Development of non-sedating antischistosomal benzodiazepines

Abstract

The neglected tropical disease schistosomiasis infects over 200 million people worldwide and is treated with just one broad spectrum antiparasitic drug (praziquantel). Alternative drugs are needed in the event of emerging praziquantel resistance or treatment failure. One promising lead that has shown efficacy in animal models and a human clinical trial is the benzodiazepine meclonazepam, discovered by Roche in the 1970's. Meclonazepam was not brought to market because of dose-limiting sedative side effects. However, the human target of meclonazepam that causes sedation (GABA_ARs) are not orthologous to the parasite targets that cause worm death. Therefore, we were interested in whether the structure of meclonazepam could be modified to produce antiparasitic benzodiazepines that do not cause host sedation. We synthesized 18 meclonazepam derivatives with modifications at different positions on the benzodiazepine ring system and tested them for in vitro antiparasitic activity. This identified five compounds that progressed to in vivo screening in a murine model, two of which cured parasite infections with comparable potency to meclonazepam. When these two compounds were administered to mice that were run on the rotarod test, both were less sedating than meclonazepam. These findings demonstrate the proof of concept that meclonazepam analogs can be designed with an improved therapeutic index, and point to the C3 position of the benzodiazepine ring system as a logical site for further structure-activity exploration to further optimize this chemical series.

Figure 1.. In vitro effects of MCLZ analogs on adult S. mansoni.

(A) Top - structure of MCLZ with the benzodiazepine ring system numbered and the modified positions highlighted in yellow. Bottom - MCLZ analogs synthesized with modifications to various positions (1, 3, 4, 7 and 2’) on the benzodiazepine ring system. Adult S. mansoni (5–10 worm pairs) were incubated with compound in vitro (30 μM for 14 hours) and the resulting phenotype is noted (paralysis indicating active compounds and motile indicating compounds with no noticeable effects on movement / morphology). (B) Concentration-response curves showing potency of compounds shown to be active in (A). Motility is normalized to movement of DMSO control treated worms (values reflect mean ± standard error of n=8 worms). (C) Comparison of the morphology of worms treated with MCLZ and the two most potent compounds, MYM-V-56 and MYM-III-10.

Figure 2.. In vivo effect of MCLZ analogs on S. mansoni.

(A) Acute in vivo antischistosomal activity of MCLZ derivatives, assayed by measuring the percentage of worms in the liver (L) as opposed to the mesenteric vasculature (M) following drug treatment (oral dose of 100 mg / kg). Open symbols reflect percentages for individual mice, solid bars indicate the mean percentage for the treatment cohort. (B) Curative activity of MCLZ derivatives following a single oral dose of 100 mg / kg. Mice were euthanized one week after drug administration and worm burden was measured. (C) Potency of MCLZ (white symbols), MYM-III-10 (black symbols) and MYM-V-56 (gray symbols) at curing infections of adult S. mansoni (mean ± standard error). (D) Curative activity of MYM-III-10 and MYM-V-56 against juvenile, liver stage parasites. (open symbols reflect the worm burden of individual mice, solid bars indicate the mean percentage for the treatment cohort).

Figure 3.. Antischistosomal activity of MYM-V-56 enantiomers.

(A) In vitro effect of purified (R) and (S)-MYM-V-56 enantiomers on adult worms (14 hour incubation). (B) Time course of the onset of contractile paralysis caused by each enantiomer (10 μM incubation for each). (C) In vivo efficacy of each MYM-V-56 enantiomer against adult parasites. Mice were dosed with various amounts of each compound six weeks post infection and euthanized one week later to assess worm burden.

Figure 4.. Antischistosomal activity of MYM-III-10 enantiomers.

(A) In vitro effect of purified (R) and (S)-MYM-III-10 enantiomers on adult worms (14 hour incubation). (B) In vivo efficacy of each MYM-III-10 enantiomer against adult parasites. Mice were dosed with various amounts of each compound six weeks post infection and euthanized one week later to assess worm burden.

Figure 5.. Sedating activity of antischistosomal benzodiazepines.

Sedation was measured by ability to complete the rotarod test. Mice were dosed with varying amounts of test compound or vehicle control and assayed for their ability to run on the device for 3 minutes (symbols denote mean ± standard error, black = MCLZ, white = MYM-III-10, purple = MYM-V-56, blue = (S)-MYM-V-56 and red = (R)-MYM-V-56.

Figure 6.. Activity of antischistosomal benzodiazepines on mammalian CNS targets.

(A) Affinity for CNS GABA_ARs was measured by assaying the ability of MCLZ (open symbols), MYM-III-10 (black symbols) or MYM-V-56 (gray symbols) to displace ³H-diazepam radioligand from rat brain membranes. (B) Positive allosteric modulator (PAM) activity of MCLZ, MYM-III-10 and MYM-V-56 was measured by recording GABA evoked Cl⁻ currents from ɑ1β2γ2 GABA_A channels recombinantly expressed in mammalian CHO-K1 cells. All three compounds displayed PAM activity. (C) Levels of MYM-III-10 and MCLZ in either mouse brain tissue or serum were measured by HPLC following oral dosing (100 mg / kg).

Figure 7.. Modeled benzodiazepine interactions with host and parasite receptors.

Left - Meclonazepam, (S)-MYM-V-56 and (S)-MYM-III-10 (cyan) docked into the benzodiazepine binding pocket of the human α1β3γ2L GABA_A receptor (cyro-EM structure 6HUP, reference(39). γ2 subunit is shown in green, α1 subunit in orange. Right - the same three benzodiazepines docked into the VSLD cavity of the S. mansoni TRP Smp_333650 (alphafold structure prediction for A0A5K4FCC0, reference(14)). Bottom - predicted potential interactions between ligands and amino acid side chains. Bold = amino acids experimentally validated by mutagenesis studies in reference (14). Panels are taken from the docked ligands provided in supplementary files 5 and 6 (.pdb files), docking scores are provided in supplementary file 7, and ligand - side chain interactions are elaborated upon in supplementary file 8.