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The Worm Institute for Research and Medicine

Therapeutics

Neglected tropical diseases (NTDs) affect over one billion people worldwide. Despite the gravity of such diseases on human health, the pharmaceutical industry has largely neglected the development of chemotherapies for NTDs, and less than 1% of new drug development over the past 30 years has related to NTD research, and no new anthelmintic classes for humans have been described within the past 15 years. This massive deficit in global effort stems mainly from the fact that these diseases affect poor people in poor regions of the world, and as such, are not viewed as viable target markets for the pharmaceutical industry. However, in recent years, many governmental, private sector and philanthropic organizations have begun to inject new funds into this area of research.

As part of this renewed effort in drug discovery for NTDs, a major goal of The Worm Institute for Research and Medicine (WIRM) is to identify potential therapeutic leads against new biological targets relevant to such diseases. In particular, our efforts have focused on onchocerciasis, or “river blindness”, a leading cause of blindness in the developing world. The disease is caused by the filarial nematode, Onchocerca volvulus, and affects more than 37 million people in Africa, Central and South America and Yemen. Currently, the only drug available for mass treatment of onchocerciasis is ivermectin (Mectizan® , Merck); however, this drug is ineffective against adult worms and drug resistance appears to be emerging. Thus, there is a crucial need to identify new drug targets and agents that effectively treat onchocerciasis.

Figure 1:  Chitin metabolism
Figure 1. Chitin metabolism.

As an untapped pathway with potential therapeutic relevance against O. volvulus, WIRM has focused on chitin metabolism. Chitin, one of the most widespread amino polysaccharides in Nature, is a major structural component of arthropod exoskeletons, fungal cell walls, and the microfilarial sheath and eggshells of parasitic nematodes; however, it is entirely lacking from vertebrates. The dynamic biosynthesis and degradation of chitin is crucial for the growth and development of these organisms, and is regulated by two classes of enzymes, chitin synthases and chitinases (Figure 1). To date, only one chitinase from O. volvulus has been identified. OvCHT1 is expressed only in the infective L3 larvae, and is stored within the granules of the cells of the esophageal glands until postinfective development, after which it is secreted and found mostly in the cuticle, or outer body covering. Although its exact mechanism is not clear, it was hypothesized to likely play roles in host transmission, ecdysis (molting) and remodeling of the L4 cuticle and casting of the L3 cuticle. Because of the critical nature of these processes in the lifecycle of the parasite, WIRM believes that probing of chitin metabolism with small, drug-like molecules should provide valuable insights into future development of this biochemical pathway as a therapeutic target in O. volvulus.

Although chitinases, in general, have been implicated in a number of human disease pathways and many highly complex natural product inhibitors have been reported, similar to NTDs, drug discovery efforts toward these targets are sparse. In the NTD field, current drug discovery strategies include piggy-back discovery (i.e. the screening of libraries that are already being assayed for a similar molecular target in another disease), de novo drug discovery and drug repositioning. Due to the time- and cost-effective nature of drug repositioning, WIRM has taken this approach for the discovery of new anti-onchocerciasis agents.

Figure 2:  Structure of closantel
Figure 2. Structure of closantel and its impact on molting in O. volvulus. The ultrastructure images (in squares) were taken to determine what stage of molting was affected by closantel. Top square: normal molting with complete separation of the L3 cuticle and the epicuticle of the newly developed L4 (separation between the black arrows). Bottom square: treatment with closantel (100 μM) causes incomplete separation between the L3 cuticle and the L4 epicuticle (no free space between the black arrows).

Recently, members of WIRM have reported their screening efforts against OvCHT1. Using The Johns Hopkins Clinical Compound Library (JHCCL), a collection of 1,514 known drugs, as a source of drugs to be repositioned, a high-throughput fluorescence-based assay was employed to screen for inhibitors of OvCHT1. From these screening efforts, one drug was discovered with potent inhibition against OvCHT1, the known veterinary anthelmintic drug closantel, with an IC50 value of 1.6 ± 0.08 μM and a competitive inhibition constant (Ki) of 468 ± 84 nM (Figure 2). This compound was also found to be highly specific for filarial family 18 chitinases compared to those from protozoans and hCHTR. Of significance, closantel almost fully inhibited the L3 to L4 molt, and ultrastructural studies revealed an interesting closantel-induced phenotype in that the separation between the L3 cuticle and the newly formed L4 cuticle was inhibited and the cuticular material in between the cuticles was not fully degraded (Figure 2). Importantly, a similar phenotype has also been observed when L3 larvae were cultured with cysteine protease inhibitors or when the transcripts corresponding to O. volvulus cysteine proteases or a serine protease inhibitor were knocked down using RNA interference. It is WIRM’s hope that closantel’s negative impact on molting could lead to new strategies for targeting the progression of O. volvulus larvae to adult worms, which are unable to be destroyed with current therapeutics.

Figure 3:  Structure of a potent fragment
Figure 3. Structure of a potent closantel fragment.

Closantel’s previously documented anthelmintic mode of action was thought to solely rely on its role as a proton ionophore and its chitinase inhibitor activity was previously unknown, hence, implicating a potential bimodal mechanism of action for its observed biochemical activity. With the newly discovered dual biochemical roles for closantel, WIRM members initiated studies toward dissecting its activity determining features. Using a “retro-fragment” based approach, a compound was identified with potency similar to closantel (IC50 of 5.8 ± 0.3 μM) (Figure 3). Using this lead fragment and its analogues, members of WIRM are currently investigating the exact required molecular features underlying the proton ionophore activity as well as the chitinase inhibitory activity in an effort to map out which molecular fragments are required for individual and/or dual activities.

In addition to studies being conducted at TSRI/WIRM, there are also ongoing collaborations with Professor Sara Lustigman of the New York Blood Center and Professor Fidelis Cho-Ngwa of the University of Buea, Cameroon.

Gloeckner, C.; Garner, A. L.; Mersha, F.; Oksov, Y.; Tricoche, N.; Eubanks, L. M.; Lustigman, S.; Kaufmann, G. F.; Janda, K. D. “Repositioning of an Exisitng Drug for the Neglected Tropical Disease Onchocerciasis.” Proc. Natl. Acad. Sci., U. S. A. 2010, 107, 3424-3429.