An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behavior

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-Antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered β-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the β-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation

Dihydrobenz[e][1,4]oxazepin-2(3H)-ones, a new anthelmintic chemotype immobilising whipworm and reducing infectivity in vivo

Trichuris trichiura is a human parasitic whipworm infecting around 500 million people globally, damaging the physical growth and educational performance of those infected. Current drug treatment options are limited and lack efficacy against the worm, preventing an eradication programme. It is therefore important to develop new treatments for trichuriasis. Using Trichuris muris, an established model for T. trichiura, we screened a library of 480 novel drug-like small molecules for compounds causing paralysis of the ex vivo adult parasite. We identified a class of dihydrobenz[e][1,4]oxazepin-2(3H)-one compounds with anthelmintic activity against T. muris. Further screening of structurally related compounds and resynthesis of the most potent molecules led to the identification of 20 active dihydrobenzoxazepinones, a class of molecule not previously implicated in nematode control. The most active immobilise adult T. muris with EC50 values around 25-50μM, comparable to the existing anthelmintic levamisole. The best compounds from this chemotype show low cytotoxicity against murine gut epithelial cells, demonstrating selectivity for the parasite. Developing a novel oral pharmaceutical treatment for a neglected disease and deploying it via mass drug administration is challenging. Interestingly, the dihydrobenzoxazepinone OX02983 reduces the ability of embryonated T. muris eggs to establish infection in the mouse host in vivo. Complementing the potential development of dihydrobenzoxazepinones as an oral anthelmintic, this supports an alternative strategy of developing a therapeutic that acts in the environment, perhaps via a spray, to interrupt the parasite lifecycle. Together these results show that the dihydrobenzoxazepinones are a new class of anthelmintic, active against both egg and adult stages of Trichuris parasites. They demonstrate encouraging selectivity for the parasite, and importantly show considerable scope for further optimisation to improve potency and pharmacokinetic properties with the aim of developing a clinical agent

2,4-Diaminothieno[3,2 d]pyrimidines, a new class of anthelmintic with activity against adult and egg stages of whipworm

The human whipworm Trichuris trichiura is a parasite that infects around 500 million people globally, with consequences including damage to physical growth and educational performance. Current drugs such as mebendazole have a notable lack of efficacy against whipworm, compared to other soil-transmitted helminths. Mass drug administration programs are therefore unlikely to achieve eradication and new treatments for trichuriasis are desperately needed. All current drug control strategies focus on post-infection eradication, targeting the parasite in vivo. Here we propose developing novel anthelmintics which target the egg stage of the parasite in the soil as an adjunct environmental strategy. As evidence in support of such an approach we describe the actions of a new class of anthelmintic compounds, the 2,4-diaminothieno[3,2-d]pyrimidines (DATPs). This compound class has found broad utility in medicinal chemistry, but has not previously been described as having anthelmintic activity. Importantly, these compounds show efficacy against not only the adult parasite, but also both the embryonated and unembryonated egg stages and thereby may enable a break in the parasite lifecycle

Resynthesized dihydrobenzoxazepinones show dose-dependent attenuation of <i>ex vivo T</i>. <i>muris</i> adult motility.

<p>Single adult worms were incubated for 24 hours in wells containing media plus compound. Motility was quantified using an automated phenotyping platform. EC₅₀ ± standard error shown in parentheses. n = 5, except OX03144 where n = 10. Curve fitted using the four parameter log-logistic model.</p

<i>T</i>. <i>muris</i> eggs treated with dihydrobenzoxazepinone OX02983 are less infective <i>in vivo</i>.

<p>(A) Experimental scheme: embryonated eggs were soaked in compound, and then used to infect mice by oral gavage. At day 15 post-infection, mice were culled and worm burden assessed. (B) Treatment with OX02983 reduced the ability of embryonated eggs to establish infection <i>in vivo</i>. Blue bar indicates median worm burden. A one-way ANOVA (worms ~ treatment) showed a significant difference between treatment groups (F(3,48) = 8.3, P< 0.0005). Differences between groups were determined using a post-hoc Tukey HSD test (** = P<0.005, *** = P<0.0001). n = 14 (DMSO), 16 (water), 10 (OX02983), 12 (OX03153). (C) Dihydrobenzoxazepinones do not act by blocking embryonation. Eggs were incubated with 100μM compounds or DMSO-alone control for 56 days. Embryonation was then quantified. No significant differences between groups were detected: one-way ANOVA (embryonation ~ treatment, F(2,12) = 0.60, P = 0.57). Blue bar indicates median percentage embryonation.</p

Structures of active dihydrobenzoxazepinones that were resynthesized, with selected calculated properties.

<p>RMM: relative molecular mass. #HBA: number of hydrogen bond acceptors. #HBD: number of hydrogen bond donors. tPSA: topological polar surface area. (Calculated using DataWarrior [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005359#pntd.0005359.ref028" target="_blank">28</a>]).</p

Schematic showing possible treatment strategies: post-infection treatment and breaking the <i>Trichuris</i> life cycle in the environment.

<p><i>Trichuris</i> infection could be prevented by using novel drugs to 1) target the prevention of egg embryonation in the external environment, 2) reduce the infectivity of embryonated eggs prior to ingestion, or 3) treat existing infections <i>in vivo</i> while worms are at larval and adult stages.</p

Elaboration of initial active compounds by characterising a diverse set of 192 dihydrobenzoxazepinones with the same core structure.

<p>(A) Screening and elaboration procedure. (B) Relationship between activity and molecular weight of each dihydrobenzoxazepinone. Activity is the minus log₂ movement reduction compared to DMSO-only controls when assayed at 100<b>μ</b>M. (C) Relationship between activity and predicted hydrophobicity (cLogP) of each dihydrobenzoxazepinone.</p

Reduced worm burden in mice given <i>T</i>. <i>muris</i> eggs that had been treated with diaminothienopyrimidines.

<p><b>(a)</b> Embryonated eggs were soaked in compound for 14 days, washed in water and then used in either <i>in vitro</i> or <i>in vivo</i> hatching assays. <b>(b)</b> Treatment with DATPs reduced the ability of embryonated eggs to hatch in <i>E</i>. <i>coli</i> bacterial suspension after 24 hours. A one-way ANOVA showed a significant difference between treatment groups (F(5,26) = 25.95 p<0.0001) with a post-hoc Dunnett’s compared to DMSO control (**** = p<0.0001) <i>n =</i> 7 (DMSO), <i>n =</i> 5 (DATP compounds) <b>(c)</b> SCID mice were infected with 40 eggs and worm burden assessed at day 15 post infection. The experiment was carried out in two batches, with n = 5 and n = 9 mice respectively in each of the control and treatment groups. Data were normalised for each batch relative to the mean of the DMSO-only control group for that batch. Blue line indicates mean for each treatment group. A two-way ANOVA showed a significant effect of treatment [F(1,24) = 9.569, P = 0.00497] but no effect of batch [F(1,24) = 0.083, P = 0.77618] or interaction [F(1,24) = 0.083 0.77618]. A post-hoc Tukey HSD test showed that the <b>OX02926</b>-treated group was significantly different from the DMSO control group (P = 0.0050).</p

Summary of the cytotoxicity in a mouse epithelial cell line of the DATP series.

<p>Mouse CMT-93 rectal epithelial cells were used for this assay. Maximum tested concentration was 100 μM. <i>n =</i> 8, error range (in parentheses) shows 95% confidence interval. EC₅₀ values in the adult <i>Trichuris</i> paralysis assay are shown for comparison.</p