Thioredoxin reductase activity in auranofin treated Brugia spp.

<p>(A) Activity of endogenous <i>Brugia</i> thioredoxin reductase from soluble worm lysates following incubation with 1% DMSO or 0.3 μM, 0.1 μM, or 0.03 μM of auranofin <i>in vitro</i>. 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.</p

TEM images of auranofin treated <i>O. ochengi</i>.

<p>Transmission electron microscopy of auranofin treated versus control female <i>Onchocerca ochengi</i> 7 days post treatment. (A) Low magnification of <i>O. ochengi</i> 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 <i>O. ochengi</i> 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).</p

Repurposing Auranofin as a Lead Candidate for Treatment of Lymphatic Filariasis and Onchocerciasis

<div><p>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 <i>Onchocerca volvulus</i> and <i>Brugia malayi</i> 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 <i>Brugia</i> spp. and <i>Onchocerca ochengi</i>, third-stage larvae (L3s) of <i>Onchocerca volvulus</i>, and the microfilariae of both <i>O. ochengi</i> and <i>Loa loa</i>. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective <i>in vitro</i> in killing both <i>Brugia</i> spp. and <i>O. ochengi</i> adult worms and in inhibiting the molting of L3s of <i>O. volvulus</i> with IC₅₀ values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC₅₀ against the microfilariae of <i>L. loa</i> compared with the IC₅₀ for adult female <i>O. ochengi</i>, which may be beneficial if used in areas where <i>Onchocerca</i> and <i>Brugia</i> are co-endemic with <i>L. loa</i>, to prevent severe adverse reactions to the drug-induced death of <i>L. loa</i> microfilariae. Further testing indicated that auranofin is also effective in reducing <i>Brugia</i> adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode.</p></div

Plasma gold levels from Brugia infected gerbils following necropsy.

<p>* Plasma taken at interim necropsy, two hours after last dose (following 14 days of treatment).</p><p>Plasma gold levels from Brugia infected gerbils following necropsy.</p

TEM images of auranofin treated <i>B. pahangi</i>.

<p>Transmission electron microscopy of auranofin treated versus control adult female <i>Brugia pahangi</i> after overnight drug treatment. (A) <i>B. pahangi</i> treated with 1 μM of auranofin. Hypodermal chord region (h) below cuticle (cu) of <i>B. pahangi</i> exhibiting vacuolation of tissue (compared to control worms, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003534#pntd.0003534.g001" target="_blank">Fig. 1F</a>). Insert; higher magnification of boxed region in (A) showing swollen mitochondria containing dark bodies (black arrows). White arrow indicates severely damaged mitochondrion. (B) <i>B. pahangi</i> 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 <i>Wolbachia</i> (w) containing dark condensed material (black arrowheads) (compared to control worms, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003534#pntd.0003534.g001" target="_blank">Fig. 1F</a>). (C) <i>B. pahangi</i> treated with 0.3 μM of auranofin. Hypodermal chord region containing <i>Wolbachia</i> (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 <i>Wolbachia</i> (black arrowhead) containing condensed material. (D) <i>B. pahangi</i> treated with 0.1 μM of auranofin. Hypodermal chord region containing <i>Wolbachia</i> (black arrows). Inset; higher magnification of boxed region in (D) showing mitochondria containing dark bodies (black arrows) as well as <i>Wolbachia</i> (black arrowhead) containing condensed material. (E) <i>B. pahangi</i> treated with 10 μM of flubendazole. Hypodermal chord region containing <i>Wolbachia</i> (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) <i>B. pahangi</i> treated with 1% DMSO. Hypodermal chord region contains numerous <i>Wolbachia</i> without condensed material observed in auranofin treated cells. Insert; higher magnification of boxed region in (F) showing <i>Wolbachia</i> (w) as well as several mitochondria (m) without the dark bodies observed in treated cells.</p

Worm retrieval from <i>B. pahangi</i> infected gerbils treated with auranofin.

<p>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. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003534#pntd.0003534.g003" target="_blank">Fig. 3A and 3C</a> 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.</p