Repositioning of an existing drug for the neglected tropical disease Onchocerciasis

Abstract

Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm's metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel's effects, a retro-fragment-based study was used to define structural elements critical for closantel's chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness.

Fig. 1.

Structure, potency, and inhibition mode of closantel. For experimental details, please see the Methods section. (A) Structure of closantel. (B) Determination of IC₅₀ for closantel. (C) Determination of mode of inhibition.

Fig. 2.

Inhibition of molting in the presence of closantel. O. volvulus L3 were cultured with closantel at 10, 25, 50, and 100 μM and 1.5 × 10⁵ normal human peripheral blood mononuclear cells for 6 d as described in the “Methods” section. The data are presented as percent molting in a total of 10 wells containing on average 5–10 larvae per well.

Fig. 3.

Ultrastructure of O. volvulus molting in 100 μM closantel-treated L3. To determine what stage of the molting process was affected by 100 μM closantel, the O. volvulus treated L3 were collected on day six and fixed for 2 h at 4 °C with 3% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) and processed for electron microscopy. In the four panels of A, the normal molting process of L3s is shown. (A) Normal separation between the L3 cuticle (arrows) and the epicuticle of the newly developed L4 (arrowheads) during the normal molting process of larvae cultured in DMSO control culture media. Please pay attention that the area between the cuticles clears during the molting process until the two cuticles are completely separated and before ecdysis; (B) incomplete separation between the L3 and L4 cuticles in 100 μM closantel-treated worms. Notably, although the cuticle of L4 was present the separation between the newly synthesized cuticle and the old L3 cuticle was never completely degraded; cuticular material is still present between the L3 cuticle and the L4 epicuticle. The regions where the cuticles of L4 and L3 separate are marked by an arrowhead and an arrow, respectively. Each bar is 500 nm.

Fig. 4.

Fragment analogues of closantel.