Drug Repositioning and Pharmacophore Identification in the Discovery of Hookworm MIF Inhibitors

SummaryThe screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new pharmacophores. Hookworms are blood-feeding, intestinal nematode parasites that infect up to 600 million people worldwide. Vaccination with recombinant Ancylostoma ceylanicum macrophage migration inhibitory factor (rAceMIF) provided partial protection from disease, thus establishing a “proof-of-concept” for targeting AceMIF to prevent or treat infection. A high-throughput screen (HTS) against rAceMIF identified six AceMIF-specific inhibitors. A nonsteroidal anti-inflammatory drug (NSAID), sodium meclofenamate, could be tested in an animal model to assess the therapeutic efficacy in treating hookworm disease. Furosemide, an FDA-approved diuretic, exhibited submicromolar inhibition of rAceMIF tautomerase activity. Structure-activity relationships of a pharmacophore based on furosemide included one analog that binds similarly to the active site, yet does not inhibit the Na-K-Cl symporter (NKCC1) responsible for diuretic activity

Extended analysis of a genome-wide association study in primary sclerosing cholangitis detects multiple novel risk loci.

A limited number of genetic risk factors have been reported in primary sclerosing cholangitis (PSC). To discover further genetic susceptibility factors for PSC, we followed up on a second tier of single nucleotide polymorphisms (SNPs) from a genome-wide association study (GWAS). We analyzed 45 SNPs in 1221 PSC cases and 3508 controls. The association results from the replication analysis and the original GWAS (715 PSC cases and 2962 controls) were combined in a meta-analysis comprising 1936 PSC cases and 6470 controls. We performed an analysis of bile microbial community composition in 39 PSC patients by 16S rRNA sequencing. Seventeen SNPs representing 12 distinct genetic loci achieved nominal significance (p(replication) <0.05) in the replication. The most robust novel association was detected at chromosome 1p36 (rs3748816; p(combined)=2.1 × 10(-8)) where the MMEL1 and TNFRSF14 genes represent potential disease genes. Eight additional novel loci showed suggestive evidence of association (p(repl) <0.05). FUT2 at chromosome 19q13 (rs602662; p(comb)=1.9 × 10(-6), rs281377; p(comb)=2.1 × 10(-6) and rs601338; p(comb)=2.7 × 10(-6)) is notable due to its implication in altered susceptibility to infectious agents. We found that FUT2 secretor status and genotype defined by rs601338 significantly influence biliary microbial community composition in PSC patients. We identify multiple new PSC risk loci by extended analysis of a PSC GWAS. FUT2 genotype needs to be taken into account when assessing the influence of microbiota on biliary pathology in PSC.Norwegian PSC Research Center German Ministry of Education and Research (BMBF) through the National Genome Research Network (NGFN) Integrated Research and Treatment Center - Transplantation 01EO0802 PopGen biobank NIH DK 8496

Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo

Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally) as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP) inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite’s resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN), Merck’s cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite’s CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN’s somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK) profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s) that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole

Odanacatib, a Cathepsin K Cysteine Protease Inhibitor, Kills Hookworm In Vivo.

Hookworm infection is chief among soil-transmitted helminthiases (STHs) for the chronic morbidly inflicted. Deworming via mass drug administration (MDA) programs most often employs single doses of benzimidazole drugs to which resistance is a constant threat. To discover new drugs, we employ a hamster model of hookworm infection with Ancylostoma ceylanicum and use albendazole (ABZ; 10 mg/kg orally) as the gold standard therapy. We previously showed that a single oral 100 mg/kg dose of the cathepsin cysteine protease (CP) inhibitor, K11777, offers near cure of infection that is associated with a 95% reduction in the parasite's resident CP activity. We confirm these findings here and demonstrate that odanacatib (ODN), Merck's cathepsin K inhibitor and post-clinical Phase III drug candidate for treatment of osteoporosis, decreases worm burden by 73% at the same dose with a 51% reduction in the parasite's CP activity. Unlike K11777, ODN is a modest inhibitor of both mammalian cathepsin B and the predominant cathepsin B-like activity measureable in hookworm extracts. ODN's somewhat unexpected efficacy, therefore, may be due to its excellent pharmacokinetic (PK) profile which allows for sustained plasma exposure and, possibly, sufficient perturbation of hookworm cathepsin B activity to be detrimental to survival. Accordingly, identifying a CP inhibitor(s) that combines the inhibition potency of K11777 and the PK attributes of ODN could lead to a drug that is effective at a lower dose. Achieving this would potentially provide an alternative or back-up to the current anti-hookworm drug, albendazole

Cure of Hookworm Infection with a Cysteine Protease Inhibitor

<div><h3>Background</h3><p>Hookworm disease is a major global health problem and principal among a number of soil-transmitted helminthiases (STHs) for the chronic disability inflicted that impacts both personal and societal productivity. Mass drug administration most often employs single-dose therapy with just two drugs of the same chemical class to which resistance is a growing concern. New chemical entities with the appropriate single-dose efficacy are needed.</p> <h3>Methods and Findings</h3><p>Using various life-cycle stages of the hookworm <em>Ancylostoma ceylanicum in vitro</em> and a hamster model of infection, we report the potent, dose-dependent cidal activities of the peptidyl cysteine protease inhibitors (CPIs) K11002 (4-mopholino-carbonyl-phenylalanyl-homophenylalanyl- vinyl sulfone phenyl) and K11777 (<em>N</em>-methylpiperazine-phenylalanyl-homophenylalanyl-vinylsulfone phenyl). The latter is in late pre-clinical testing for submission as an Investigational New Drug (IND) with the US Federal Drug Administration as an anti-chagasic. <em>In vitro</em>, K11002 killed hookworm eggs but was without activity against first-stage larvae. The reverse was true for K11777 with a larvicidal potency equal to that of the current anti-hookworm drug, albendazole (ABZ). Both CPIs produced morbidity in <em>ex vivo</em> adult hookworms with the activity of K11777 again being at least the equivalent of ABZ. Combinations of either CPI with ABZ enhanced morbidity compared to single compounds. Strikingly, oral treatment of infected hamsters with 100 mg/kg K11777 <em>b.i.d.</em> (i.e., a total daily dose of 200 mg/kg) for one day cured infection: a single 100 mg/kg treatment removed >90% of worms. Treatment also reversed the otherwise fatal decrease in blood hemoglobin levels and body weights of hosts. Consistent with its mechanism of action, K11777 decreased by >95% the resident CP activity in parasites harvested from hamsters 8 h post-treatment with a single 100 mg/kg oral dose.</p> <h3>Conclusion</h3><p>A new, oral single-dose anthelmintic that is active in an animal model of hookworm infection and that possesses a distinct mechanism of action from current anthelmintics is discovered. The data highlight both the possibility of repurposing the anti-chagasic K11777 as a treatment for hookworm infection and the opportunity to further develop CPIs as a novel anthelmintic class to target hookworms and, possibly, other helminths.</p> </div

Oral administration of K11777 cures <i>A. ceylanicum</i> infection and improves blood hemoglobin levels.

<p>Groups of Syrian hamsters (n = 6) were infected with 75 third stage <i>A. ceylanicum</i> larvae and followed for 24 days to monitor blood hemoglobin levels and weight gain. At 17 days post-infection (DPI) hamsters were treated orally with K11777 (100 mg/kg <i>q.d.</i> or <i>b.i.d.</i>), ABZ (10 mg/kg <i>q.d</i>.) or vehicle alone as indicated. At 18 DPI, one group of hamsters was treated again with K11777 (100 mg/kg <i>b.i.d</i>.) and one group was treated with vehicle alone. At 24 DPI, all hamsters were sacrificed and intestinal worms counted. Compared to infected vehicle controls, hamsters treated <i>q.d.</i> or <i>b.i.d.</i> with K11777 did not show significantly improved weight gain (Panel A), but levels of blood hemoglobin were significantly higher (Panel B; P<0.001 and P<0.001 at 21 and 24 DPI, respectively). Dosing <i>q.d.</i> significantly decreased worm burdens by 90.1% (P<0.001) whereas dosing <i>b.i.d.</i> resulted in cure of infection (Panel C).</p

K11002 and K11777 cause morbidity in <i>ex vivo</i> cultured <i>A. ceylanicum</i> adult worms.

<p>Adult hookworms were recovered from infected hamsters and used in an adult worm killing assay to measure the effect of K11002 (Panel A) and K11777 (Panel B) on adult worm viability. Worms were scored individually using the 5 point morbidity scale (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001680#pntd.0001680.s001" target="_blank">Figure S1</a>) at 120 hours post-treatment (HPT). Control wells were treated with ABZ (50 µM) or equivalent volumes of DMSO carrier alone. Data are expressed as means ± SD of triplicate measurements. The results presented are representative of two separate experiments.</p

ABZ combined with K11002 or K11777 enhances morbidity in <i>ex vivo</i> cultured <i>A. ceylanicum</i> adult worms.

<p>Adult hookworms were recovered from infected hamsters and used in an adult worm killing assay to measure the effect of combining ABZ with K11002 (Panel A) or K11777 (Panel B) on adult worm survival and morbidity. Worms were scored individually using the 5 point morbidity scale (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001680#pntd.0001680.s001" target="_blank">Fig S1</a>) at different time points post-treatment. Hookworms were incubated with each compound at 25 µM. Control wells were treated with equivalent volumes of DMSO vehicle alone. Data are expressed as means ± SD of triplicate measurements. The results presented are representative of two separate experiments.</p