Non-sedating benzodiazepines cause contractile paralysis and tissue damage in the parasitic blood fluke Schistosoma mansoni

Parasitic flatworm infections (e.g. tapeworms and fluke worms) are treated by a limited number of drugs. In most cases, control is reliant upon praziquantel (PZQ) monotherapy. However, PZQ is ineffective against sexually immature parasites, and there have also been several concerning reports of cestode and trematode infections with poor PZQ cure-rates, emphasizing the need for alternative therapies to treat these infections. We have revisited a series of benzodiazepines, given the known anti-schistosomal activity of meclonazepam (MCLZ). MCLZ was discovered in the 1970’s but was not brought to market due to dose-limiting sedative side effects. However, in the decades since there have been advances in our understanding of the benzodiazepine GABAA receptor sub-types that drive sedation and the development of sub-type selective, non-sedating ligands. Additionally, the sequencing of flatworm genomes reveals that parasitic trematodes and cestodes have lost GABAAR-like ligand gated anion channels, indicating that MCLZ’s anti-parasitic target is likely distinct from the human receptors that drive sedation. Therefore, we screened a library of classical and non-sedating 1,4-benzodiazepines against Schistosoma mansoni and identified a series of imidazobenzodiazepines that immobilize worms in vitro. One of these hits, Xhe-II-048 also disrupted the parasite tegument, causing extensive vacuole formation beneath the apical membrane. The imidazobenzodiazepine compound series identified has a dramatically lower (∼1 log) affinity for human central benzodiazepine binding site and is a promising starting point for the development of novel anti-schistosomal benzodiazepines with minimal host side-effects. Author Summary Over 200 million people are infected with schistosomiasis, yet there are limited therapeutic options available to treat this disease. The benzodiazepine meclonazepam is known to cure both intestinal and urinary schistosomiasis in animal and human studies, but dose-limiting sedation has been a barrier to its development. Little is known about the structure-activity relationship of meclonazepam and other benzodiazepines on schistosomes, or the identity of the parasite receptor for these compounds. However, schistosomes lack obvious homologs to the human GABAARs that cause sedation. This indicates that the parasite target of this drug is distinct from the host receptors that underpin dose-limiting side effects of meclonazepam, and raises the possibility that benzodiazepines with poor GABAAR affinity may still retain anti-parasitic effects. Here, we report an in vitro screen of various benzodiazepines against schistosomes, and the identification of hit compounds that are active against worms yet possess reduced affinity for the human GABAARs that cause sedation.


Introduction
Over 200 million people are infected with the parasitic blood flukes that cause the neglected tropical disease schistosomiasis (1). Over 90% of infections occur in sub-Saharan Africa, where the disease kills 300,000 persons / year (2). The pathology associated with chronic infection adds to the disease burden, putting the socioeconomic cost of schistosomiasis (70 million disability adjusted life years) near HIV/AIDs, malaria or tuberculosis (1,3). However, despite these enormous costs treatment relies on just one broad-spectrum drug, praziquantel (PZQ) (4). PZQ treatment has high cure rates of 70-90% (5, 6), but it is concerning that a subset of infections in human and animal populations appear to be refractory to treatment (7)(8)(9), either due to PZQ's lack of efficacy against recently acquired, immature parasites worms (10,11) or standing genetic variation in parasite populations. The latter possibility is especially concerning in regard to the potential emergence of PZQ-resistant parasites, and consideration needs given to whether PZQ-monotherapy will be sufficient to achieve schistosomiasis elimination (12).
One lead compound with proven anti-schistosomal activity is the benzodiazepine meclonazepam ((S)-3-methylclonazepam, MCLZ). MCLZ was discovered in the 1970's and found to cure both the mature and immature parasites that cause urinary and intestinal forms of schistosomiasis (13). Development of this lead stalled in the 1980's due to dose-limiting sedation in human trials (14)(15)(16)(17). However, we have revisited benzodiazepines as potential antiparasitic leads given advances in two areas. First, it is now understood that the sedative effects of benzodiazepines are driven by α 1-subunit containing GABA A Rs (18). This has enabled the design of various benzodiazepines with reduced affinity towards α 1-containing GABA A Rs to treat various conditions involving GABA A Rs that contain other α subunits such as asthma and schizophrenia (19). Second, with recent advances in the sequencing of parasitic helminth genomes there is an abundance of data available to establish whether flatworm parasites possess GABA A Rs (20). If GABA A Rs are not present in parasitic worms, then it is possible that the structure-activity requirements of anti-parasitic compounds may differ significantly from those mediating benzodiazepine binding to host GABA A Rs, offering the opportunity to develop ligands with increased parasite selectivity. Here, we have profiled the repertoire of S. mansoni ligand gated ion channels and, having found no obvious parasite GABA A Rs, screened a library of benzodiazepines to identify compounds that display anti-parasitic activity and exhibit reduced mammalian GABA A R affinity.
6 workflow used to quantify movement from these video recordings is shown in Supplemental Figure 1. Maximum intensity projections were generated for the entire stack of images (241 frames for a 1-minute recording) and integrated pixel values were measured for the resulting composite image, allowing normalization of movement relative to DMSO control treated worms. and post-fixed for 2 hours on ice in reduced 1% osmium tetraoxide. Worms were then 2 x 10 minutes in distilled water and stained overnight at 4 o C in alcoholic Uranyl Acetate. Worms were rinsed in distilled water, dehydrated in 50%, 75% and 95% MeOH, followed by successive 10 minute rinses in 100% MeOH and acetonitrile. Worms were incubated in a 1:1 mix of acetonitrile and epoxy resin for 1 hour prior to 2 x 1 hour incubations in epoxy resin. Worms were then cut transversely and embedded overnight in epoxy (60 o C). Ultra-thin sections (70 nm) were cut onto bare 200-mesh copper grids and stained in aqueous lead citrate for 1 minute and sections were imaged on a Hitachi H-600 electron microscope fitted with a Hamamatsu C4742-95 digital camera) operating at an accelerating voltage of 75 kV. GABA A R Modeling. Benzodiazepines were docked to the human GABA A R cryo-EM structure (PDB ID 6HUO) (27) using Schrödinger Maestro suite v2019-1. 3-D ligand structures were generated using the LigPrep module. Water molecules further than 5 Å from the protein surface were removed, hydrogen bonds were optimized at pH 7.0 and the structure was minimized in the OPLS3e forcefield. Ligands were docked into a 10x 10x10 Å grid centered on the bound alprazolam molecule in the GABA A R structure using Glide (28) in ExtraPrecision (XP) mode, and output poses were ranked by XP GlideScore. Figures were generated using PyMOL 2.3.0.

GABA A Rs are not the schistosome targets of meclonazepam.
The benzodiazepine meclonazepam (MCLZ) is an effective anti-schistosomal drug, but the sedative side effects of MCLZ coincide with the anti-parasitic dose (14). The sedative side effects of MCLZ are likely driven by human GABA A Rs -specifically those heteromeric receptors that contain the α 1 subunit (18). These receptors account for approximately 60% -80% of brain GABA A Rs (18,29). However, the parasite target of MCLZ remains unknown. Since the earliest reports of MCLZ's anti-schistosomal activity it has been noted that these effects are not replicated by other benzodiazepines that also bind GABA A Rs with high affinity (13,30).
Therefore, we considered that the parasite target of MCLZ may be distinct from GABA A Rs, since it is not even clear whether flatworms possess this class of ligand-gated ion channel (LGIC) (31,32).
In order to comprehensively search the repertoire of flatworm LGICs, we generated  Table 1). Previously reported schistosome GluCls (31) and cholinergic receptors (33) clustered alongside sequences 8 consistent with their functional characterization. GABA A Rs are clearly present in the mollusk A. californica and free-living flatworm M. lignano, but they appear to have been lost in free-living planarians and the parasitic flatworms E. multilocularis and S. mansoni ( Figure 1).

Non-sedating imidazobenzodiazepines cause parasite contractile paralysis
If schistosomes lack GABA A Rs, then the parasite target of MCLZ may have different structural requirements for ligand binding than the MCLZ -GABA A R interaction that drives sedation. If so, then it should be possible to identify parasite-selective benzodiazepines that retain anti-schistosomal activity but lack affinity at mammalian GABA A Rs that contain

Imidazobenzodiazepine Xhe-II-048 causes structural damage to parasite tissue
Given the parasite-selectivity of imidazobenzodiazepines (Figure 3, blue) relative to MCLZ-like compounds (Figure 3, red), we investigated the effects of XliHeII-048, Xhe-II-048 and SH-I-055 on schistosome tissues in more detail. Specifically, we were interested in drug-evoked damage to the parasite tegument, which is a feature of many anti-schistosomal compounds (35). Worms treated with DMSO vehicle control, MCLZ (5 µM) or various imidazobenzodiazepines (10µM) overnight, fixed and processed for imaging by transmission electron microcopy (TEM). Imaging transverse schistosome cross sections revealed a typical body wall structure in DMSO treated worms, with alternating layers of schistosome muscle, followed by the tegument basal membrane, tegument syncytium, and tegument apical membrane. In MCLZ treated worms, tissue layers exhibit are disrupted, with pervasive vacuolization of the tegument ( Figure 4A). The tegument of Xhe-II-048 treated worms displayed a similar pattern of extensive vacuole distribution beneath the apical membrane, while worms 1 0 treated with XliHeII-048 and the less potent imidazobenzodiazepine SH-I-055 displayed normal tissue ultrastructure ( Figure 4A).
MCLZ is a schistocidal compound, and worms do not recover movement following drug washout ( Figure 4B). Similarly, Xhe-II-048 treated worms did not recover movement up to one day following drug washout, consistent with the extensive ultrastructural damage caused by this compound ( Figure 4A). Compounds XliHeII-048 and SH-I-055, which did not cause pervasive tegument damage, evoked only a transient paralysis, recovering by 1 day after drug washout.

Discussion
The anti-schistosomal activity of the benzodiazepine meclonazepam (MCLZ) was discovered in the 1970's, but development of this lead as a human therapy for schistosomiasis stalled due to sedative side effects that coincide with the dose of drug needed to clear infections (13,14). This is expected, given the structural similarity of MCLZ to centrally acting benzodiazepines such as CLZ and NIT that are clinically used as anxiolytics and display well known sedating effects. Some attempts were made in the 1980's to antagonize the sedative effects of MCLZ using the GABA A R antagonist flumazenil. While flumazenil did not impair the anti-schistosomal effect of MCLZ (36), pharmacokinetic differences between flumazenil (administered by IV due to poor bioavailability, <1 hour elimination half-life (37)) and MCLZ (orally bioavailable, half-life up to 80 hours (38)) precluded the development of an admixture as a viable non-sedating therapeutic approach (15,39). Research on MCLZ as an antischistosomal lead has slowed over the past several decades. However, we have revisited this compound based on recent helminth genomic data indicating that parasitic flatworms lack GABA A Rs (Figure 1), advances in our understanding of mammalian GABA A R subtypes that account for sedative side effects (18,40), and advances in the development of non-sedating benzodiazepines with selectivity towards various GABA A R sub-types (22-25).

Schistosome genomes lack GABA A Rs
Sequenced genomes of parasitic trematode (S. mansoni) and cestode (E. multilocularis) flatworms appear to lack GABA A Rs, the benzodiazepine targets that cause sedation in humans

2
Modification of this N1 is also observed in the non-sedating antiviral benzodiazepine BDAA (47), although smaller alkyl groups are likely tolerated, such as the methyl on diazepam and MYM-II-53. Additionally, the large TMS-acetylene group at the C7 position of the imidazobenzodiazepine hit series may not be tolerated within the GABA A R binding pocket, where the MCLZ C7 nitro group is predicted to interact with the γ N60 sidechain ( Figure 5).
While these ligands phenocopy MCLZ to a degree, it is unclear whether they act via the same schistosome receptor -or if they do bind the same receptor, whether they share a common binding pose. The target of MCLZ will need to be identified to generate hypothesis into ligand-receptor structure-activity relationships, as the structures of MCLZ and the TMSacetylene imidazobenzodiazepines appear quite different. However, we can observe some similarities and differences in the SAR of the two series.
Two interesting positions are (i) the benzodiazepine C3 position, which is typically unmodified in classical benzodiazepines but contains a chiral methyl group in these two series, and (ii) the phenyl C2' position, which is commonly halogenated in benzodiazepines with GABA A R affinity. The benzodiazepine C3 position is essential for MCLZ activity, the key difference between clonazepam and MCLZ is that clonazepam lacks a C3 methyl group. This results in a roughly 1 log decrease in potency (Figure 3). Chirality of this position is also important for 3-methylclonazepam, since the (R) enantiomer reportedly exhibits reduced antiparasitic efficacy (34). On the other hand, C3 methylation decreased activity of XliHeII-048. The (S)-methylated compound SH-I-055 was ~3 times less potent than XliHeII-048, and the (R)methylated compound SH-I-060 was essentially inactive. This schistosome SAR is distinct from the binding of imidazobenzodiazepines to mammalian GABA A Rs, where (S) and (R) isomers have roughly equivalent binding affinities (49). The halogen on the phenyl C2' position of MCLZ seems important for schistosome activity, given that nitrazepam (inactive against parasites) differs from clonazepam (movement IC 50 1.9 µM) in that the phenyl ring is unsubstituted.
However, XliHeII-048 (which contains a fluorine at this position) and Xhe-II-048 (which possess an unsubstituted phenyl ring) appear equipotent ( Figure 2C). In fact, the unsubstituted compound Xhe-II-048 was unique in evoking structural damage to the parasite tegument ( Figure   4A).
Development of the Xhe-II-048 hit compound into a bona fide anti-schistosomal lead will likely require the modifications to improve metabolic stability in vivo. Specifically, the ester and 1 3 TMS-acetylene groups will likely require substitution with bioisosteres. The imidazole ester group is likely rapidly hydrolyzed by carboxylesterases during first pass metabolism, and the TMS-acetylene group is also likely to be unstable in vivo (50). From the SAR shown in Figure   2C, is it apparent that loss of this group dramatically decreased potency. Nevertheless, this is the first report of non-sedating benzodiazepines screened against schistosomes. MCLZ has been shown to have anti-schistosomal activity in human clinical trials, but with an extremely narrow therapeutic index; the effective anti-parasitic dose (0.2 -0.3 mg/kg) coincided with the dose at which sedation was reported (14). This work has identified benzodiazepine hits that exhibit potent anti-schistosomal effects in vitro and dramatically lower affinity for host α 1GABA A Rs (Figure 3). Given the scarcity of new lead compounds to treat schistosomiasis, these data are valuable in advancing a pharmacophore that retains anti-schistosomal activity while displaying reduced sedation.  and two distinct clades of GluCl subunits (green) that include a previously reported flatwormspecific group of receptors (31). Bootstrap percentage is shown at nodes (500 replicates).      Table. Structures and phenotypes of benzodiazepines screened against S. mansoni.
Data from the primary screen shown in Figure 2A. SMILES IDs are provided for all compounds screened, and phenotypes are shown for worms exposed to 30 µM test compound overnight.
Blue highlighted rows indicate hits at 30 µM, red highlighted rows indicate hits at 10 µM.