseases caused by filariid nematodes that, together, affect an estimated 145 million people worldwide in mostly poor, developing countries [1,2]. River blindness, caused by the filariid nematode Onchocerca volvulus, is a chronic, debilitating disease and a major cause of infectious blindness. The adult worms, or macrofilariae, reside in subcutaneous tissues where females release the early larval stage, microfilariae, into the skin. Adult worms can reproduce for up to 10–14 years, releasing millions of microfilariae over an infected individual’s lifetime [3]. Microfilariae migrate throughout the tissues and those that accumulate in the eyes induce an inflammatory response that eventually leads to blindness [4]. LF is caused by several species of filariid nematodes: Wuchereria bancrofti, Brugia malayi and B. timori. The adult worms reside in the lymphatic tissues where females release microfilariae into the circulation. The microfilariae are then ingested by mosquitoes and develop into the infectious larval stage. With LF, the chronic condition is characterized by pain and severe lymphedema often involving the arms, legs, breasts and genitalia, as well as elephantiasis, all of which may lead to social stigma and economic loss to those afflicted [4,5].

Currently, global health programs that aim to eliminate these diseases distribute ivermectin, diethylcarbamazine (DEC), and albendazole through mass drug administration (MDA) to reduce transmission and ideally break the cycle of infection [6]. However, these drugs mainly target the microfilarial stage of the parasite, leaving the adult worms to continue to reproduce. DEC can cause adverse effects in patients infected with O. volvulus, so it can only be used to treat LF in areas where onchocerciasis is not endemic [4,6]. There is also an increased risk of serious adverse events, including encephalopathy and death, in those individuals who are treated with ivermectin or DEC and are co-infected with Loa loa with high microfilaraemia (greater than 30,000 microfilariae per mL) [7-10]. Recently, the veterinary drug, moxidectin has been investigated as a potential new therapeutic for filarial infection. Awadzi et al (2014) found that moxidectin was an effective microfilaricidal drug in a small-scale study, but it could not be concluded that moxidectin was macrofilaricidal or caused sterility in adult worms [11]. The antibiotic, doxycycline, has been shown to be safe and efficacious in treating both lymphatic filariasis and onchocerciasis, and can sterilize and eventually kill adult worms. However, doxycycline requires long treatment periods of upwards of 4–6 weeks, which is unlikely to be feasible for MDA [4]. These factors, in addition to the difficulty of attaining sufficient coverage through MDA, make discovering effective macrofilaricidal treatments to cure infections a high priority in stopping the transmission of filariasis. An ideal drug candidate is one that has high specificity for Onchocerca and Wuchereria/Brugia macrofilariae, but has little to no effect on the microfilariae of L. loa.

The overall goal of our program is to identify lead candidates for the treatment of river blindness and LF. Previously, we developed an in vitro worm assay [12] using Brugia pahangi and B. malayi as a primary screen to identify compounds that inhibit worm motility. The WormAssay apparatus and computer software (Worminator) enables us to screen compounds against adult Brugia in 24-well plates in less than one minute and assess worm killing in an objective manner. Compounds that strongly inhibited adult worm motility in a 3-day assay were then tested against molting O. volvulus third-stage larvae (L3) and adult O. ochengi. Adult O. ochengi, which naturally infect cows and develop in subcutaneous nodules, serve as a model organism for O. volvulus, which only infects humans and non-human primates [13-15].

In this study, we screened a library of over 2,000 FDA-approved compounds and found that auranofin was highly effective in inhibiting adult Brugia motility. Auranofin is an FDA-approved, gold-containing compound (2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranosato-S (triethylphosphine) gold) that has been used to treat rheumatoid arthritis for over 25 years [16,17]. Orally dosed auranofin is rapidly metabolized in vivo but its active metabolite is not known. It has been suggested that triethylphosphine gold or deacetylated auranofin could be the biologically active metabolites and that some form of the gold from auranofin circulates bound to plasma protein [18-20]. Since gold is known to be necessary for auranofin’s drug activity, studies of its pharmacokinetics employ elemental analysis for gold [19,21-24]. Previous studies have shown that the likely target of auranofin is thioredoxin reductase (TrxR) [25,26], which is a key enzyme involved in reducing oxidative damage in cells. We also found that auranofin is effective in killing adult Brugia in an in vivo gerbil model and that TrxR is most likely the target of auranofin in Brugia.

Methods

Drug screening of adult Brugia worms in vitro

Adult female and male Brugia (B. malayi and B. pahangi) were shipped from TRS Labs Inc., Athens, GA and assayed using methods described by Marcellino et al. (2012) [12]. Individual females were placed in each well of a 24-well plate with media (RPMI-1640 with 25 mM HEPES, 2.0 g/L NaHCO3, 5% heat inactivated FBS, and 1X Antibiotic/Antimycotic solution). Excess media was removed from plates using a Biomek FxP, leaving 500 μL of media per well. Initial screening of a library of FDA-approved compounds, compiled by the Small Molecule Discovery Center at the University of California San Francisco, was conducted at 10 μM per compound, and 1% DMSO was used as a negative control. All drugs including auranofin (Enzo Life Sciences, Farmingdale, NY) were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO) and 10 mM stock solutions were stored at -20°C. Four worms were used as replicates for each concentration and worm plates were kept in a 37°C, 5% CO2 incubator for four days. Auranofin was also tested against male Brugia worms under the same conditions after initial screening against female Brugia revealed its high level of inhibitory activity.

To determine the effect of a compound on worm motility, individual worm movements were counted as the number of pixels displaced per second by each worm in each well using the Worminator. Each plate of worms was video recorded for approximately 60 seconds, and mean movement units (MMUs) were determined for individual worms. Percent inhibition of motility was calculated by dividing the MMUs of the treated worms by the control average MMUs, subtracting the value from 1.0, flooring the values to zero and multiplying by 100%. Videos were recorded for 4 days, including the first day of the assay (Day 0). IC50 determinations were conducted at 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM and 0.03 μM, with 1% DMSO used as a control. IC50 assays were repeated at the same concentrations and at six point, three-fold dilutions from 1 μM to 0.003 μM or 3 μM to 0.001 μM to ensure that activity was consistent between assays. Prism 4.0 was used to calculate IC50 values using a non-linear regression curve fit. The means of all IC50s with R2 values greater than or equal to 0.7 are reported.

Drug screening of adult Onchocerca ochengi in vitro

Cows that had grazed in northern Cameroon where O. ochengi is highly endemic were brought to abattoirs located in Douala, Cameroon. Subcutaneous nodules containing adult O. ochengi worms were identified on the umbilical skin of infected cows. Adult worm masses containing one viable, adult female and zero to several adult males were carefully recovered by dissection of the nodule with a sterile razor blade. The masses were then incubated in 4 mL of complete culture medium (CCM), which was comprised of RPMI-1640 (Sigma-Aldrich), 5% newborn calf serum, 200 units/mL penicillin, 200 μg/mL streptomycin and 2.5 μg/mL amphotericin B (Sigma-Aldrich), in standard 12-well culture plates. Masses were maintained in the medium in a 37°C, 5% CO2 incubator overnight during which period most of the smaller and more agile adult males migrated out of the masses while the females remained in the nodules. Worm viability was checked microscopically by observing the movement of adult male worms or emergence of viable microfilariae from the nodular masses. The next day, 2 mL of the CCM was removed and replaced with 60 μM auranofin in 2 mL CCM in each well to generate a final drug concentration of 30 μM. The compound and controls were tested in quadruplicate at each concentration and the experiments were repeated twice on different days. The negative control wells received only 1% DMSO. Cultures were terminated on day 7 post addition of drug. Adult male worm viability was visually scored on day 5 as percent reduction of motility ranging from 100% (complete inhibition of motility), 90% (only head or tail of worm moving or vibrating), 75% (worm very sluggish), 50% (worm sluggish), 25% (little change in motility), to 0% (no observable reduction in motility). Adult female worm viability was assessed on day 7 by the standard MTT/formazan assay in which each nodular mass was placed in a well of a 48-well microtiter plate containing 500 μL of 0.5 mg/mL MTT (Sigma-Aldrich) in incomplete culture medium, and then incubated in the dark at 37°C for 30 minutes. Adult female worm viability was evaluated visually by the extent to which the female worm mass was stained with MTT. Mean percent inhibition of formazan formation was calculated relative to the negative control worm masses after 7 days in culture. Adult worm death positively correlated with inhibition of formazan formation.

To calculate the IC50 of auranofin, quadruplicate worm masses were incubated with final concentrations of 30 μM, 10 μM, 3 μM, 1 μM, 0.3 μM, 0.1 μM and 0.03 μM and assays were conducted as described above. Prism 4.0 for Windows was used to calculate IC50s.

Drug screening of Onchocerca ochengi and Loa loa microfilariae in vitro

O. ochengi microfilariae were obtained from the umbilical skin of infected cattle and cultured on confluent monkey kidney epithelial cells for drug testing as previously described [27].

Loa loa microfilariae were purified from the blood of a heavily infected subject (having approximately 10,000 microfilariae/mL of blood) using Percoll (GE Healthcare, Piscataway, NJ) gradient centrifugation. Venous blood (10 mL) was collected from consenting, infected individuals in an EDTA tube. The blood was layered on a step-wise Percoll gradient (46% and 43% Percoll prepared in CCM) followed by centrifugation at 400 rcf for 20 minutes. The L. loa microfilariae were recovered in the 43% layer, washed 3 times in CCM and counted.

Microfilariae (10–15 per well) were cultured in 96-well culture plates in duplicate under the same conditions and drug concentrations as were used for the adult O. ochengi, except that 10 μg/mL ivermectin was used as a positive control. Microfilariae viability was visually scored based on motility reduction using the same scale described above for adult male O. ochengi. Scores were recorded every 24 hours after the addition of drugs for 5 days using an inverted microscope.

Drug screening of Onchocerca volvulus L3 molting in vitro

L3 stage larvae previously collected and cryopreserved in Cameroon were rapidly thawed in a 37°C water bath and washed in incomplete media comprised of a 1:1 ratio of Medium NCTC-109 and IMDM + GlutaMax-I containing 1X glutamine, penicillin, and streptomycin (all from Gibco by Life Technologies, Grand Island, NY). The number of worms was adjusted to about 10 worms per 50 μL in complete medium containing 20% heat inactivated FCS. Worms were distributed into the wells of a 96-well plate containing 50 μL of 1.5 × 105 normal human PBMCs. 100 μL of 2X auranofin (final concentrations of 30 μM, 10 μM, 3 μM, 1 μM and 0.3 μM) were added to each well for a final volume of 200 μL. Each concentration was tested in triplicate. Controls included 0.05% DMSO in complete medium and complete medium only with neither DMSO nor compound added. The 96-well plates were then incubated at 37°C in a 5% CO2 incubator for 6 days, then molting was assessed using an inverted microscope. Molting was determined in each well by counting the presence of fourth-stage larvae (L4) and empty casts of the L3. The percent inhibition of molting was calculated based on the number of treated larvae that were able to molt in comparison to the number of control larvae that had successfully molted. Prism 4.0 for Windows was used to calculate IC50s.

Ultrastructure of auranofin-treated adult female Brugia and O. ochengi

Adult female B. pahangi worms were incubated with either 1 μM, 0.3 μM, or 0.1 μM auranofin, 10 μM flubendazole (as a positive control [28]), or 1% DMSO overnight, then cut into 3 segments separating the anterior, middle and posterior sections. The middle sections were further cut into 1 mm sized pieces in fixative (2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3–7.4) and stored at 4°C. Middle sections were subsequently treated with 1% tannic acid for 1 hour, followed by three buffer washes before post fixation staining with 2% osmium tetroxide for 1 hour. The samples were washed three times in buffer before dehydration in an ethanol series. Worm sections were then infiltrated with propylene oxide, embedded in epon 812 resin and polymerized in a vacuum oven at 60°C overnight. Ultrathin sections were cut using an RMC MTX ultramicrotome with a Diatome diamond knife followed by post staining of the grids with saturated ethanolic uranyl acetate and Reynolds lead citrate. Samples were imaged on a FEI Tecnai 12 spirit TEM operated at 80 kV. A similar procedure was performed on adult female O. ochengi worm masses that were cultured for 7 days with 10 μM auranofin before fixation of cut pieces of the adult female mass. Untreated adult female masses cultured for 7 days and fixed by the same procedure served as the control.

In vivo studies of Brugia pahangi infected gerbils

Animal studies were performed under IACUC approval #AN085723–02 to test the efficacy of auranofin in vivo. Male Mongolian gerbils (Meriones unguiculatus, Charles River Laboratories International, Inc., Wilmington, MA) were injected intraperitoneally (IP) with 300 B. pahangi L3 (Filariatech, Inc., Athens, GA) and treated 3 months post-infection. Auranofin was dissolved in 100% ethanol at 4 mg/mL and mixed 1:1 with PBS. Vehicle doses consisted of the same mixture of ethanol and PBS but without auranofin. Doses (up to 200 μl) were given to gerbils orally at 5 mg/kg body weight, BID weekdays and SID weekends for a total of 48 doses over 4 weeks.

Two studies (Study 1 and Study 2) were conducted using the same protocols and the same dosing schedule except that in Study 1, two gerbils from the auranofin treatment group and two gerbils from the vehicle group were treated for 14 days and were necropsied 2 hours after their last dose (interim necropsy) to determine plasma gold levels (from auranofin). The remaining gerbils in Study 1 were treated for 28 days and were necropsied 11, 14, or 16 days after the end of dosing. In Study 2, all gerbils were treated for 28 days and were necropsied 16 days after the end of dosing. For both of these in vivo studies, worms were collected from the gerbil’s peritoneal cavity, counted, sexed and examined under a dissecting microscope. For each study a two-tailed Student’s T-test assuming equal variance was conducted using Microsoft Excel to determine the statistical significance of the difference in mean worm retrieval between the auranofin treated and vehicle treated groups. Gerbil blood was collected by cardiac puncture and plasma was sent to NMS Labs, Willow Grove, PA to determine plasma gold levels (elemental gold analysis) by graphite furnace atomic absorption spectroscopy.

Target validation of auranofin-treated worms in vitro and in vivo

Thioredoxin reductase activity of worm lysates was assayed using female B. malayi treated in vitro with either 0.3 μM, 0.1 μM, or 0.03 μM auranofin or 1% DMSO. After 5 hours of treatment, worm motility was measured using the Worminator, and then worms (24 in each group) were pooled, washed three times in PBS, and lysed by douncing in a glass homogenizer in assay buffer (Abcam Thioredoxin Reductase Assay kit, ab83463) with 1 mM PMSF. The crude lysates were centrifuged at 10,000 rcf for 15 minutes at 4°C to pellet insoluble material. The total protein concentrations of soluble lysates were measured using the Bradford assay. The soluble lysates were incubated for 20 minutes in assay buffer or assay buffer with a proprietary thioredoxin reductase specific inhibitor before adding a specific substrate, DTNB (5, 5′-dithiobis (2-nitrobenzoic) acid), and measuring activity at 20 second intervals for 40 minutes using the SpectraMax Plus Microplate Reader (Molecular Devices, Sunnyvale, CA) at λ = 412 nm. Lysates were tested in duplicate. TrxR activity was calculated based on the linear amount of TNB produced per minute per mg of total protein and adjusted for background activity from enzymes other than TrxR in the lysates.

Thioredoxin reductase activity was also analyzed in worms that were treated with auranofin or vehicle in vivo. Adult male and female worms were transplanted intraperitoneally, and gerbils were treated with auranofin or vehicle for 28 days as was done in the previous in vivo studies. Gerbils were necropsied 16 days after the final dose, and lysates were prepared from recovered worms and assayed as above.

Expression of recombinant Brugia malayi thioredoxin reductase

The open reading frame for B. malayi TrxR (XM_001898694.1) was synthesized (GenScript) with codons optimized for expression in Escherichia coli. The two C-terminal amino acids (selenocysteine (Sec)-Gly), missing in XM_001898694.1, were added along with a bacterial SECIS (selenocysteine insertion sequence) to allow expression of the Sec protein in E. coli in pET100 (Invitrogen by Life Technologies) [29]. For PCR, the reverse primer was 5’-GGCCGCATAGGTTAACGATTGGTGCAGACCTGCAACCGATTATTAACCTCAGCATCCCGTTGCTTTC-3’ and forward primer was 5’-CACCATGCTGCTGCGTTCCAATGC-3’.

To determine if the B. malayi TrxR is a selenoprotein, as are some known thioredoxin reductases, a bioinformatics search was conducted to find a SECIS in the B. malayi genome near the thioredoxin reductase gene. For a detailed description, please see supplementary information (S1 Text).

Recombinant 6-His-tagged B. malayi TrxR (rBmTrxR) was produced in E. coli BL21(DE3) in the presence of pSUABC [29] in LB medium supplemented with 20 μg/mL riboflavin under conditions for optimal selenoprotein expression [30]. An overnight starter culture in LB with 50 μg/mL ampicillin and 34 μg/mL chloramphenicol was diluted (1:100) into in LB medium with the same antibiotics. When the culture reached an OD600 = 0.8, the medium was supplemented with 5 μM sodium selenite and 100 μg/mL L-cysteine. When the culture OD600 = 2, riboflavin (20 μg/mL) was added and protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (50 μM). At this point the cultures were shifted to 24°C and incubated for 24 hr. Cells were collected by centrifugation, lysed by alternative freeze-thaw cycles, and resuspended in lysis buffer (50 mM potassium phosphate, pH 7.8, 500 mM NaCl, 30 mM imidazole, 1 mg/mL lysozyme, 1 mM phenylmethanesulfonylfluoride) supplemented with 100 μM flavin adenine dinucleotide. The sample was sonicated and cellular debris pelleted at 25,000 x g at 4°C for 25 min. The supernatant was collected and filtered through a 0.45 μm filter before purification by immobilized metal ion affinity chromatography using a His-Trap FF column (GE Healthcare). The column was washed with 10 column volumes binding buffer (50 mM potassium phosphate, pH 7.8, 500 mM NaCl, 30 mM imidazole) and then with 5 column volumes of buffer A (binding buffer with 100 mM imidazole). TrxR protein was eluted in 3 × 1 mL buffer B (binding buffer with 500 mM imidazole).Protein was concentrated (Amicon Ultra-4 10K) and purity was verified by SDS-PAGE and quantified by absorbance at 280 nm (ε = 69.76 mM-1 cm-1).

rBmTrxR activity was assayed in 0.1 M potassium phosphate (pH 7.2) with 10 mM EDTA and 25 nM rBmTrxR. rBmTxrR was pre-incubated for 20 min with NADPH (100 μM) and auranofin (ICN Pharmaceuticals, now Valeant Pharmaceuticals, Bridgewater, NJ) or aurothioglucose (USP Reference Standards, Rockville, MD) in DMSO in 100 μL, followed by addition of an equal volume of buffer with NADPH (200 μM) and DTNB (6 mM) with reaction progress monitored at λ = 412 nm for TNB production. The concentration of DMSO in all reactions was 3.5%.

Ethics statement

The Loa loa microfilariae donors were all adult male and female patients, aged 21 or older, residing in the Edea Health District of the Littoral Region of Cameroon. Ethical and administrative clearances were obtained from the Cameroon National Ethics Committee (N°2013/11/371/L/CNERSH/SP) and the Cameroon Ministry of Health, respectively. Written and signed informed consent was obtained from each participating patient, and all of them had 2000 L. loa microfilariae per mL of blood or greater. The patients were employed in the study as microfilariae donors only.

Animal studies were performed under the University of California San Francisco Institutional Animal Care and Use Committee (IACUC) approval #AN085723–02 and adhere to guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals and the USDA Animal Care Policies. Animals were euthanized by carbon dioxide inhalation followed by bilateral thoracotomy.

Results

Identification of auranofin as an effective drug that kills Brugia and Onchocerca worms in vitro

Results of the adult worm assay showed that the motility of female B. pahangi and B. malayi was inhibited by 97% within 18 hours of incubation with 3 μM of auranofin. Following our prescribed screening funnel, after this primary screen, auranofin was then assayed with adult female and male O. ochengi, O. volvulus L3, and O. ochengi and L. loa microfilariae.

Auranofin was highly effective in killing both male and female adult Brugia and Onchocerca worms and inhibiting molting of O. volvulus third-stage larvae to the fourth stage with IC50 values less than or equal to 1.1 μM (Table 1). Auranofin, however, was not very effective in killing O. ochengi and L. loa microfilariae. Auranofin’s IC50 value for adult female O. ochengi was 10 times lower than its IC50 value for O. ochengi microfilariae and 42.7 times lower than its IC50 value for L. loa microfilariae. This is an important consideration when treating individuals with auranofin in L. loa endemic areas.

Table 1

Effect of auranofin on filarial worms in vitro.

Species	Sex	Stage	Day	IC50 (uM)
B. malayi	Female	Adult	Day 3	1.1
	Male	Adult	Day 3	0.3
B. pahangi	Female	Adult	Day 3	0.5
	Male	Adult	Day 3	0.1
O. ochengi	Female	Adult	Day 7	0.3
	Male	Adult	Day 5	0.4
O. volvulus	-	L3	Day 6	0.3
O. ochengi	-	Microfilariae	Day 5	3.0
L. loa	-	Microfilariae	Day 5	12.8

Structural features of auranofin-treated B. pahangi and O. ochengi adult worms

Adult female B. pahangi incubated with 1 μM, 0.3 μM, or 0.1 μM auranofin overnight and adult female O. ochengi worms encapsulated in nodules incubated with 10 μM auranofin for 7 days were subjected to transmission electron microscopy to compare the internal morphology with their respective control female worms. Auranofin-treated B. pahangi worms showed considerable damage in the hypodermal region compared to control worms (Figs. 1a-1d). The hypodermal area of treated worms was highly vacuolated with remnants of swollen mitochondria containing dark bodies as well as shrunken Wolbachia containing dark condensed material. The hypodermal chord region of B. pahangi female worms treated with 10 μM of flubendazole contained normal Wolbachia with very few mitochondria containing dark bodies (Fig. 1e). In contrast, the hypodermal chord region in control worms (Fig. 1f) contained numerous Wolbachia without the condensed material observed in auranofin treated worms.

Fig 1

TEM images of auranofin treated B. pahangi.

Transmission electron microscopy of auranofin treated versus control adult female Brugia pahangi after overnight drug treatment. (A) B. pahangi treated with 1 μM of auranofin. Hypodermal chord region (h) below cuticle (cu) of B. pahangi exhibiting vacuolation of tissue (compared to control worms, Fig. 1F). Insert; higher magnification of boxed region in (A) showing swollen mitochondria containing dark bodies (black arrows). White arrow indicates severely damaged mitochondrion. (B) B. pahangi treated with 1 μM of auranofin. High magnification of hypodermal chord region showing numerous swollen mitochondria containing dark bodies (black arrows) as well as shrunken Wolbachia (w) containing dark condensed material (black arrowheads) (compared to control worms, Fig. 1F). (C) B. pahangi treated with 0.3 μM of auranofin. Hypodermal chord region containing Wolbachia (black arrows) and dark bodies (white arrows). Insert; higher magnification of boxed region in (C) showing mitochondria containing dark bodies (black arrows) as well as Wolbachia (black arrowhead) containing condensed material. (D) B. pahangi treated with 0.1 μM of auranofin. Hypodermal chord region containing Wolbachia (black arrows). Inset; higher magnification of boxed region in (D) showing mitochondria containing dark bodies (black arrows) as well as Wolbachia (black arrowhead) containing condensed material. (E) B. pahangi treated with 10 μM of flubendazole. Hypodermal chord region containing Wolbachia (black arrows) and numerous mitochondria containing dark bodies (black concave arrows). Insert; higher magnification of boxed region in (E) showing a mitochondrion containing dark bodies (black arrows). (F) B. pahangi treated with 1% DMSO. Hypodermal chord region contains numerous Wolbachia without condensed material observed in auranofin treated cells. Insert; higher magnification of boxed region in (F) showing Wolbachia (w) as well as several mitochondria (m) without the dark bodies observed in treated cells.

Similar morphology was also observed in the O. ochengi auranofin treated worms (Fig. 2). Numerous vacuoles with inclusion bodies were observed in the muscle tissue below the hypodermal chord. Numerous vacuoles and a complete lack of mitochondria were also observed in the hypodermal chord region directly below the cuticle.

Fig 2

TEM images of auranofin treated O. ochengi.

Transmission electron microscopy of auranofin treated versus control female Onchocerca ochengi 7 days post treatment. (A) Low magnification of O. ochengi treated with 10 μM auranofin. Numerous vacuoles with inclusion bodies (black arrows) were observed in the muscle tissue (mu) below the hypodermal chord (h). (B) High magnification of hypodermal chord region directly below the cuticle (cu). Numerous vacuoles (black arrows) were observed as was a complete absence of mitochondria. (C) Untreated O. ochengi exhibiting the typical arrangement of muscle (mu) and hypodermal chord (h) tissue below the cuticle (cu). (D) High magnification of hypodermal chord region directly below the cuticle showing numerous mitochondria (m).

Efficacy of auranofin on Brugia worms in vivo

Two in vivo studies were performed using the same dosing regimen of 5 mg/kg BID weekdays and SID weekends for 28 days (for a total of 48 doses). Study 1 and Study 2 are replicate studies, except that in Study 1 an interim necropsy was conducted to determine the plasma levels and level of infection 14 days after the first dose. The number of worms collected from these vehicle treated gerbils was 43 (13 male worms and 30 female worms, a ratio of approximately 1:2) and the total number of worms from the auranofin treated gerbils was 11 (4 males and 7 females, a ratio of approximately 1:2).

In Study 1, the average number of worms from all vehicle treated animals (n = 7) was 9.4 worms and the average number of worms from all treated animals (n = 9) was 4.0 worms (Fig. 3A). There was a 58% overall reduction in worm burden in the auranofin treated group in comparison with the vehicle treated group but difference between the two groups was not statistically significant (p > 0.05). In the control group the ratio of male to female worms at terminal necropsy was 1:2, similar to the ratios found in the control group and treated group at the interim necropsy. In the treated group however, the ratio of male to female worms was 12:1 at terminal necropsy. This sex ratio bias was also observed in the auranofin treated group in Study 2 (Fig. 3B).

Fig 3

Worm retrieval from B. pahangi infected gerbils treated with auranofin.

Total worms recovered from (A) Study 1 and (B) Study 2 of gerbils treated with 5 mg/kg auranofin or vehicle with 48 doses for 28 days. Fig. 3A and 3C also include worms recovered from interim necropsy gerbils treated for 14 days. The difference in total worm retrieval between auranofin treated and vehicle treated gerbils in Study 2 was statistically significant (p < 0.05). Male and female worms recovered from (C) Study 1 and (D) Study 2.

In Study 2, there was a 91% reduction in worm burden in the auranofin treated group compared to the control group, which was statistically significant (p = 0.01) in a Student’s T-Test. There were 161 total worms recovered from the vehicle group (mean = 32 worms per gerbil), of which 55 were males and 106 were females (ratio of 1:2). In the auranofin treated group, there were a total of 12 worms recovered (mean = 3 worms per gerbil): 11 were males and only 1 was a female worm (ratio of 11:1) (Fig. 3D). This remaining female was encapsulated with host tissue.

Plasma collected from the necropsies from Study 1 and Study 2 was submitted for elemental gold analysis (Table 2). Gold was not detected in the vehicle group. Plasma taken 2 hours after gerbils were given an auranofin dose (but had been treated for 14 days) had gold levels of 5.08 μM and 8.63 μM. In Study 1 and Study 2, the mean plasma gold levels 16 days after the last dose were 701 nM and 609 nM, respectively. There were 2 animals in each of the treatment groups that did not have detectable levels of gold in their plasma but this may be due to the limit of detection in the assay, where any value less than 100 μg/L (508 nM) gold is given as zero.

Table 2

Plasma gold levels from Brugia infected gerbils following necropsy.

		Time after last dose	Plasma gold levels (ug/L)	Concentration of gold (uM)
Study #1	Aur1	2 hours*	1700	8.631
	Aur2	2 hours*	1000	5.077
	Aur3	11 days	270	1.371
	Aur4	14 days	340	1.726
	Aur5	16 days	160	0.812
	Aur6	16 days	170	0.863
	Aur7	16 days	0	0
	Aur8	16 days	170	0.863
	Aur9	16 days	190	0.965
Study #2	Aur1	16 days	130	0.660
	Aur2	16 days	190	0.965
	Aur3	16 days	0	0
	Aur4	16 days	160	0.812

* Plasma taken at interim necropsy, two hours after last dose (following 14 days of treatment).

TrxR activity decreased in adult female Brugia after treatment with auranofin in vitro and in vivo

Thioredoxin reductase activity in Brugia females cultured for 5 hours with 0.3 μM, 0.1 μM or 0.03 μM of auranofin in vitro was significantly reduced (p < 0.05) to 15%, 33% and 69% of endogenous activity, respectively, compared to the activity in DMSO-treated worms (Fig. 4A).

Fig 4

Thioredoxin reductase activity in auranofin treated Brugia spp.

(A) Activity of endogenous Brugia thioredoxin reductase from soluble worm lysates following incubation with 1% DMSO or 0.3 μM, 0.1 μM, or 0.03 μM of auranofin in vitro. Percentages indicate the percent activity of TrxR compared to DMSO controls. (B) Enzymatic activity of worms collected 16 days after the last dose from gerbils treated with auranofin or vehicle. The lysate of worms taken from gerbils treated with auranofin shows 49% less thioredoxin reductase activity than those taken from gerbils treated with vehicle only. Percentages indicate the percent activity of TrxR compared to vehicle controls.

When Brugia worms were removed 16 days after the last dose from gerbils treated with auranofin in vivo, endogenous enzyme activity was reduced significantly (p < 0.05) by 49% compared to worms collected from vehicle treated gerbils (Fig. 4B). These data further suggest that endogenous Brugia TrxR is specifically inhibited by auranofin.

Activity of recombinant B. malayi TrxR is inhibited by auranofin

Recombinant B. malayi TrxR (rBmTrxR) was overexpressed in E. coli at approximately 10 mg of protein per liter of culture following His-Trap affinity chromatography. Two organic gold compounds, auranofin and aurothioglucose, were assayed with rBmTrxR and both were found to be effective inhibitors suggesting that gold is the active component of auranofin as expected from previous studies with TrxR and thioredoxin glutathione reductase [24,25]. Both compounds had inhibitory activity in the low nanomolar range with auranofin IC50 = 3 nM and aurothioglucose IC50 = 9 nM. Production of eukaryotic Sec-proteins in bacteria is not 100% efficient. The misreading of the Sec codon (UGA) results in premature termination of the peptide resulting in an enzymatically inactive product [29]. Since the active and inactive proteins both bind metal affinity resins and differ in size by only two amino acids, recombinant protein is a mixture of both active and inactive enzyme forms. Based on previous studies [29-31] between 10% and 20% of the protein is active, with the remainder inactive. The inhibitory activity of both compounds indicates that they irreversibly inhibit rBmTrxR at a one-to-one molar ratio, with potencies similar to those found for other TrxR and thioredoxin glutathione reductase enzymes [32,33].

Discussion

The main goal of our study was to identify macrofilaricidal drugs for the treatment of onchocerciasis and LF. Two major challenges in developing new drugs for these neglected diseases are finding suitable animal models for preclinical studies and limiting the costs of drug development and production. To date, the only animals in which O. volvulus can develop to patency are chimpanzees and mangabey monkeys [34-36]. O. ochengi, which infects cows, is thought to be closely related to O. volvulus [37], and previous studies have used O. ochengi as a model for O. volvulus infection [13-15]. Brugia malayi and B. pahangi, as members of the Filariidae family, are also closely related to O. volvulus [38]. Because of the large number of compounds required to identify preclinical candidates and with the accessibility of large numbers of adult worms that can be collected from gerbils, we selected adult Brugia for our primary screens. Following our funneling scheme, we first identify compounds screened with adult female Brugia in the Worminator assays. Compounds that inhibit motility by 75% compared with control worms are then screened against O. volvulus molting larvae and O. ochengi adult worms in an MTT assay and motility assay.

In an effort to identify candidate drugs that could be more rapidly moved into clinical trials, we screened an FDA-approved library of compounds and found that auranofin was effective in killing adult Brugia and O. ochengi worms and in inhibiting larval O. volvulus from molting from L3s to L4s in vitro. Microfilariae of O. ochengi and L. loa were used in a counter screen to determine the effects of auranofin on the microfilarial stage. We found that the IC50s for O. ochengi and L. loa microfilariae were approximately 10 and 42.7 times higher, respectively, compared with the IC50s of adult female O. ochengi. These results may have important implications, should auranofin be used for treatment in areas endemic for both onchocerciasis and loaiasis to avoid severe adverse events.

Auranofin was then tested for its efficacy in secondary screens with infected gerbils. Results of the in vivo studies showed that dosing animals for 28 days at 5 mg/kg was effective in reducing worm burden by 58% and 91% in the two studies. Gold plasma levels in gerbils obtained at 2 hours post-dose after 2 weeks of treatment indicated that the plasma gold levels were in the micromolar range (5.08 μM and 8.63 μM), approximately 5 to 10-fold higher than the IC50s from the in vitro worm assays. These gerbils continued to maintain gold levels in their blood approximately 2 weeks after the last dose (0.66 μM) which may suggest that a sustained level of gold is necessary for worm killing.

Transmission electron micrographs of adult Brugia incubated overnight with 1 μM auranofin showed that there was extensive damage to Wolbachia in the hypodermal area, in contrast to worms treated with 10 μM flubendazole. Flubendazole at this concentration did not cause vacuolization but only minor changes to the mitochondria, which appeared to contain black bodies. Loss of integrity in muscle tissue and the hypodermal chord were also observed when O. ochengi adults were incubated with auranofin at 10 μM for 7 days. Thus, the structural damage caused by auranofin is similar in both species, except that presumably due to the large size of O. ochengi, auranofin takes a much longer time and higher concentrations of drug to have an effect.

Auranofin is an FDA-approved drug that was originally developed to treat rheumatoid arthritis. There is strong evidence in several species of parasites that thioredoxin reductase and a similar enzyme, thioredoxin glutathione reductase (TGR), are targeted by auranofin [26,33,39-41]. Previous studies have shown that this drug is an effective antiparasitic agent against a number of organisms, including Schistosoma mansoni and S. japonicum [33,42], Echinococcus granulosus [43], Taenia crassiceps [44], Plasmodium falciparum [45], Leishmania spp. [46], Trypanosoma brucei [47], Giardia lamblia [39,48] and Toxoplasma gondii [49]. In animal studies, auranofin was highly efficacious in treating amoebic colitis in mice and amoebic liver abscesses in hamsters [26]. Auranofin treatment also significantly decreased worm burdens in mice infected with S. mansoni [33] and suppressed footpad lesion formation and reduced existing lesions in a mouse model of cutaneous leishmaniasis [46].

The thioredoxin system is integral to maintaining a reduced state and managing oxidative stress within the cell, which makes this system critical for organism survival [50]. Thioredoxin, which is reduced by thioredoxin reductase, is a substrate for redox enzymes including peroxidases in filarial worms [18,51]. Inhibition of TrxR by auranofin alters the redox state of the cell leading to an increased production of hydrogen peroxide and oxidation of the components of the thioredoxin system thereby enhancing apoptosis [52]. Sayed et al (2006) found that silencing peroxiredoxins, downstream redox partners of TrxR, in schistosomes led to detectable protein and lipid oxidation [53]. Inhibition of Brugia TrxR by auranofin may disrupt this process in filarial worms, which can then lead to worm death. Interestingly, there were significantly fewer female worms than male worms from gerbils treated with auranofin. The preferential killing of female worms may be due to the host’s immune response against females when they release microfilariae [54,55]. It is also possible that as female worms develop and molt from the larval stage to the adult stage, they elicit an immune response that, together with auranofin, preferentially kills female worms over males.

The mode of action of auranofin is thought to be a specific inhibition of the selenoenzymes thioredoxin reductase (TrxR) and thioredoxin glutathione reductase (TGR). No TGRs from Brugia have been identified thus far. Kuntz et al (2007) showed that auranofin inhibited TGR in adult schistosomes in vitro but had no effect on the activities of another selenoenzyme, glutathione peroxidase, or the abundant enzyme lactate dehydrogenase [33]. Loss of TGR activity preceded parasite death, indicating that specific inhibition of TGR by auranofin was responsible for parasite death in schistosomes.

Auranofin inhibition has also been shown to be less specific to glutathione peroxidase and glutathione reductase, which have about 1000-fold higher IC50s compared to TrxR isolated from human placenta [32]. Other thioenzymes, such as the cysteine protease cathepsin B, also had significantly higher IC50s when tested with auranofin (approximately 250 μM) [56] compared with the IC50 of auranofin with rBmTrxR.

Thioredoxin reductase enzyme activity of B. malayi adult worms treated with auranofin was significantly lower compared to with vehicle-treated worms in the in vitro assays. TrxR activity was also decreased by 49% in worms removed from gerbils 16 days after treatment with auranofin, supporting the hypothesis that auranofin specifically targets TrxR in these worms.

Targeting the thioredoxin system by inhibiting thioredoxin reductase may be a promising strategy for treating filarial infections, since the enzyme appears to be necessary for worm survival. It is possible that auranofin treatment increases the susceptibility of the parasite to oxidative damage, which in turn allows the host’s immune system to eliminate the parasite.

Since auranofin is already an FDA-approved drug, the path to clinical trials is streamlined. Patients with rheumatoid arthritis who were treated with auranofin for an average of 6 months had few side effects, with the most common side effect being diarrhea [20]. In the present study auranofin was shown to be efficacious in the Brugia/gerbil model when given for 28 days. Additional studies will be conducted to determine efficacy with shorter treatment regimens and to obtain pharmacokinetic data. Auranofin will also be evaluated for any synergistic effects with other drugs such as doxycycline and for its use as part of a macrofilaricidal cocktail.

This file contains detailed information on the process used to verify that B. malayi TrxR is a selenoprotein.

(DOC)

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