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

Abstract

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.

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.

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).

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.

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.