Allergen delivery inhibitors: Characterisation of potent and selective inhibitors of der p 1 and their attenuation of airway responses to house dust mite allergens

International Journal of

Molecular Sciences

Article

Allergen Delivery Inhibitors: Characterisation of

Potent and Selective Inhibitors of Der p 1 and Their

Attenuation of Airway Responses to House Dust

Mite Allergens

Jihui Zhang ^1,2, Jie Chen ¹, Jie Zuo ^1,3, Gary K. Newton ⁴, Mark R. Stewart ⁴, Trevor R. Perrior ⁴,

David R. Garrod ⁵and Clive Robinson ^1,

*

1

Institute for Infection & Immunity, St George’s, University of London, Cranmer Terrace,

London SW17 0RE, UK; zhang.jihui@im.ac.cn (J.Z.); jchen99556688@outlook.com (J.C.);

mrzuojie@126.com (J.Z.)

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences,

Beijing 100101, China

BOE Technology Center, BOE Technology Group Co., Ltd., Beijing 100176, China

Domainex Ltd., Chesterford Research Park, Little Chesterford, Saffron Walden, Essex CB10 1XL, UK;

Gary.Newton@domainex.co.uk (G.K.N.); Mark.Stewart@domainex.co.uk (M.R.S.);

Trevor.Perrior@domainex.co.uk (T.R.P.)

Faculty of Biology Medicine and Health, University of Manchester, Manchester M13 9PL, UK;

d.garrod@manchester.ac.uk

2

3

4

5

*

Correspondence: c.robinson@sgul.ac.uk; Tel.: +44-(0)20-8725-5620

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Received: 31 August 2018; Accepted: 5 October 2018; Published: 15 October 2018

Abstract: Group 1 allergens of house dust mites (HDM) are globally significant triggers of allergic disease.

They are considered as initiator allergens because their protease activity enables the development of

allergy to a spectrum of unrelated allergens from various sources. This initiator-perpetuator function

identiﬁes Group 1 HDM allergens as attractive drug design targets for the ﬁrst small-molecule

approach directed towards a non-human, root cause trigger of allergic disease. The purpose of

this study was to: (i) identify exemplar inhibitors of these allergens using Der p 1 as a design

template, and (ii) characterise the pharmacological proﬁles of these compounds using in vitro and

in vivo models relevant to allergy. Potent inhibitors representing four different chemotypes and

differentiated by mechanism of action were investigated. These compounds prevented the ab initio

development of allergy to the full spectrum of HDM allergens and in established allergy they inhibited

the recruitment of inﬂammatory cells and blunted acute allergic bronchoconstriction following aerosol

challenge with the full HDM allergen repertoire. Collectively, the data obtained in these experiments

demonstrate that the selective pharmacological targeting of Der p 1 achieves an attractive range of

beneﬁts against exposure to all HDM allergens, consistent with the initiator-perpetuator function of

this allergen.

Keywords: Der p 1; airway inﬂammation; eosinophil; house dust mite; allergens; allergen delivery

inhibitor

1. Introduction

A compelling case demonstrates that as well as binding allergen-speciﬁc IgE, some allergens

possess bioactivities which are essential to the development and maintenance of sensitisation to a wide

range of unrelated allergens [

1,2]. These bioactivities deﬁne the function of the allergenic protein in

its host source, but upon interaction with structural elements of human mucosal surfaces they can

Int. J. Mol. Sci. 2018, 19, 3166; doi:10.3390/ijms19103166

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Int. J. Mol. Sci. 2018, 19, 3166

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cause disease, especially in individuals possessing an underlying genetic predisposition. Consequently,

the broader ranging these bioactivities are, the greater their inﬂuence on the pathogenesis of allergic

disease. Such allergens typically exert serodominance, generating high titre allergen-speciﬁc IgE,

but by being permissive for the effects of unrelated allergens they also serve as wide-ranging

initiator-perpetuators of allergy [1]. This ‘functionalist’ concept of a cadre of allergens that act as

bioinitiators of sensitisation to other, comparatively effete, allergens is well established in experimental

models that have given insights into the mechanisms of immunological and non-immunological

collateral priming, adjuvancy, or bystander effects [3–6]. The functionalist view of allergens is also

understood from the perspective of longitudinal epidemiological surveys of polysensitisation where

a temporal progression in reactivity to different allergens is recognised, and where discrete initiator

allergens are linked strongly to the development of disease [7–10].

Group 1 allergens from HDM have been most extensively investigated as initiator allergens [2 ,11 ,12 ]

In HDM, they function as digestive enzymes and are excreted in faecal pellets which are inhalable by

humans. Impaction of inhaled HDM faecal pellets on the airway surface releases the protease allergen,

which is resistant to inactivation by natural antiprotease defences of the airways, achieving a high

localised concentration of the enzyme. The cysteine protease activity of these allergens increases the

probability of detection by dendritic antigen presenting cells creates a signalling environment in the

airways which favours the programming and then maintenance of immune responses with an allergic

phenotype [1,11,13].

.

The detection of inhaled allergens by antigen presenting cells is facilitated by Group 1 HDM

allergens because they cleave interepithelial tight junctions (TJs) by directly attacking the extracellular

domains of the TJ adhesion proteins [14]. The ensuing non-selective increase in epithelial permeability

enhances the probability of contact between any inhaled allergen and antigen presenting cell networks

whose dendrites become increasingly exposed to the milieu of the airway lumen [12]. A further

consequence is that dendritic and other cells accumulate in increasing numbers in the airway [13].

Additionally, the effects on TJs enable allergens to engage receptors of the innate immune system

which ordinarily exhibit basolateral distribution. Ligation of these receptors activates signalling which

upregulates the expression of inﬂammatory cytokines and facilitates the release of other mediators.

Some pro-allergic cytokines, notably IL-13, downregulate the expression of TJ adhesion proteins

thus exacerbating the cycle of allergen delivery to antigen presenting cells and innate signalling

receptors [15]. Similar permeability events have been reported for the nasal epithelium and skin [16,17]

and linkage to allergy is suggested by claudin-1 expression being inversely proportional to the

Th2 polarisation of immune responses [18,19]. The recent discovery that Group 1 HDM allergens

have prothrombinase activity provides new insight into how this polarisation to allergic responses

occurs and suggests a novel mechanism which might contribute to epithelial–mesenchymal cell

transition and airway remodelling driven primarily by the innate effects of certain allergens [2,12,20,21].

The combination of allergen delivery, polarisation of adaptive responses and immunodominance by

Group 1 HDM allergens is consistent with them being initiators of allergic responses to themselves

and to unrelated allergens through a process of collateral priming of immune responses [2,11,12,14].

The identiﬁcation and characterisation of initiator allergens creates opportunities for the design of

small molecule interventions designed to target the agents whose activities promote and sustain

allergic disease [2,11,12,22]. A prominent appeal of such an approach is that the targeting of

a non-human, root cause trigger of disease should combine an attractive safety proﬁle with a

signiﬁcantly wider range of efﬁcacy beneﬁts than can be achieved by targeting discrete downstream

effector pathways (e.g., leukotriene antagonists, cytokine-speciﬁc biologics), or by the less selective

action of corticosteroids [12

biological advances in understanding the molecular basis of allergenicity, and chemical advances in

structure-based protease inhibitor design [ 11 12 22]. Thus, for the ﬁrst time since the invention of

,22]. Attempting this type of intervention is now feasible following

2

, , ,

allergen immunotherapy by Noon and Freeman in 1911, is it possible to envisage small-molecule

pharmacotherapy aimed directly at the causative, non-human, aspect of allergy [23].

Int. J. Mol. Sci. 2018, 19, 3166

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We have recently disclosed the design and synthesis of novel inhibitors of Group 1 HDM allergens

which target the allergy initiator-sustainer mechanisms by inhibiting their proteolytic activity [ 12 22].

2

,

Conveniently, the high degree of identity or similarity in the amino acid sequences of the Group 1

allergens from dust mites of different species means that they comprise a single target for the purposes

of inhibitor design [2,11,12]. As inhalant allergens with dominant roles as initiator-sustainers of

allergic asthma and perennial allergic rhinitis, direct administration of the inhibitors to the airways

should neutralise inhaled allergen molecules before signiﬁcant interactions with the airway lining can

occur. For these reasons, our inhibitor design programme sought to identify potent molecules which

possessed the pharmaceutical credentials for delivery by inhalation, together with other attributes

(e.g., selectivity, safety, and endurance) necessary for clinical developability. These compounds have

been named ‘allergen delivery inhibitors’ (ADIs), reﬂecting an important facet of the bioactivity of

Group 1 HDM allergens [2,11,12,22].

In the present paper, we compare the effects of exemplar inhibitors arising from this drug design

programme. Additionally, we describe the properties of a range of novel reagents and analytical

methods that will facilitate further investigations of the Group 1 HDM allergens using approaches

which offer substantial improvements over tool compounds available hitherto.

2. Results

2.1. Degradation of ADZ 50,059 by Group 1 HDM Allergens and Its Inhibition

Initially, we investigated the cleavage of ADZ 50,059 by Der p 1 and Der f 1 to establish whether the

substrate exhibited similar behaviour against Group 1 allergens from two globally-signiﬁcant species

of HDM. We were also interested in comparing the susceptibility of the substrate to a range of other

enzymes of interest. Functional similarity of the allergens was conﬁrmed by their comparable, low

micromolar, KM values (Table 1), consistent with predictions from their aligned amino acid sequences.

ADZ 50,059 was a poor substrate for the serine proteases trypsin and chymotrypsin but, as anticipated

from bioinformatics and computational chemistry, a more effective substrate for off-target human

cysteine peptidases related to Der p 1 and Der f 1. That cathepsin B was less favoured than cathepsins

K and S was also anticipated because of the combined effects of its slightly lower degree of sequence

identity with the HDM allergens (~25% for mature sequence) compared to cathepsins K or S (31–34%)

and differences in the size of substrate binding pockets in these enzymes.

Figure 1A,B shows that the rate of degradation of ADZ 50,059 by Der p 1 was retarded in the

presence of ADZ 50,000 or Compound 3. The ability of ADZ 50,000 to inhibit Der p 1, Der f 1 and

other proteases was quantiﬁed by comparison of pseudo ﬁrst order rate constants (^k^obs). As expected,

[I]

ADZ 50,000 was highly reactive with Der p 1 and Der f 1, but essentially inactive against selected

serine peptidases (Table 2). Against cathepsins B and S it showed limited (<10-fold) intrinsic selectivity

over Der p 1, whereas against cathepsins H, K, and L the difference in reactivity was considerable

(~100–10,000-fold). For comparison, the generic cysteine peptidase inhibitor E-64, which has been used

by others as a Der p 1 inhibitor tool, is signiﬁcantly less reactive with Der p 1 than ADZ 50,000 (^k

(3.4 ± 0.23) × 10⁵M⁻¹s⁻¹) and has little selectivity.

obs

[I]

A snapshot of ADZ 50,000 compared to a range of ‘standard’ protease inhibitors is shown in

Figure 1C which depicts their effects on the cleavage of substrate ADZ 50,059 by a natural mixture of

HDM allergens (i.e., containing both cysteine and serine peptidase activity). Most of the proteolytic

activity was inhibited by ADZ 50,000, whereas E-64 was only a moderate inhibitor. While we did not

elect to ensure that either compound completely titrated the catalytic sites of Der p 1 in the mixture,

their relative performance in this assay is consistent with pseudo ﬁrst order kinetic rate constant

measurements reported above. Antipain and chymostatin, which discriminate poorly between serine

and cysteine proteases, were effective inhibitors of substrate cleavage, whereas TLCK and TPCK,

which show marginally better preference but not clear selectivity for serine proteases, were less

effective and were augmented by ADZ 50,000 but not E-64.

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Figure 1. Proteolysis of ADZ 50,059 by Der p 1 or mixed HDM allergens and its inhibition. (A) Progress

curves for cleavage by Der p 1 in the absence or presence of a range of concentrations of ADZ 50,000.

) Inhibition of Der p 1 by Compound . In both ( ) and ( ), the grey lines show data in the absence of

added enzyme. Progress curves are depicted as a single data set which was replicated twice on a single

96-well plate and >3 times for each compound on separate occasions. ( ) Proteolysis of ADZ 50,059 by

mixed HDM allergens containing 40 nM Der p 1 and the effects of inhibitors tested at the following

concentrations: ADZ 50,000 (50 M); E-64 (200 M); antipain (200 M); TLCK (1 mM); chymostatin

(50 M); TPCK (50 M). Data are mean s.e. (n = 4). ( ) Example progress curves for the cleavage

of N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide by chymotrypsin (green triangles) and its modiﬁcation

by 400 M AEBSF (purple squares) or 40 M ADZ 50,000 (blue squares). Corresponding data are

presented for native Der p 1 (black circles) with AEBSF (orange circles) or ADZ 50,000 (magenta

triangles) showing that this substrate is insensitive to pure Der p 1. ( ) Example initial rates of

reaction as determined from an experiment similar to that depicted in ( ) conﬁrming the resistance

to degradation of the chymotrypsin substrate and the lack of inhibitory activity of ADZ 50,000 on

chymotrypsin. ( ) Processing of N-Bz-Phe-Val-Arg-p-nitroanilide by trypsin (blue), native Der p 1

(green), and recombinant Der p 1 (yellow) and the effects of 80 µM AEBSF or 8 µM ADZ 50,000.

(B

4

A

B

C

µ

±

D

µ

E

D

F

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Although not optimised for selectivity, it is clear that ADZ 50,000 lacks the indiscriminate

behaviour of common inhibitor tools and highlights the limitations of those compounds in the study

of HDM protease allergens. As shown in Figure 1D,E it does not inhibit the initial degradation of

N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide by chymotrypsin, whereas AEBSF is, as expected, a potent

inhibitor. In contrast, Der p 1 is inactive against this substrate (Figure 1D,E). Figure 1F illustrates the

degradation of N-Bz-Phe-Val-Arg-p-nitroanilide by trypsin which is strongly inhibited by AEBSF but

not by ADZ 50,000. This substrate is also degraded by both native Der p 1 and recombinant Der p 1 in

reactions which are insensitive to AEBSF but fully attenuated by ADZ 50,000. These experiments show

that despite a lack of formal optimisation and having a mechanism which involves an electrophilic

attack on the catalytic residue, ADZ 50,000 discriminates well between its intended Der p 1 target and

serine peptidases. Furthermore, there is no serine protease activity associated with Der p 1.

Table 1. Measured KM values for ADZ 50,059 against Der p 1 and a panel of cysteine and

serine peptidases.

Enzyme

KM (µM)

Der p 1

Der f 1

Cathepsin B

Cathepsin L

Cathepsin S

Trypsin

15.4 ± 2.1

29.0 ± 2.6

40.9 ± 4.5

8.0 ± 1.3

12.3 ± 1.8

46.6 ± 2.9

132.1 ± 22.7

Chymotrypsin

Data are mean ± s.e. mean from 4 determinations.

Table 2. Measured activity of ADZ 50,000 against Der p 1, Der f 1, and a panel of off-target cysteine

and serine peptidases.

−1 −1

)

Enzyme

K_obs/[I] (M

s

2 × 10⁸

Der p 1

Der f 1

1.9 × 10⁸

3.7 × 10⁷

2.1 × 10⁷

1.7 × 10⁶

1.5 × 10⁶

1.6 × 10⁴

Cathepsin B

Cathepsin S

Cathepsin L

Cathepsin H

Cathepsin K

Thrombin

inactive at ≤50 µM

Trypsin

Chymotrypsin

2.2. Effects of Der p 1 and Der p 2 on Barrier Properties of Human Airway Epithelial Cells and Its Modulation

by ADZ 50,000

At conﬂuence, Calu-3 cells developed an appreciable TER (>1000 Ohms.cm²) consistent with

the well-established properties of these cells [24]. Application of Der p 1 to the apical side of the

monolayers signiﬁcantly reduced TER, whereas Der p 2 in greater molar equivalence neither had any

effect itself nor altered the response to Der p 1 (Figure 2A). Pre-treatment of Der p 1 with ADZ 50,000

fully prevented the reduction in TER in monolayers treated with either Der p 1 alone or a combination

of Der p 1 and Der p 2 (Figure 2A).

Addition of Der p 1 to conﬂuent monolayers of Calu-3 cells with a well-developed TER was

associated with transepithelial passage of the allergen such that it was detectable in the basolateral

medium in the Transwell

™ (Figure 2B). Consistent with the inability of Der p 2 to inﬂuence the

TER changes evoked by Der p 1, the extent of Der p 1 transepithelial permeation was unaffected by

Der p 2 (Figure 2B). However, addition of ADZ 50,000 signiﬁcantly reduced the amount of Der p 1

recoverable in the basolateral medium (Figure 2B). In cells treated with Der p 2 alone, a small amount of

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immunoreactive material was detected in the basolateral compartment but this ﬁnding was similar to

data from untreated control cells, suggesting that it may be due to cross-reactive material released from

the cells (Figure 2C). Der p 2 only became detectable in the basolateral compartment in appreciable

amounts when mixtures of Der p 1 and Der p 2 were added to the apical side of the chamber, and this

response was signiﬁcantly reduced by ADZ 50,000 (Figure 2 C). These effects mirror the action of

Der p 1 on TER and its own transepithelial disposition. The tendency to a larger ﬂux of Der p 2 reﬂects

the fact that it was present in excess over Der p 1 and that as a smaller molecule, and in the absence of

other factors, its permeability would be expected to be greater than that of Der p 1.

Figure 2. Effect of ADZ 50,000 on TER and the transepithelial disposition of Der p 1 and Der p 2 in

the Calu-3 human airway epithelial cell line. (

A) Contrasting actions of Der p 1 (2

µM) and Der p 2

(2.8 M) on TER and the inhibition of Der p 1-mediated effects by ADZ 50,000 (1

µ

µM). * p < 0.001,

†

p < 0.01 vs. vehicle control. Vehicle comprised serum-free EMEM containing 5 mM cysteine.

(

B) Immunoreactive Der p 1 (i-Der p 1) detected in the basolateral chamber of Transwells after 2 h

incubation with apically-applied Der p 1, Der p 2 or Der p 1 with Der p 2 and the effect of ADZ 50,000.

* p < 0.001 vs. vehicle control; p < 0.01 vs. Der p 1 and Der p 2 in the absence of ADZ 50,000. ( ) as

), but measuring i-Der p 2, * p < 0.01 vs. vehicle control; p < 0.01 vs. Der p 1/Der p 2 mixture.

) Detection of contaminant serine peptidase activity in a commercial preparation of native Der p 2

M). Data are mean s.e. mean on 4 replicate tests on a single

batch of allergen using N-Bz-Phe-Val-Arg-p-nitroanilide as substrate. * p < 0.05. ( ) measurement of

†

C

(

B

D

†

and its inhibition by AEBSF (100

µ

±

E

basolateral i-Der p 1 after the application of a mixture of Der p 1 and Der p 2 to the apical surface of

calu-3 cells showing that AEBSF has an inhibitory effect on the serine peptidase contaminant associated

with Der p 2. * p < 0.001 vs. vehicle control;

AEBSF. In ( ) and in ( ), data are shown as values from individual experiments with the mean

s.e. indicated by the cross-hair and whiskers.

† p < 0.001 vs. Der p 1 and Der p 2 in the absence of

A–

C

E

±

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In passing, we observed that some batches of commercially-sourced Der p 2 were associated

with protease activity which could be inhibited by treatment with AEBSF (Figure 2D). As Der p 2

is not proteolytic per se, this most likely reﬂects the presence of contaminants with the potential to

confound investigations of the bioactivity of Der p 2. An example of this is shown in Figure 2E where

the transepithelial ﬂux of Der p 1 evoked by a mixture of Der p 1 and Der p 2 was partially inhibited

by AEBSF, which is not an inhibitor of Der p 1 (Figure 1F).

2.3. Cleavage of CLD1.1 by Der p 1

Our next aim was to study the susceptibility of the ﬁrst extracellular domain of claudin 1 to

cleavage by Der p 1. This domain was chosen because claudin 1 is expressed in the airways and is

a useful representative of claudins with a ‘sealing type’ function [12]. Incubation of CLD1.1 with

Der p 1 resulted in the generation of multiple peptide fragments which are summarised in Table 3.

These sites are well conserved in the claudin repertoire of human lung. Unexpectedly, inspection

of the fragments generated revealed similarities to a facile cleavage site identiﬁed by us in occludin

(viz. Leu-Leu) [14,25] suggesting that it could be cleavage hot spot in both families of TJ adhesion

protein. Formation of all claudin cleavage products was prevented by inactivation of Der p 1 with

ADZ 50,000, but not by its treatment with AEBSF.

Table 3. Cleavage sites for Der p 1 identiﬁed in CLD1.1. Peptides identiﬁed by LC-MS-MS were used to

identify positional cleavages indicated by single letter amino acid notation in the ﬁrst column. Implied

0

subsite speciﬁcity of Der p 1 is indicated for positions S₄–S₄. Similarity with potential cleavage sites

in the ﬁrst extracellular domain of occludin are highlighted in yellow. Grey and blue shading denote

non-prime and prime subsite interactions of Der p 1 relative to the cleavage site (Schecter and Berger

nomenclature).

0

S₄

TJ Loop and Identiﬁed Cleavage

CLD1.1

S₄

S₃

S₂

S₁

S₂

S₃

SLLN.LSST

FDSL.LNLS

LSST.LQAT

IVTA.QAMY

IQCK.VFDS

RIYS.YAGD

CVSQ.STGQ

VTAQ.AMYE

Ser

Phe

Leu

Ile

Leu

Asp

Ser

Val

Gln

Ile

Leu

Ser

Thr

Cys

Tyr

Ser

Ala

Asn

Leu

Thr

Ala

Lys

Ser

Leu

Gln

Val

Tyr

Ser

Ala

Ser

Asn

Gln

Ala

Phe

Ala

Thr

Met

Ser

Leu

Ala

Met

Asp

Gly

Tyr

Thr

Ser

Thr

Tyr

Ser

Asp

Gln

Glu

Ile

Arg

Cys

Val

Thr

Gln

Occludin 1.1

GTSL.LGGS

Gly

Thr

Ser

Leu

Gly

Ser

2.4. Cytokine Release from Airway Epithelial Cells and Its Modulation By ADZ 50,000

We next investigated the ability of Der p 1 to evoke the release of IL-6 and IL-8 from airway

epithelial cells and the effect of ADZ 50,000 (Figure 3 A–C). The choice of the A549 cell line for these

experiments was informed by literature precedent which had established them as a basis for the

investigation of cytokine release by Der p 1 [26]. However, the release of both cytokines showed

large variation between experiments, with some cells being refractory to activation whereas others

were not. The reasons for this separation into responders and non-responders are unknown but are

not related to the proteolytic competence of the Der p 1 preparations. Under identical conditions,

Der p 2 was inactive in all tests and was similarly lacking in efﬁcacy when other treatment protocols

were evaluated in both A549 and calu-3 cells. Thrombin and trypsin, representative activators of

PAR-1/PAR-4 and PAR-2, respectively, elicited no detectable change in IL-8 release. However, IL-6 was

detectable after addition of thrombin (Figure 3 A), implying the existence of PAR-1/PAR-4-dependent

signal transduction in its release. Similar results to those described above were obtained when cells

were incubated with these enzymes for up to 24 h and when studies were performed in calu-3 cells.

Despite the variation in cytokine release overall, those experiments which were characterised by

Int. J. Mol. Sci. 2018, 19, 3166

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responder cells enabled us to examine whether these effects were dependent on Der p 1. Figure 3C

shows the inhibitory effect of ADZ 50,000 on the release of IL-6 and IL-8 for cell passages identiﬁed as

cytokine responders, consistent with the protease activity of Der p 1 in enabling their release.

Figure 3. Release of IL-8 or IL-6 from cultured A549 cells and the effect of ADZ 50,000. (A,B) Release

of IL-8 or IL-6 following stimulation by Der p 1 (20 nM), Der p 2 (14 nM), TPCK-treated trypsin

(9 nM), or thrombin (0.5 U/mL) all applied apically. Cells were exposed for 8 h to serum-free EMEM

containing the treatments, supplemented with 5 mM cysteine in the case of Der p 1. Cells were then

washed and incubated with serum-free EMEM alone for a further 16 h prior to collection of samples.

Data are shown as results from individual experiments with cells of different passage number. * p < 0.05

vs. vehicle treatments in experiments where cells were designated as responders. (

50,000 treatment on cytokine release (IL-8, black; IL-6, red) elicited by Der p 1 in designated responder

experiments. Data from individual experiments are shown. * p < 0.05, p < 0.01 vs. corresponding

C) Effect of ADZ

†

inhibitor-free control. In all cases, cross-hairs and whiskers denote mean ± s.e. mean, respectively.

2.5. Lung Function and Cell Recruitment Studies in Sensitized Rats

In brown Norway rats, sensitisation to mixed HDM allergens produced variable effects on

post-study total serum IgE compared to the vehicle-treated group (Figure 4A) but there were

consistent elevations in serum allergen-speciﬁc IgE and IgG_2acompared to the vehicle-treated group

(Figure 4B,C).

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Figure 4. Effects of allergen delivery inhibitors on airway responses to either an extract of mixed HDM

allergens or Der p 1 in brown Norway rats. ( ) Post-sensitisation serum concentrations of IgE,

allergen-speciﬁc IgE and allergen-speciﬁc IgG_2ain animals assigned to the treatment groups for the lung

function investigations performed in ( ). Assignment to drug treatment was made blind with respect

to these data which were obtained after the conclusion of the lung function studies. * p < 0.001–0.05

vs. control animals assigned to sham sensitisation with vehicle (shown in grey). ( ) Changes in

airway resistance following challenge of HDM-sensitised animals with an i.t. aerosol of mixed HDM

allergens (10 g Der p 1 equivalent) or vehicle. Animals were treated with ADZ 50,000 ( 10 molar

ratio to Der p 1), Compound 250 molar ratio to Der p 1), Compound 200 molar ratio to

Der p 1), Compound 8 molar ratio to Der p 1) prior to HDM challenge. * p < 0.05 vs. control

animals assigned to sham sensitisation and challenge with vehicle (data shown in grey). p < 0.01–0.05

vs. challenged with mixed HDM allergens. ( ) Changes in airway resistance to acetylcholine and

adenosine, respectively, measured 1 h after allergen challenge in the six treatment groups. ( ) BAL

cell proﬁles and BAL ﬂuid recovery in animals sensitised to mixed HDM allergens and challenged

with Der p 1 (10 g in 100 L) with or without prior treatment with ADZ 50,000 ( 2.5 molar ratio to

Der p 1) or Compound 8 molar ratio to Der p 1). * p < 0.01–0.05 vs. control animals assigned to

sham sensitisation and challenge with vehicle or vehicle challenge in animals sensitised to mixed HDM

(data shown in grey). p < 0.05 vs. animals challenged with Der p 1. Data are shown as results for

individual animals with mean ± s.e. indicated by cross-hair and whiskers.

A–C

D–F

D

µ

×

1

(

×

2

(

×

3

(

×

†

E,F

G–J

µ

×

1

(

×

†

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10 of 34

Challenge of sensitised rats with an intratracheal aerosol of mixed HDM allergens (HDM 10)

evoked an acute increase in lung resistance (Figure 4D) which returned to baseline within 60 min.

This effect of HDM challenge was signiﬁcantly greater than the response to vehicle seen in sensitised

animals. In contrast, lung resistance changes in animals challenged with HDM allergens treated with

ADZ 50,000 or Compounds 1, 2, and 3, were signiﬁcantly reduced compared to the control challenge

with HDM 10 (Figure 4D). As a further component of the study, we investigated whether challenge

was associated with a change in airways reactivity to ACh or Ado. Although both stimuli suggested a

tendency to increased airways reactivity 1 h after HDM challenge, these effects were not signiﬁcant

when compared to sensitised animals challenged with vehicle due to the high level of variability in

responses. A similar pattern was evident in animals challenged simultaneously with HDM allergens

and ADI compounds (Figure 4E,F). There was no change in airways reactivity to ACh or Ado in

animals tested 24 h after HDM allergen challenge.

In the next study, we investigated the effect of two ADI compounds on the Der p 1-dependent

recruitment of cells to the airways of rats sensitised to the full extract of HDM allergens. Our previous

investigations had shown that the optimum time for monitoring the eosinophil component of this

response, in which we were most interested, was 48 h following aerosol delivery of the allergen

challenge. Electively, we therefore performed BAL at this time after challenge. The total number of

nucleated cells in recovered BAL ﬂuid was increased in animals challenged with Der p 1 compared

to vehicle-challenged, HDM-sensitised rats, or naïve animals receiving only the BAL procedure.

This response was partially attenuated in animals challenged with Der p 1 that had been treated with

either ADZ 50,000 or Compound

3 (Figure 4G). A similar outcome was seen when eosinophil numbers

were considered, with their recruitment signiﬁcantly inhibited by the ADIs (Figure 4H). In contrast,

the neutrophil recruitment response was not attenuated by ADIs (Figure 4I), but it should be noted

that the cell sampling time in this protocol is not optimal for neutrophils whose airway inﬁltration

and its resolution is more rapid than that of eosinophils. The number of macrophage/monocytes was

similar regardless of the treatment group assigned. Figure 4 J conﬁrms that the recoveries of BAL ﬂuid

were comparable in the study groups.

2.6. Cell Recruitment Studies in Mice

In the ﬁrst of these we investigated the effect of ADZ 50,000 on the development of allergic

sensitisation to Der p 1. As shown in Figure 5A, post-study levels of total serum IgE were elevated

compared to the vehicle-treated mice. In contrast, animals receiving Der p 1 treated with ADZ 50,000

had serum IgE levels like the control group. A similar pattern was seen when levels of Der p 1-speciﬁc

IgE were measured (Figure 5 B). However, the Der p 1-dependent increase in allergen-speciﬁc IgG₁

was not affected by pre-treatment of Der p 1 with ADZ 50,000 (Figure 5 C). Unlike Der p 1, Der p 2

administered alone failed to produce an elevation in total serum IgE (Figure 5A). A further aspect of

this study was to test the prophylactic effect of ADZ 50,000 on mice being sensitised to the full extract

of HDM allergens. As shown in Figure 5D, the presence of ADZ 50,000 during sensitisation prevented

elevation of total serum IgE and the development of allergen-speciﬁc IgE to any HDM allergen

(Figure 5E). The additional presence of the serine peptidase inhibitor produced no effect beyond that

of ADZ 50,000 alone (Figure 5D,E). Like the study in brown Norway rats (Figure 4), we found that the

assay for allergen-speciﬁc IgE and IgG₁(or IgG_2ain the case of rats) detected a variable background

in vehicle-treated animals, but in spite of this it was possible to distinguish the effect of sensitisation.

We do not believe the background immunoreactivity represents prior sensitisation because the mice

used in all these studies were all fully isolator-maintained and, moreover, allergen challenge did not

evoke obvious allergic responses in these control animals.

Int. J. Mol. Sci. 2018, 19, 3166

11 of 34

Figure 5. Prophylactic action of ADZ 50,000 against sensitisation to Der p 1 or an extract of mixed HDM

allergens. ( ). Serum concentrations of IgE, allergen-speciﬁc IgE and allergen-speciﬁc IgG₁in Balb/c

mice following intranasal immunisation (10 L/head) with vehicle, Der p 1 or Der p 2 and the effect of

ADZ 50,000 on responses to Der p 1. * p < 0.001–0.05 vs. control animals assigned to sham sensitisation.

p < 0.01 vs. animals sensitised with Der p 1 (5 g/head). ( ) Total and HDM allergen-speciﬁc

serum IgE in Balb/c mice immunised intranasally with vehicle or mixed HDM allergens (normalised

to 5 g Der p 1) and the effect of co-administered ADZ 50,000 and AEBSF formulated in a 20:1 and

A–C

µ

†

µ

D,E

µ

25:1 molar ratio to Der p 1 content. * p < 0.001–0.01 vs. control animals assigned to sham sensitisation.

† p < 0.001–0.01 vs. animals sensitised with mixed HDM allergens. In all cases, data are shown as

results for individual animals with mean ± s.e. indicated by cross-hair and whiskers.

In the next study, we evaluated the effect of another ADI, Compound 4, on the development of

allergic sensitisation by comparing pre-immune and post-sensitisation IgE levels in individual animals.

Figure 6A,B shows that intratracheal aerosol delivery of mixed HDM allergens in the absence of any

added adjuvant increased total serum IgE and resulted in the development of allergen-speciﬁc IgE.

However, inclusion of the ADI compound in the immunisation mixture prevented both responses,

whereas the development of allergen-speciﬁc IgG₁was unaffected by ADI treatment (Figure 6C).

Int. J. Mol. Sci. 2018, 19, 3166

12 of 34

Figure 6. Prophylactic action of Compound

4 against i.t. aerosol sensitisation to an extract of

mixed HDM allergens and its inhibition of acute allergic and innate cell recruitment in C57BL/6

mice challenged with mixed HDM allergens (25 L, i.t., 10 g equivalent Der p 1). ( ) Pre- and

post-immune serum concentrations of IgE, HDM allergen-speciﬁc IgE and allergen-speciﬁc IgG₁in

mice with and without ADI treatment (Compound , 10:1 molar ratio to Der p 1) during the intranasal

sensitisation protocol. Responses for individual animals are shown. ( ) BAL cell proﬁles in C57BL/6

mice sensitised to mixed HDM allergens following challenge with vehicle or mixed HDM allergens

and the modulating effect of Compound . The ADI was administered (25 L at 40 M, i.t.) 120 min

prior to i.t. allergen provocation (25 L, i.t.). Test compound was dosed at a 10:1 molar ratio with

respect to Der p 1. The mixed HDM preparation contained a dose of 10 g Der p 1. * p < 0.001–0.01

vs. vehicle challenge; p < 0.01 vs. HDM challenge. ( ) BAL leukocyte proﬁles in unsensitised

C57BL/6 mice following i.t. challenge with vehicle or mixed HDM allergens and the modulating

effect of ADI Compound on the innate recruitment of leukocytes. The ADI was administered (25

at 40 M, i.t.) 15 min prior to i.t. allergen provocation. * p < 0.001, p < 0.05 vs. vehicle challenge;

† p < 0.001–0.05 vs. HDM challenge. ( ) BAL ﬂuid returns in the study groups. * p < 0.01–0.05 vs. other

µ

A–C

4

D–F

4

µ

†

G–I

4

µL

µ

‡

J

treatments. Data for individual animals are shown in (

s.e. mean, respectively.

D–

J

). Cross-hairs and whiskers denote mean

±

Int. J. Mol. Sci. 2018, 19, 3166

13 of 34

We then investigated the recruitment of inﬂammatory cells to the airways in mice sensitised to

mixed HDM allergens following challenge with the same allergens. The primary purpose was to

discover how these responses were affected by treatment with Compound 4 given 2 h before HDM

allergen provocation. In mice challenged with HDM allergens administered by i.t. aerosol there

was a signiﬁcant increase in the numbers of nucleated cells recovered by BAL compared to animals

which had been sham-treated with vehicle alone. This was most marked in mice actively sensitised to

HDM allergens. Pre-treatment with Compound 4 prior to challenge attenuated the cell recruitment

(Figure 6 D). These overall effects were reﬂected when speciﬁc effects on eosinophils and neutrophils

were considered, with the allergen-dependent recruitment of both being signiﬁcantly inhibited by the

single dose of Compound 4 (Figure 6E,F).

An interesting feature evident in Figure 6D–F was the ability of the mixed HDM allergens to

evoke cellular responses in sham-sensitised animals that were not evident in mice challenged with

the vehicle. We therefore investigated whether pre-treatment with Compound

4 inﬂuenced these

innate responses also. As depicted in Figure 6 G–I there were signiﬁcant reductions in the overall

recruitment of cells, and speciﬁcally eosinophils and neutrophils. The ADI compound itself had no

effect on baseline cell numbers in BAL ﬂuid in unchallenged animals (Figure 6 G–I). Recovery of BAL

ﬂuid in the treatment groups comprising the studies depicted in Figure 6 were generally consistent

except in the case of sensitised animals that were HDM challenged in whom ﬂuid return was reduced

(Figure 6J).

To explore whether the inhibitory effects of ADI compounds on cell recruitment were due to a

direct effect on Der p 1, as intended by the drug design campaign, we conducted a further study in

mice sensitised to and challenged with OVA. As shown in Figure 7A,B, Compound 5 failed to attenuate

the cell recruitment response following acute challenge with OVA in monosensitised mice, consistent

with exemplar ADI compounds exerting their beneﬁcial effects in HDM challenge by selective action

on Der p 1.

Figure 7. An allergen delivery inhibitor directed against Der p 1 (Compound

responses to OVA in OVA-monosensitised Balb/c mice. (

) Recovery of CCR3⁺eosinophils in BAL

ﬂuid of OVA-challenged animals (10 g, i.t. aerosol) treated 2 h prior to OVA with saline vehicle or

Compound (50 g/kg, i.t.) ( ) Corresponding data for dendritic cells. Cross hairs and whiskers show

mean ± s.e. mean. * p < 0.05 vs. control group of unsensitised animals.

5) does not affect

A

µ

5

µ

B

Int. J. Mol. Sci. 2018, 19, 3166

14 of 34

3. Discussion

This paper presents novel insights into the pharmacology of protease inhibitors designed to target

Group 1 allergens of HDM. We refer to this diverse class of compounds as allergen delivery inhibitors,

although it will be clear from the ﬁndings presented herein that the scope of their pharmacological

proﬁle is signiﬁcantly broader than implied by that name. Using exemplar ADIs from four distinct

chemotypes and with differing mechanisms of inhibitory action, our studies reveal signiﬁcant

modulation of events ranging from innate immune responses to the recruitment of inﬂammatory

cells to the airways following acute challenge with a mixture of HDM allergens. Unexpectedly,

one feature of acute treatment with chemically diverse ADI compounds was the inhibition of acute

allergic bronchoconstriction. These actions arise from a designed interaction with the Group 1 HDM

allergen target and not through speciﬁc immunosuppression or bronchodilatation.

The importance of protease activity to allergenicity and the progression of allergic disease is

supported by a growing range of evidence which may be brieﬂy summarised thus: (i) facilitation of

inhalant allergen detection by antigen presenting cells; (ii) activation of signal transduction mechanisms

which drive innate immune responses to break tolerance and promote the acquisition of allergic

sensitisation; and (iii) more chronically, activation of IgE-independent mechanisms which result in

cellular transitions and airways pathophysiology of variable reversibility. In the case of (i) and (ii),

it would be appropriate to consider these actions as being forms of adjuvant behaviour. These topics,

speciﬁcally the target validation case for Group 1 HDM allergens, have been the subject of recent

review [2,11,12].

The compounds used in these experiments are directed against Group 1 HDM allergens. As these

are inhalant allergens which evoke respiratory symptoms it is therefore appropriate to consider

inhalation of inhibitor drugs as a preferred route of delivery for asthma therapy. Accordingly,

compounds produced in this discovery campaign were required to comply with ‘inhalation by design’

criteria during lead optimisation [2,22]. However, this is not to ignore non-respiratory effects of

these allergens and compounds have been identiﬁed which may be developable for systemic use and

therapeutic indications beyond asthma [22]. For asthma, because the inhibitory engagement between

drug and the allergen target occurs at the luminal face of the airway epithelium, one of several factors

relevant to this interaction is the composition of airway surface liquid. Airway surface liquid is rich in

reducing agents, principally glutathione, which provides a means to maximise the proteolytic activity

of the allergen. The pH of airway surface liquid is estimated to be in the range 6.8–7.6 [27–29] which

is optimal for Der p 1 [30]. In severe asthma, exhaled breath condensate measurements suggest that

pH may be reduced [31], but not below levels likely to materially impair enzymatic activity of Der p 1

based on assessment of its pH optimum. In fact, any such acidiﬁcation would be expected to augment

the protease-dependent effects of Der p 1 mediated through the generation of reactive oxygen and

nitrogen species [2,32].

Using two chemically and mechanistically distinct ADIs, we showed that inhibition of Der p 1

protease activity attenuated the development of allergic sensitisation. The signiﬁcance of adjuvancy

provided by the protease activity of the allergen was underscored in the study using Compound

4

where mice were sensitised and challenged with the full spectrum of HDM allergens. These data clearly

demonstrate that Der p 1 is an enabler/initiator of allergy to proteins of diverse structure-function.

Previous studies have shown that Der p 1 facilitates sensitisation to ovalbumin and that the potentiation

of allergenicity is dependent on the protease activity of Der p 1 [4,5,33]. Although capable of initiating

IgE synthesis, ovalbumin differs markedly from Der p 1: The former is a comparatively effete allergen

which requires an adjuvant to overcome tolerance and provide a means to cross epithelial barriers

when delivered to the airways. The clear implication of our data is that protease activity of Group 1

HDM must act similarly for unrelated allergens of mite origin. Analogous ﬁndings have been made

in fungi with Aspergillus species where protease-containing extracts of A. fumigatus or A. oryzae

convert tolerogenic reactions to ovalbumin into allergic responses and, more speciﬁcally where the

serine protease allergen Asp f 13 facilitates the development of allergy to the non-protease allergen

Int. J. Mol. Sci. 2018, 19, 3166

15 of 34

Asp f 2

[

3,34

,35]. Given the extent and diversity of the HDM allergen repertoire, and considering that

all allergens generally fall within <2% of protein families [

1

], it can be inferred that the adjuvant effect

of Group 1 HDM allergens on the overall sensitisation to HDM allergens is a microcosm of their ability

to promote allergic responses to allergens of non-HDM origin. Moreover, the signiﬁcance of HDM

as allergenic sources indicates that HDM protease allergens are well justiﬁed as exemplars of those

allergens which enjoy exclusivity as initiators [8,10].

Whereas inhibition of the protease activity of Der p 1 attenuated the development of

allergen-speciﬁc IgE and the recruitment of eosinophils following allergen challenge, it was notable

that in mice neither ADZ 50,000 nor Compound 4 prevented the development of allergen-speciﬁc IgG₁.

While this may initially seem counter to the concept that Der p 1 cleaves interepithelial junctions to

increase the probability of allergen detection by DCs, there are two important considerations which

have a bearing on these studies. Firstly, to minimise off-target effects of non-optimised test compounds,

animals were electively treated with doses incapable of completely inhibiting the amount of Der p 1

target delivered to the airways and so some immunological response could be anticipated. Secondly,

the serine peptidase allergen components of the HDM allergen repertoire were not targeted and these

are known to contribute to allergen delivery through an ability to cleave TJ adhesion proteins [25].

An inference from these studies is that the cysteine peptidase activity of Der p 1 is central to

the initiation of allergy because its bioactivity drives both allergen delivery and the polarisation

of immune responses towards IgE production. The unrestrained contribution from HDM serine

peptidase allergens is, however, insufﬁcient to drive these responses when Der p 1 is inhibited,

because endogenous serine peptidase inhibitors which are known to be vulnerable to degradation

by Der p 1

Other than providing a biosignature of allergen exposure, understanding the functional consequence

of elevated allergen-speciﬁc IgG₁is complex because mouse IgG₁signals through both CD16 (Fc RIII),

a stimulatory molecular recognition system, and CD32B (Fc RIIB), which is inhibitory, and both are

widely expressed on murine haematopoietic cells [38 39]. Nevertheless, and regardless of the nuanced

[36,37] provide protection against them when the activity of Der p 1 is inhibited.

γ

,

effects of IgG₁, our data demonstrate that inhibition of Der p 1 evidently associates with a prophylactic

reduction in the development of IgE on allergen exposure and usefully attenuates hallmark allergic

effector responses when allergic sensitisation has already been established.

Aerosol exposure of rats or mice to allergen challenge elicited an inﬂammatory cell response

which was observed by sampling of BAL ﬂuid. Pre-treatment with ADZ 50,000 or Compounds

3 or 4

attenuated the response to Der p 1 or mixed allergens, conﬁrming their efﬁcacy against the puriﬁed

target and the more realistic, tougher, test posed by the allergen mixture. These experiments were

undertaken with a sampling time which was optimised for the study of eosinophil recruitment, but it

was pleasing to see that neutrophil recruitment was also inhibited.

The mechanism(s) by which ADIs inhibit the acute bronchoconstriction resulting from challenge

with mixed HDM allergens is presently unclear. Of potential relevance is that Der p 1 degranulates

human mast cells in a protease-dependent, IgE-independent manner [40]. The basis of this effect

remains unknown but, interestingly, mast cells express the orphan receptor MRGPRX1 of the

mas-related G-protein coupled receptor family and in airway epithelial cells this receptor has recently

been suggested as a potential target of Der p 1 [41]. By analogy, the effects of the cysteine protease

mucunain (the active pain-producing substance from cowhage (Mucuna pruriens)), is known to be a highly

mast cell dependent, pseudo-allergic response [12

mas-related G-protein coupled receptors may be the target(s) of these proteases in mast cells [12 ].

The prophylactic effect of protease inhibitors such as Compounds and in preventing

,42 ]. These lines of evidence suggest that one or more

3

4

sensitisation to HDM allergens raises questions about the underlying mechanism. We have

previously presented evidence that small quantities of Der p 1 increase epithelial permeability to

well-characterised marker solutes, suggesting that this would increase the probability of allergen

detection by DCs [14

,25,43,44] and showed that this effect was due, in part, to proteolysis of the

extracellular domain of the TJ adhesion protein, occludin [14]. Using recombinant CLD1.1 as a model

Int. J. Mol. Sci. 2018, 19, 3166

16 of 34

we now demonstrate that claudin family adhesion proteins possess cleavage sites for Der p 1 with

similarities to principal sites identiﬁed in the MARVEL family member, occludin [14]. The choice of

claudin 1 for exempliﬁcation is justiﬁed because it is present in all airway epithelial cells and archetypal

of claudin family proteins which have a ‘sealing function’. Its ﬁrst extracellular domain demonstrates

good (40–70%) identity with the corresponding domains from most other family members (Table 4),

including claudin-18, a non-classical claudin with a lung-restricted expression [15,45,46]. It shares

less identity with those classical claudins (e.g., claudin 10b, claudin 15) which may be categorised as

forming cation-selective components of TJs [12]. Accordingly, the Leu-Leu cleavage site for Der p 1,

which is common to claudin 1 and occludin, is well represented by identity or similarity in the majority

of other claudins; indeed the dyad is well conserved in the claudin repertoire (claudins 1, 3, 4, 5, 7, and

18) (Figure 7) of human bronchial and bronchiolar epithelial cells [47]. However, the cleavage sites

are absent in the other TJ adhesion proteins tricellulin and MARVELD3 which are thought to have

different functions from occludin and claudins (Figure 8).

Consistent with the ability of Der p 1 to attack TJ adhesion proteins we demonstrated that

Der p 1 reduced TER and that this was inhibited by ADZ 50,000. This is anticipated from the

prediction that claudins are cleavage targets for Der p 1 because claudins have a controlling inﬂuence

on electrical resistance, whereas occludin regulates epithelial permeability to non-ionic solutes [48–50].

The transepithelial ﬂux of Der p 1 was inhibited by ADZ 50,000 and this was accompanied by a

reduction in the transepithelial ﬂux of Der p 2, implying that the Group 2 allergen depended on the

activity of Der p 1 to augment its transepithelial passage. In support of this, we noted that Der p 2 had

no effect on TER in calu-3 cells.

A further feature arising from this programme is the characterisation of useful tool compounds

for the investigation of HDM protease allergens. This is important because much previous work has

relied on reagents which lack potency, selectivity, and stability. Compound ADZ 50,059 has been

identiﬁed as a useful substrate for Group 1 HDM allergens offering high sensitivity and good selectivity.

Illustratively, its measured K_Mof 15.4

µM contrasts with 250–280

µM for Boc-Gln-Ala-Arg-AMC,

a substrate previously used to study Der p 1 [30

,36]. Compound ADZ 50,000 is a potent irreversible

inhibitor of Group I HDM allergens which other investigations performed by us show to be a useful

active site titrant for the quantiﬁcation of functional active site concentration. To date, E-64 has been

the principal inhibitor used to investigate the protease activity of Der p 1, but as shown here its broad

spectrum of actions against other enzymes, including some serine peptidases, and only moderate

reactivity with Der p 1 limit its utility. The novel reagents used by this study in tandem with a

range of other inhibitors provides an emphatic functional rebuttal to the suggestion that Der p 1 is a

multifunctional protease with both cysteine and serine peptidase activity [36

claim has been confusion in the literature and consequent uncertainty about the meaning of data in an

unknown number of studies beyond those reporting enzyme multifunctionality [36 51]. This rebuttal is

the ﬁrst involving direct functional data with a selective active site titrant and supports key inferences

drawn when the crystal structure of Der p 1 was solved [52 53]. It seems likely that the reports of

,51]. A consequence of this

,

multifunctionality were consequences of using an inadequately puriﬁed protein. The easy potential

for impurities in allergen preparations to lead to erroneous conclusions was highlighted in the present

study where contamination of Der p 2 by proteases was shown to affect its transepithelial disposition

and is reminiscent of other work in which a particularly intransigent serine protease contaminant was

found associated with the major cat allergen, Fel d 1 [54].

In summary, the exemplar ADI compounds described in this study produce effects which suggest

that an allergen-directed, inhalable small-molecule can achieve a broad range of beneﬁts. The essential

key to achieving this spectrum of desirable effects is the identiﬁcation of a therapeutic target which

belongs to an exclusive cadre of initiator allergens. The combination of a non-human target and an

inhalable inhibitor with root cause intervention potential offers a future line of attack on the Th2 high

asthma phenotype and related allergic diseases using a strategy which has a conceptually simple basis

and an attractive safety proﬁle.

Int. J. Mol. Sci. 2018, 19, 3166

17 of 34

Table 4. Similarity matrix for ﬁrst extracellular domains of human claudin (CL) family adhesion proteins created using Clustal Omega. Grey shading indicates

claudins expressed in human bronchi and bronchioles. Note that: (i) some evidence indicates that claudin-7 may be an anion channel-forming rather than a sealing

type claudin; (ii) the similarity data for claudin 10 are based on the canonical claudin 10b sequence; (iii) the data for claudin 18 are based on the canonical sequence of

the A1 isoform (UniProt: P56856).

CL

1

2

3

4

5

6

7

8

9

10

11

12

14

15

16

17

18

19

20

21

22

23

25

1

2

3

4

5

6

7

8

9

10

11

12

14

15

16

17

18

19

20

21

22

23

25

100

43

61

62

57

60

70

51

60

37

29

24

49

38

26

42

44

64

38

34

25

30

43

100

45

42

49

47

40

49

35

29

22

58

30

26

36

42

55

40

24

19

26

61

45

100

94

67

80

59

71

80

44

32

25

61

42

31

63

42

57

45

40

24

31

62

45

94

100

74

85

55

68

83

42

35

28

62

40

34

58

46

53

43

38

25

34

57

42

67

74

100

72

60

57

70

40

37

24

53

36

34

43

40

49

45

36

25

30

60

49

80

85

72

100

53

66

98

40

35

26

59

32

53

42

57

43

40

23

34

70

47

59

55

60

53

100

49

53

42

33

26

49

38

32

43

42

60

45

36

25

28

51

40

71

68

57

66

49

100

66

33

31

28

49

32

34

70

37

45

32

40

25

32

60

49

80

83

70

98

53

66

100

40

35

26

58

32

53

40

57

43

40

23

34

37

35

44

42

40

42

33

40

100

35

18

37

64

30

31

29

42

37

30

29

13

28

29

32

35

37

35

33

31

35

100

17

37

24

27

33

32

38

27

26

19

27

24

22

25

28

24

26

28

26

18

17

100

22

23

27

20

27

24

22

21

24

19

49

58

61

62

53

59

49

58

37

22

100

34

45

38

47

32

23

30

38

30

42

40

36

32

38

32

64

24

23

34

100

27

34

28

38

32

22

16

20

26

31

34

32

34

32

30

27

34

27

100

30

25

28

26

32

31

19

26

42

36

63

58

43

53

43

70

53

31

27

20

45

34

30

100

31

47

36

25

32

44

42

46

40

42

37

40

29

33

27

38

28

25

31

100

38

35

28

27

29

28

64

55

57

53

49

57

60

45

57

42

32

24

47

38

28

47

38

100

43

32

27

28

38

40

45

43

45

43

45

32

43

37

38

22

47

32

26

36

35

43

100

24

21

28

34

24

40

38

36

40

36

40

30

27

21

32

22

32

36

28

32

24

100

24

62

34

24

40

38

36

40

36

40

29

26

21

32

22

31

36

27

32

24

100

25

62

25

19

24

25

23

25

23

13

19

24

23

16

19

25

29

27

21

24

25

100

18

30

26

31

34

30

34

28

32

34

28

27

19

30

20

26

32

28

62

18

100

Int. J. Mol. Sci. 2018, 19, 3166

18 of 34

Figure 8.

(A) Multiple sequence alignments of the ﬁrst extracellular domain of claudins (CLD)

expressed in human lung (claudins 1, 3, 4, 5, 7, and 18) and their consensus sequence created

using Clustal Omega and Jalview. Colour shading denotes residues scored for conservation and

quality. Arrows denote cleavage sites for Der p 1 identiﬁed in this study. (B) Sequence alignments

of TJ-associated MARVEL domain adhesion proteins (occludin (OCL), tricellulin (TRI), MARVELD3

(MALD3)). Two isoforms of MARVELD3 are shown. Arrows indicate occludin residues cleaved by

Der p 1 based on partial scanning of the ﬁrst extracellular domain [14]. In both panels, the numbers

appended after the protein identiﬁer indicate the number of residues in each domain. Dyads bounded

by red boxes denote putative cleavage hot spots for Der p 1 and other Group 1 HDM allergens based

on the present study and previous work [14].

4. Methods and Materials

4.1. Materials

Cell culture media and reagents were obtained from Life Technologies, Sigma-Aldrich (Poole,

Dorset, UK), LGC (Teddington, Middlesex, UK), and GE Healthcare, Little Chalfont, Buckinghamshire,

UK. All other materials were sourced as indicated below. Unless otherwise stated, reagents and

solvents for chemical synthesis were readily available from commercial suppliers.

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Analytical LC-MS was performed using a Waters ZQ instrument and purity assessed using a

diode array detector. Chromatography was performed using a Phenomenex Gemini 5.0 3.0 mm,

m, C₁₈column with a ﬂow rate 1 mL/min using solvents A and solvent B according to the following

×

5

µ

gradient: Solvents: A: 0.1% v/v formic acid/water; B: 0.1% v/v form ic acid/MeCN; % B: ramping from

5% B to 95% B between 0 min and 3.5 min and then continuing at 95% B for 3.5–5 min.

Compounds were typically puriﬁed by standard ﬂash column chromatography using BDH silica

gel, purchased from VWR, or using a Flash Master Personal with Isolute SPE cartridges, purchased

from Biotage. Where necessary, reversed phase preparative LC-MS was performed using the Waters

ZQ instrument. The structures and purities of compounds were assigned using analytical LC-MS and

¹H NMR. ¹H NMR spectra were run on a Bruker 400 MHz instrument and spectra were analysed using

MestReC. Chemical shifts are given in ppm and coupling constants (J) are quoted in Hz.

4.2. Synthesis of Der p 1 Substrate and Speciﬁc Inhibitors

(3S,6S,9S,12S,15S,18S)-1-(2-aminophenyl)-9-butyl-18-carbamoyl-15-(4-hydroxy-3-nitrobenzyl)-

12-(hydroxymethyl)-3-isopropyl-6-methyl-1,4,7,10,13,16-hexaoxo-2,5,8,11,14,17-hexaazaicosan-20-oic

acid (ADZ 50,059) was identiﬁed by high throughput screening (HTS) of a positional scanning library

and prepared by solid-phase synthesis using conventional chemistry [55].

The following tripeptides were synthesized on Wang resin using FMoc protected amino acids

and standard amide coupling procedures [55]. They were subsequently used for the synthesis of ADZ

50,000, and Compounds 1 and 2.

ADZ 50,000, Compounds

1 and 2 required the synthesis of the corresponding bromomethyl

ketones as described below (Scheme 1):

To a solution of the corresponding peptide (1 equivalent) in dry tetrahydrofuran (THF)

(2 mL/50 mg of peptide) and dry dimethylformamide (0.2–0.4 mL/50 mg peptide depending upon

amount required to dissolve the peptide) at

−

20 ^◦C (MeOH/ice) was added isobutylchloroformate

(3 equivalents) and N-methylmorpholine (3 equivalents) and the reaction mixture stirred at

◦

−

20 C for 30 min. A solution of diazomethane in diethylether (approx. 6 equivalents; 0.15 M

synthesised from Diazogen, made in accordance with Aldrich Technical Bulletin AL-180 https://ww w .

sigmaaldrich.com/content/dam/sigma-aldrich/docs/Aldrich/Bulletin/al_techbull_al180.pdf) was

added. The reaction mixture was stirred at <0 ^◦C for 1 h then allowed to warm to room temperature

and stirred for a further 1 h. A 1:1 mixture of 50% v/v HBr (aqueous) and acetic acid (9 equivalents,

based on amount of HBr added) was then added and the reaction mixture stirred at room temperature

for 30 min. Note that nitrogen is evolved, and the yellow colour disappears. The reaction mixture was

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subsequently diluted with ethyl acetate and washed with an equal volume of water and a saturated

aqueous solution of NaHCO₃. The organic layer was dried over MgSO₄and evaporated to give the

desired compound as a white solid. This was used without further puriﬁcation to generate ADZ 50,000,

Compound 1 and Compound 2.

Scheme 1. Schematic illustration.

Compounds

1 and 2 were synthesised from the corresponding bromomethyl ketone by stirring

with the requisite amine according to Scheme 2.

Scheme 2. Schematic illustration.

1, an amino ketone, (N-((2R)-1-(((2S)-1-((1-(dimethylamino)-2-oxoheptan-3-yl)amino)-

Compound

1-oxopropan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)benzamide) was prepared via Scheme 2 as

described as follows:

To a solution of the corresponding bromomethyl ketone (1.28 g, 1.5 mmol) in dry THF (60 mL)

under nitrogen, was added a solution of dimethylamine (3.8 mL, 2M in THF) and the reaction stirred

at room temperature for 6 h prior to evaporation. The crude residue was puriﬁed by reversed phased

preparative LC-MS to give the title compound as a 1:1 mixture of diastereomers at the centre adjacent

to the ketone, as its formate salt (269 mg, 36% over two steps); ¹H NMR (400 MHz; d₆-DMSO)

δ 8.72

(1H, d, J = 7.3), 8.68 (1H, d, J = 7.6), 8.43 (1H, d, J = 7.3), 8.41 (1H, d, J = 7.3), 8.18 (2H, s), 8.02 (1H, d,

J = 7.6), 7.99 (1H, d, J = 7.8), 7.84–7.78 (4H, m), 7.55–7.49 (2H, m), 7.48–7.41 (4H, m), 7.38–7.33 (4H, m),

7.30–7.24 (4H, m), 7.21–7.15 (2H, m), 4.69–4.60 (2H, m), 4.36–4.20 (4H, m), 3.45 (1H, d, J = 17.7), 3.30

(1H, d, J = 17.7), 3.22 (1H, d, J = 17.7), 3.15 (1H, d, J = 17.7), 3.09–3.01 (4H, m), 2.15 (6H, s), 2.07 (6H, s),

1.76–1.64 (2H, m), 1.64–1.41 (2H, m), 1.34–1.10 (14H, m), 0.86–0.81 (3H, m), 0.79–0.74 (3H, m); LC-MS

R_t1.81 min; purity 97.6%; MS m/z 495 (MH)⁺.

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Compound 2, also an amino ketone, (N-((2S)-1-oxo-1-(((2S)-1-oxo-1-((2-oxo-1-(pyrrolidin-1-yl)

heptan-3-yl)amino)propan-2-yl)amino)-3-phenylpropan-2-yl)benzamide) was prepared via Scheme 2

as follows:

To a solution of the corresponding bromomethyl ketone (1.16 g, 2.2 mmol) in dry DCM (35 mL)

was added pyrrolidine (0.40 mL, 4.8 mmol), and the reaction mixture stirred at room temperature

for 4 h after which time the solvent was removed and the crude residue puriﬁed by mass-directed

preparative HPLC to give the title compound as an orange solid, and a 1:1 mixture of epimers at the

centre adjacent to the ketone, as a formate salt (716 mg, 58%); ¹H NMR (400 MHz; d₆-DMSO)

δ 8.60 (1H,

d, J = 8.3), 8.59 (1H, d, J = 8.3), 8.35 (1H, d, J = 7.3), 8.33 (1H, d, J = 7.6), 8.27 (1H, d, J = 7.6), 8.23–8.19

(3H, m), 7.79–7.75 (4H, m), 7.54–7.49 (2H, m), 7.47–7.41 (4H, m), 7.39–7.35 (4H, m), 7.29–7.23 (4H, m),

7.19–7.13 (2H, m), 4.76–4.67 (2H, m), 4.39–4.25 (4H, m), 3.70 (1H, d, J = 17.7), 3.69 (1H, d, J = 17.7), 3.52

(1H, d, J = 17.7), 3.48 (1H, d, J = 17.7), 3.18–3.07 (2H, m), 3.00–2.95 (2H, m), 2.66–2.55 (8H, m), 1.77–1.64

(10H, m), 1.57–1.44 (2H, m), 1.34–1.18 (14H, m), 0.90–0.80 (6H, m); LC-MS R_t1.80 min; purity 98.3%;

MS m/z 521 (MH)⁺.

ADZ 50,000 ((S)-3-((S)-2-((S)-2-benzamido-3-methylbutanamido)propanamido)-2-oxoheptyl

2,6-bis(triﬂuoromethyl)benzoate) was prepared according to Scheme 3:

Scheme 3. Schematic illustration.

To a solution of potassium ﬂuoride (14 mg, 0.2 mmol, thre equivalents) and 2,6 bis-

(triﬂuoromethyl)benzoic acid (42 mg, 0.16 mmol, two equivalents) in dry DMF (1 mL) over 4Å

molecular sieve was added a solution of the bromomethyl ketone (40 mg, 0.08 mmol, one equivalent)

in dry DMF (1 mL) and the reaction mixture stirred at room temperature for 3 h. The reaction mixture

was then ﬁltered through a plug of silica, washed with 10% v/v methanol/DCM and then evaporated

to dryness. The crude residue was puriﬁed by reversed phase preparative HPLC to give ADZ 50,000

(8 mg, 15%); MS m/z 660 (MH)⁺.

Compound

3 was synthesised according to the following route (Scheme 4) from Bz-Phe-AlaOH

which had been prepared using standard Fmoc solid phase peptide synthesis.

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Scheme 4. Schematic illustration.

The intermediate N-((S)-1-(((S)-1-(((S)-1-hydroxyhexan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-

oxo-3-phenylpropan-2-yl)benzamide was synthesised as follows:

To a solution of Bz-Phe-AlaOH (374 mg, 1.1 mmol) in dry THF (12 mL) and dry DMF (0.5 mL)

at

1.2 mmol). The mixture was stirred at this temperature for 1 h then S-(+)-2-amino-1-hexanol (142 mg,

1.2 mmol) in dry DMF (0.5 mL) was added. The mixture was stirred at

20 ^◦C for 2 h then allowed

−

20 ^◦C was added N-methylmorpholine (375

µL, 3.4 mmol) and isobutylchloroformate (157 µL,

−

to gradually warm to room temperature and stirred for 18 h. The reaction mixture was subsequently

diluted with ethyl acetate, washed with a saturated aqueous solution of NaHCO₃, brine and dried over

MgSO₄. The ﬁltrate was evaporated and the crude residue puriﬁed by ﬂash chromatography on silica

eluting with ethyl acetate to give the title compound as a white solid (360 mg, 82%); ¹H NMR (400 MHz;

d₆-DMSO)

δ 8.59 (1H, J = 8.4), 8.24 (1H, J = 7.3), 7.79–7.75 (2H, m), 7.54–7.48 (2H, m), 7.47–7.41 (2H, m),

7.39–7.35 (2H, m), 7.29–7.23 (2H, m), 7.19–7.13 (1H, m), 4.74–4.60 (1H, m), 4.64 (1H, t, J = 5.6), 4.32–4.24

(1H, m), 3.70–3.61 (1H, m), 3.36–3.29 (1H, m), 3.29–3.20 (1H, m), 3.11 (1H, dd, J = 13.6, 3.8), 2.98 (1H,

dd, J = 13.6, 11.1), 1.60–1.58 (1H, m), 1.32–1.14 (8H, m), 0.86–0.79 (3H, m); LC-MS: R_t2.96 min; purity >

95%; MS m/z 440(MH)⁺.

Compound

3, (N-((S)-1-oxo-1-(((S)-1-oxo-1-(((S)-1-oxohexan-2-yl)amino)propan-2-yl)amino)-3-

phenylpropan-2-yl)benzamide) was prepared as described below:

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To a solution of N-((S)-1-(((S)-1-(((S)-1-hydroxyhexan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-

oxo-3-phenylpropan-2-yl)benzamide (180 mg, 0.32 mmol) in dry DCM (5 mL) and DMF (0.9 mL) was

added a solution of Dess–Martin periodinane (15% wt solution in DCM, 1.9 mL, 0.65 mmol) dropwise

and the mixture stirred at room temperature for 1 h. The reaction mixture was diluted with ethyl

acetate (20 mL) and a solution of sodium thiosulphate (1 g) in a saturated aqueous solution of NaHCO₃

(10 mL) added. The mixture was stirred at room temperature for 15 min, the organic layer separated

and then washed with saturated NaHCO₃(aq.) (2

ﬁnally evaporated to dryness. The resulting off-white solid was triturated with ether and dried under

vacuum to give the title compound as a white solid (115 mg, 64%); ¹H NMR (400 MHz; d₆-DMSO)

×

20 mL), brine (2

×

20 mL), dried over MgSO₄and

δ

9.40 (1H, s), 8.59 (1H, d, J = 8.1), 8.34 (1H, d, J = 7.3), 8.28 (1H, d, J = 7.1), 7.80–7.75 (2H, m), 7.54–7.49

(1H, m), 7.47–7.41 (2H, m), 7.40–7.35 (2H, m), 7.29–7.23 (2H, m), 7.19–7.13 (1H, m), 4.76–4.67 (1H, m),

4.41–4.33 (1H, m), 4.10–4.02 (1H, m), 3.17–3.10 (1H, m), 3.04–2.94 (1H, m), 1.79–1.69 (1H, m), 1.55–1.43

(1H, m), 1.34–1.20 (7H, m), 0.89–0.80 (3H, m); Purity by ¹H NMR >95%; MS m/z 538 (MH)⁺.

N-{(S)-1-[(S)-1-((S)-1-cyclohexylaminooxalyl-2-methyl-propylcarbamoyl)-ethylcarbamoyl]-2,2-

dimethyl-propyl}-isonicotinamide (Compound 4) was prepared by sequential synthesis involving

an aldehyde intermediate (Scheme 5). To generate this intermediate, a dipeptide acid ((S)-2-{(S)-3,3-

dimethyl-2-[(pyridine-4-carbonyl)-amino]-butyrylamino}-propionic acid), was ﬁrst prepared

by solution-phase synthesis from an amino acid ester and Boc-(t-Bu)Gly-OH using methods

previously described by us [22]. The dipeptide acid was then coupled with an amino alcohol in

the presence of isobutyl chloroformate/N-methylmorpholine and oxidation with Dess–Martin

periodinane performed to yield an aldehyde product. A modiﬁed Passerini reaction was then

employed to produce an α-hydroxyamide from which the desired target compound, N-{(S)-1-[(S)-

1-((S)-1-cyclohexylaminooxalyl-2-methyl-propylcarbamoyl)-ethylcarbamoyl]-2,2-dimethyl-propyl}-

isonicotinamide, was prepared by Dess-Martin periodinane oxidation at ambient temperature until full

conversion to product had occurred, as judged by LC-MS. The reaction was quenched using saturated

sodium bicarbonate and sodium thiosulphate and stirred before being extracted into ethyl acetate.

The extract was washed sequentially with sodium bicarbonate, deionised water, and brine before being

dried and evaporated prior to puriﬁcation by reversed-phase preparative HPLC. Desired product

1

(Compound

4

) was characterised as: [M + H]⁺516. H NMR ((CD₃)₂SO, 400 MHz):

δ

8.75 8.72 (2H,

−

m, ArH), 8.57 (1H, d, J = 8.3 Hz, NH), 8.38 (1H, d, J = 9.4 Hz, NH), 8.24 (1H, d, J = 7.1 Hz), 8.02 (1H,

d, J = 7.8 Hz, NH), 7.81 7.78 (2H, m, ArH), 5.04 (1H, dd, J = 7.8 and 5.3 Hz, CHCO), 4.52 (1H, d,

J = 9.4 Hz, CHCO), 4.47 4.40 (1H, m, CHCO), 3.60 3.45 (1H, m, NHCH(cyclohexyl), 2.22 2.12 (1H,

m, CHMe₂), 1.72 1.64 (4H, m), 1.61 1.54 (1H, m), 1.34 1.17 (7H, m), 1.12 1.04 (1H, m), 1.00 (9H,

s,t-Bu), 0.89 (3H, d, J = 6.8 Hz, CH₃), 0.80 (3H, d, J = 6.8 Hz,CH₃).

−

N-{(S)-1-[(S)-1-((S)-1-Benzylaminooxalyl-2-methyl-propylcarbamoyl)-ethylcarbamoyl]-2,2-

dimethyl-propyl}-benzamide (Compound 5) was synthesised as described in our previous work [22].

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Scheme 5. Schematic illustration.

4.3. Preparation of HDM Allergen and Puriﬁcation of Der p 1

◦

Continuous solid-phase culture at 25 C and 75% relative humidity in custom-engineered

containment was used to produce populations of Dermatophagoides pteronyssinus. Native HDM

allergen mixtures were prepared from spent culture medium according to our standard operating

procedures [56]. While the conditions under which HDM are grown are known to inﬂuence the exact

composition of HDM allergen mixtures, the extract produced by these procedures is designed to

preserve labile bioactivity and is, to the best of our knowledge, representative of the full allergenic

repertoire of HDM. The Der p 1 content of the mixtures was determined by ELISA measurement

(Indoor Biotechnologies, Cardiff, UK) while proteolytic activity was measured using ADZ 50,059

as substrate. Where appropriate, ADZ 50,000 was used as a titrant to measure the concentration of

functional active sites. For most in vivo studies, we used mixed HDM allergens as the reagent for

sensitisation and challenge because this presentation is most representative of the material to which

the airways are subjected in life. HDM mixtures were normalised by reference to Der p 1 content

expressed as

µg/mL as previously described [22]. According to this procedure, ‘HDM 1’ means

1

µg/mL Der p 1 and so forth. Batch-wise consistency in the activity of Der p 1 was ensured by the

inclusion of a reducing agent (cysteine or dithiothreitol) in buffers or culture medium. The appropriate

agent was present also in vehicle controls.

The mixed HDM allergens were also used as feedstock for the puriﬁcation of Der p 1. High-purity

Der p 1 was required for the characterisation of the Der p 1 substrate and for the testing of novel

molecular entities designed as inhibitors of its proteolytic activity. General methods for this puriﬁcation

have been described in full elsewhere [56]. In brief, 2–3 volumes of Dulbecco’s PBS were added to mixed

◦

HDM allergens with overnight stirring. After centrifugation (30 min, 24,000

×

g, 4 C), the supernatant

was aspirated, and solid ammonium sulphate added to 50% saturation in the presence of 1 mM

EDTA. After precipitation for >2 h the pellets were collected, and insoluble matter removed from

the reconstituted solution. The soluble fraction then underwent size exclusion chromatography at

pH 7.4 on an ÄktaPuriﬁer system (HiPrep 16/60 Sephacryl S-200 HR, GE Healthcare, UK) with an

elution buffer comprising 0.2 M sodium phosphate containing 0.5 M sodium chloride and 1 mM EDTA.

A fraction containing Der p 1 was collected and then reﬁned using a soybean trypsin inhibitor (SBTI)

column. This cycle of size exclusion and serine peptidase removal was repeated and the ﬁnal eluate

passed through an Amicon ultraﬁltration cell (Millipore, Bedford, MA, USA) with a 10 kDa cut-off

membrane to concentrate and desalt the preparation. After dilution in 20 mM Tris-HCl buffer, pH 8.0,

the sample was then chromatographed on Resource Q (GE Healthcare) using an ÄktaPuriﬁer. Der p 1

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was eluted by gradient chromatography with 0–0.5 M NaCl. Peaks containing Der p 1 were analysed

by SDS-PAGE and MALDI-TOF mass spectrometry (Kratos Axima, Kratos Analytical, UK or Bruker

Flex, Bruker, UK) and combined. Der p 1 was quantiﬁed by ultraviolet absorbance spectrometry in a

quartz cuvette at 280 nm using an extinction coefﬁcient of 47,705 M⁻¹cm⁻¹

.

4.4. Der p 1 and Der f 1 Enzyme Activity Assays

These were performed in 96-well plates using a PerkinElmer Multiprobe II Plus HTS EX robot

(PerkinElmer, Seer Green, Buckinghamshire, UK). Reactions consisted of substrate (10 L at 12.5

ﬁnal concentration), reaction buffer (70 L potassium phosphate buffer, pH 8.25 containing 1 mM

EDTA) and dithiothreitol (DTT, 10 L with a ﬁnal concentration of 1 mM). Catalysis was initiated

µ

µM

µ

with the addition of 10

µ

L Der p 1 or Der f 1 dissolved in reaction buffer at 2.5

µ

g/mL and

◦

the reaction followed at 30 C by measurement of ﬂuorescence (excitation/emission 330/420 nm)

using either a Fusion Alpha-FP or Envision plate reader ﬁtted with a temperature-controlled carrier

(PerkinElmer, UK).

Using a modiﬁcation of the procedure described above with mixed HDM allergens containing the

equivalent of 40 nM Der p 1, studies were undertaken to compare the effects of ADZ 50,000 with a

range of standard enzyme inhibitors (Sigma-Aldrich).

Enzymatic activity in a commercial Der p 2 preparation (Indoor Biotechnologies, UK) was assayed

at ambient temperature using 200

µM N-Bz-Phe-Val-Arg-p-nitroanilide hydrochloride (Sigma-Aldrich,

UK) and 3 M Der p 2 in PBS containing 1 mM DTT and in the absence and presence of 100 µM

µ

4-(2-aminoethyl) benzenesulphonyl ﬂuoride hydrochloride (AEBSF, Sigma-Aldrich, UK). A₄₀₅nm was

measured 15 min after reaction.

4.5. Assay of Cathepsin B

Assays comprised 10

UK, 5.9 M ﬁnal concentration), 70

and 10 L of DTT (2.5 mM ﬁnal). Reactions were initiated by adding 10

µ

L Abz-Gly-Ile-Val-Arg-Ala-Lys-DNP-OH (Merck, Watford, Hertfordshire,

L of reaction buffer (0.1 M NaAc-HAc, pH 4.5, 0.2 M NaCl),

L of human liver cathepsin B

µ

dissolved in reaction buffer to give ﬁnal concentration of 0.5 nM. Reactions were performed at 30 ^◦C

and followed kinetically by excitation/emission at 320/420 nm. For inhibitor studies, the reaction

buffer volume was 60 µL and inhibitor added in 10 µL aliquots.

4.6. Assay of Cathepsin H

Assays comprised 10

reaction buffer (37.5 mM KH₂PO₄, 37.5 mM K₂HPO₄, 1 mM EDTA, pH 6.8), and 10

ﬁnal). Reactions were initiated by adding 10 L of human liver cathepsin H dissolved in reaction buffer

at a ﬁnal concentration of 0.4

g mL⁻¹. Reactions were monitored at 30 C with excitation/emission at

µL L-Arg-7-AMC (Sigma-Aldrich, 150

µM ﬁnal concentration), 70

µL of

µL cysteine (3 mM

µ

◦

µ

360/460 nm. Buffer volume was 60 µL when using an inhibitor (10 µL).

4.7. Assay of Cathepsin K

Assays comprised 10

µL (Z-Leu-Arg)₂–Rhodamine 110 (Merck) (1.25

µM ﬁnal), 70 µL of reaction

buffer (50 mM MES pH 5.5, 2.5 mM EDTA, 10% v/v DMSO), and 10

µL DTT (2.5 mM ﬁnal). Reactions

were started by adding 10 µL of cathepsin K dissolved in reaction buffer to give a ﬁnal concentration of

0.075 nM. Reactions were monitored at 30 ^◦C with excitation/emission at 485/535 nm. Buffer volume

was 60 µL when using an inhibitor (10 µL).

4.8. Assays for Cathepsin L and Cathepsin S

Assays comprised 10

in the cathepsin L assay, 20

acetate buffer, pH 5.5 containing 4 mM EDTA for cathepsin L and 0.1 M sodium phosphate buffer,

µL of Z-Phe-Arg-AMC substrate (Sigma-Aldrich, 10

µM ﬁnal concentration

µ

M in the cathepsin S assay), 70 L reaction buffer (400 mM sodium

µ

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pH 7.5 containing 2 mM EDTA for cathepsin S), and 10

µL DTT (8 mM and 2 mM ﬁnal concentrations,

respectively). Reactions were initiated by adding 10 µL of human liver cathepsin L or recombinant

human cathepsin S dissolved in reaction buffer to give ﬁnal concentrations of 0.29 nM and 2.5 nM,

respectively. Reactions were monitored at 30 ^◦C with excitation/emission at 360/460 nm.

4.9. Assay of Thrombin

Assays comprised 10

reaction buffer (10 mM HEPES, pH 8.0, 5 mM CaCl₂, 0.02% v/v NaN₃) and started by adding 10

human plasma thrombin dissolved in reaction buffer (1 nM ﬁnal). Reactions were performed at 30 ^◦C

µL Bz-Phe-Val-Arg-AMC (Merck, 73

µM ﬁnal concentration), 70

µL of

and followed by excitation/emission at 370/450 nm. Inhibitor was added in 10 µL aliquots, as above.

4.10. Assay of Trypsin

This comprised 10

ﬁnal concentration) and 70

10 L of trypsin (2 nM ﬁnal) and monitored by A₄₀₅nm. Inhibitor was added in 10

For comparative studies using trypsin (2 g/mL) and Der p 1 (20 g/mL), the above conditions were

modiﬁed to permit the inclusion of 10 L DTT (1 mM ﬁnal) and in these experiments AEBSF (80 M)

µL N-Bz-Phe-Val-Arg-p-nitroanilide hydrochloride (Sigma-Aldrich, 200 µM

◦

µ

L of reaction buffer (PBS). Reactions were started at 30 C by adding

µ

µL aliquots.

µ

or ADZ 50,000 (80 µM) were used as inhibitors.

4.11. Assay of Chymotrypsin

Assay mixtures comprised 10

M ﬁnal), 70 L of reaction buffer (0.1 M Tris-HCl, 10 mM CaCl₂, 250 mM NaCl, pH 8.0).

Reactions were monitored by A₄₀₅nm at 30 ^◦C after adding 10

L of chymotrypsin (TLCK-treated,

Sigma-Aldrich) in reaction buffer (2 nM ﬁnal). Inhibitor was added in 10 L aliquots, as above.

For comparative studies using chymotrypsin (2 g/mL) and Der p 1 (20 g/mL), the above conditions

were modiﬁed to permit the inclusion of 10 L DTT (1 mM ﬁnal) and in these experiments AEBSF

µL N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Sigma-Aldrich,

200

µ

(400 µM) or ADZ 50,000 (40 µM) were used as inhibitors.

4.12. Analysis of Inhibitor Kinetics

Inhibitor kinetics were analysed by the progress curve method essentially as described [57].

For irreversible inhibitors, data were ﬁtted by computational non-linear regression and the apparent

inactivation rate constant (k_obs) was calculated from the Equation (1):

ꢀ

ꢁ

V_z

k_obs

_obs·t

[P]_t=

1 − e^−k

(1)

where [P]_tis the product concentration at time t.

V_zis the velocity of the uninhibited reaction.

k_obsis a pseudo ﬁrst order rate constant.

For reversible inhibitors, IC₅₀values were calculated using conventional procedures.

4.13. Expression of Claudin 1.1

RNA was extracted from conﬂuent cultures of Calu-3 cells grown as previously

described [12,21,24,58] and cDNA synthesised according to manufacturer protocol (Qiagen,

Manchester, UK). cDNA corresponding to the ﬁrst extracellular domain of claudin 1, henceforth

designated CLD1.1, was ampliﬁed using 5⁰-TAC ATA TGC AGT GGA GGA TTT ACT CCT ATG

CCG GCG A-3⁰as forward primer and 5⁰-ACG GAT CCT TAA CGG GTT GCT TGC AAT GTG CTG

CTC AGA-3’ as reverse primer. PCR products were sub-cloned into pET 22b+ and transformed into

E. coli expression strain BL21DE3 plySs. After induction by 1 mM IPTG this yielded a 6 kDa peptide

on SDS-Tricine PAGE consistent with that predicted for CLD1.1 and which was absent in negative

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controls lacking IPTG. The CLD1.1 band was subjected to in-gel digestion by trypsin and the resultant

fragments analysed by MALDI-TOF in positive reﬂectron mode. The resulting mass spectrum revealed

two major fragments (m/z 620.2 and m/z 1764.6) which correspond to the sequences ¹Met-Gln-Trp-Arg⁴

and ³⁹Val-Phe-Asp-Ser-Leu-Leu-Asn-Leu-Ser-Ser-Thr-Leu-Gln-Ala-Thr-Arg⁵⁴conﬁrming identity with

a residue coverage of 37%.

To study its proteolysis by Der p 1, recombinant CLD1.1 was separated by SDS-Tricine PAGE and

the band containing the peptide excised from the gel. The gel slices were successively washed with

10 mM ammonium bicarbonate buffer (pH 8.9), 50% MeCN and 100% MeCN and then freeze-dried.

Reactions were performed by exposing the dried gel slices to Der p 1 in PBS containing 1 mM DTT

and incubating at room temperature. Inhibitor studies were performed by pre-treating Der p 1 (4

with either ADZ 50,000 (400 M) or AEBSF (4 mM) for 10 min prior to addition to CLD1.1 with

a 1:10 v/v dilution. Negative controls comprised Der p 1 or CLD1.1 incubated alone in reaction

buffer. After treatment with Der p 1, 10 L of supernatant from each reaction mixture was analysed

by LC-MS. Capillary liquid chromatography was performed using a 5 m reversed-phase column

(BioBasic-18, 100 0.18 mm; ThermoElectron, Hemel Hempstead, Hertfordshire, UK) at 2 L/min

µM)

µ

×

µ

using a Surveyor MS pump coupled to the electrospray source of an ion trap mass analyser (LCQ

Deca XP Plus, ThermoElectron, UK). Samples were eluted by gradient chromatography (0.1–30%

B in A over 35 min, 30–50% B in A over 10 min followed by 50–80% B in A over 5 min, where A

was aqueous 0.1% v/v formic acid and B was 0.1% v/v formic acid in MeCN. Mass spectra were

acquired in full scan mode (m/z 300–1800) and the three most abundant ions subjected to MS/MS

analysis in data-dependent acquisition mode with dynamic exclusion. Acquired MS/MS spectral

data were searched against a custom database created for this experiment using BioworksBrowser

and the TurboSEQUEST algorithm. Search parameters were declared as free cysteine and undeﬁned

enzyme. The molecular mass range was set to 600–3500 Da and ﬁlters applied as follows (

∆

cn:

≥

0.1;

Xc (± 1, 2, 3) 1.5, 2.0, 2.5; protein probability:

≤1 ×

10⁻³. Fragments were evaluated according to their

scores to verify the characterisation.

4.14. Cell Culture

Calu-3 cells (American Type Culture Collection) were cultured as previously described by us and

is a favoured model system because they are of human origin, retain effective tight junctions (TJs) and

form polarised monolayers which develop substantial transepithelial resistance [24,58]. These cells are

well characterised as responding to HDM allergens through various mechanisms present in primary

cultures of human airway epithelial cells [12,20,21,58].

For experiments involving measurements of permeability and transepithelial electrical resistance

(TER), calu-3 cells were grown to conﬂuence on 6.5 mm diameter, 0.1 µm pore polycarbonate Transwell

ﬁlters (Corning^®Costar^®) as described [14]. Treatments in serum-free minimum essential medium with

Earle’_◦s salts (EMEM) were applied to the apical compartment of each well and the plates incubated

at 37 C in a humidiﬁed 5% CO₂/air atmosphere. TER was measured using an EVOM epithelial

volt/ohm meter (World Precision Instruments, Sarasota, FL, USA) and medium harvested from both

compartments at the end of the experimental treatment.

A549 cells were cultured in 12-well plates (Corning^®Costar^®) using methods described

elsewhere [58]. At conﬂuence, cells were washed with serum-free EMEM and experimental treatments

applied for 8 h at 37 ^◦C in a ﬁnal volume of 1 mL. Treatment medium was harvested and replaced by

1 mL serum-free medium and incubation continued for 16 h. The medium was harvested, centrifuged

(10,000

×

g, 30 s) to sediment non-adherent cells and the supernatants stored at

−

20 ^◦C. Whole cell

protein extracts were prepared by the addition of 1 mL boiling 2% SDS w/v in 312 mM Tris, pH 6.8).

Protein concentration was measured using the Biuret method.

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4.15. Studies Performed In Vivo

These studies were compliant with requirements of the Animals Scientiﬁc Procedures Act

(UK) in an AAALAC-accredited facility and were subject to prior ethical review. Acute tolerability

investigations performed with the test substances prior to conduct of these studies did not reveal any

obvious adverse events over a 24 h period following dosing.

4.16. Allergic Responses in Rats

Animals (Brown Norway, 250–350 g, Charles River, Ormiston, UK) were housed under isolation

and randomly assigned to treatment groups. Rats were sensitised to HDM allergen mixture,

with aluminium hydroxide adjuvant, or vehicle on days 0, 7, and 14 by intraperitoneal (i.p.) injection

(0.5 mL). On day 17 the animals were brieﬂy anaesthetised with isoﬂurane in oxygen and sensitisation

treatments administered as intratracheal aerosols (delivery volume to airways 100

Penn-Century IA-1C/FMJ-250 dosing device.

µL) using a

For measurement of acute allergic bronchoconstriction and airways hyperreactivity, animals were

anaesthetised on day 21 with pentobarbitone (100 mg/kg i.p.) and ventilated via a tracheal cannula

(approximately 7 mL/kg, 1 Hz) with a 50:50 v/v mixture of oxygen in air. The anaesthetised, ventilated

animals were then paralysed with norcuron (4 mg/kg i.m., MSD, Hoddesdon, Hertfordshire, UK).

Ventilation was monitored using a ﬂow transducer (Fleisch, type 0000) in-line with the respiratory

pump. Coincident pressure changes within the thorax were monitored directly using an intrathoracic

cannula, enabling display of the pressure differential between trachea and thorax. Airways resistance

(R_L) and dynamic compliance (C_dyn) were calculated for each respiratory cycle on a digital electronic

respiratory analyser (PMS, Mumed Ltd., London, UK). Blood pressure and heart rate were also

routinely recorded. Animals were challenged by vehicle of HDM allergen mixture (with or without

ADI compounds) using a Penn-Century aerosoliser inserted into the trachea. Dose delivery volume to

the airways was 100

as the change in R_Lfrom baseline. One hour after challenge the responses to a dose of acetylcholine

(100 g/kg, 1 mL/kg) and then to a dose of adenosine (10 mg/kg, 1 mL/kg) were recorded. At the end

µL. Acute bronchoconstrictor responses arising from the challenge were measured

µ

of the study, the animals were euthanised by pentobarbitone overdose. As a further control, a group of

sensitised animals was challenged by i.t. aerosol on day 21 and airways hyperreactivity measurements

made 24 h later as described above.

For studies of allergen-induced leukocyte accumulation, animals were brieﬂy anaesthetised

(isoﬂurane in oxygen) on day 21 after the beginning of the sensitisation procedure and vehicle, HDM

allergen mixture or HDM allergen mixture with ADI compound delivered from a Penn-Century

aerosoliser as previously described. Animals were then allowed to recover from anaesthetic to enable

assessment of cell recruitment to the lungs to be made 48 h after challenge. At this point animals

were killed with pentobarbitone (250 mg/kg i.p.) and the lungs lavaged via a tracheal cannula using

3

×

4 mL aliquots of Hanks’ balanced salt solution (HBSS) containing 10 mM EDTA and 25 mM

HEPES. The recovered cells from each animal were pooled and the total volume recovered adjusted

to 12 mL using HBSS. Total cells were counted (ADVIA , Bayer Healthcare, Diagnostic Division,

®

Reading, Berkshire, UK) and smears made by diluting recovered ﬂuid (to approximately 106 cells/mL)

and pipetting an aliquot (100 mL) into a centrifuge (Cytospin, Shandon, UK). Smears were air-dried,

ﬁxed for 10 s using methanol, and stained with buffered eosin (10 s) and methylene blue/Azur

(5 s) (Speedy-Diff, Clin-Tech Ltd., Guildford, Surrey, UK) to differentiate eosinophils, neutrophils,

macrophages/monocytes, and lymphocytes. A total of 500 cells in each sample were counted by light

microscopy at ×1000 using an oil immersion objective.

In both studies, a terminal blood sample was taken from each animal and serum prepared for the

measurement of immunoglobulins.

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4.17. Allergic Sensitization Studies in Mice

Mice (female Balb/c or C57BL/6 depending on study, 20

±

2 g, Charles River/BioLASCO) were

maintained in an isolator facility using individually ventilated cages (Allentown IVC Racks, 36 Mini

Isolator System, Allentown, NJ, USA) which had been autoclaved before use. The environmental

◦

regime was 22–24 C/60–80% relative humidity on a 12 h light/dark cycle. There was ad libitum access

to reverse osmosis-puriﬁed water and food (MF-18 laboratory rodent diet). Where the investigation

required both pre- and post-study serum samples, the pre-study samples were taken from the

retro-orbital sinus on acclimatisation in the isolator. Animals were randomly assigned to groups

and sensitised to a natural mixture of HDM allergens, Der p 1 alone, Der p 2 alone, or treated

with vehicle on day 0, 7, and 14. The route of administration, i.p. or intranasal, was according to

predetermined study protocol. Animals were challenged under anaesthetic cover by i.t. aerosol on

day 21 using a Penn-Century IA-1C/FMJ-250 aerosoliser (20

were anaesthetised with propofol (AstraZeneca, 10 mg/mL, 50

samples taken from the retro-orbital sinus. Then BAL was performed using 2

aliquots of PBS according to study design. The returns were combined, ﬂuid volumes measured, and

cells enumerated (Sysmex XT-1800iV, Sysmex, Kobe, Japan). A similar approach was used in studies

involving ovalbumin (OVA) challenge in animals sensitised i.p. to OVA with Al(OH)₃as adjuvant.

µ

L/mouse). On day 22 the animals

L/mouse, i.v.) and terminal blood

0.25 mL or 3 0.5 mL

µ

×

4.18. ELISA Measurements

Cytokines were assayed using kits and according to manufacturer protocol (Biosource Europe,

Nivelles, Belgium). Der p 1 and Der p 2 were quantiﬁed by ELISA using reagents obtained from Indoor

Biotechnologies, UK.

Total IgE was measured by sandwich ELISA in Corning^®Costar^®high binding 96-well

microplates. For rat serum, plates were coated overnight with mouse anti-rat IgE (clone B41-1,

BD Biosciences Europe, Wokingham, Berkshire, UK) in carbonate-bicarbonate buffer and blocked

using DPBS with 0.05% v/v Tween^®20 (DPBST) containing 10% v/v foetal bovine serum. Rat serum

samples or IgE standards (BD Biosciences) were diluted in Can Get Signal^®Solution 1 (2B Scientiﬁc,

◦

Upper Heyford, Oxfordshire, UK) and incubated on the plate for 15 h at 4 C. After washing,

the secondary antibody (biotin-conjugated mouse anti-rat, clone B41-3, BD Biosciences) and

streptavidin-horseradish peroxidase were added in Can Get Signal^®Solution 2 and the plate incubated

at room temperature for 3.5 h. After washing, substrate solution (3,3⁰,5,5⁰-tetramethylbenzidine

dihydrochloride in phosphate-citrate perborate buffer) was added to each well and the reaction

terminated by addition of 2 M sulphuric acid after incubation in the dark for 5–15 min. Plates were

read at 450 nm using an automated plate analyser and data quantiﬁed by interpolation from standard

curves. A similar procedure was used for the measurement of mouse IgE using an OptEIA

™ set

(BD Biosciences).

Allergen-speciﬁc immunoglobulin assays were performed by indirect ELISA. Plates were coated

overnight at 4 ^◦C with Der p 1 or mixed HDM allergens in carbonate-bicarbonate buffer and blocked

according to our standard procedures. Samples of serum were then added and incubated as described

before addition of the appropriate rat (RG7/1.30 or B41-3, BD Biosciences) or mouse (BD OptEIA or

MCA336P, AbD Serotec, Kidlington, UK) secondary antibodies diluted in Can Get Signal^®Solution 2.

Assays were developed as described above and data reported as absorbance at 450 nm.

4.19. BAL Analysis by Flow Cytometry

Polychromatic ﬂow cytometry of BAL ﬂuid samples was performed on a BD FACSAria

instrument (Becton Dickinson Biosciences, Franklin Lakes, NJ, USA) with data acquisition and

analysis performed using FACSDiva software. Unless stated otherwise, antibodies for these

™

studies were obtained from BD Pharmingen and comprised MHC class II-FITC conjugate (antibody

2G9), CD11c-allophycocyanin conjugate (antibody HL-3), CD11c-phycoerythrin/Cy7 conjugate,

Int. J. Mol. Sci. 2018, 19, 3166

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CD3-phycoerythrin/Cy5 conjugate (antibody 145-2C11), B220 (CD45R)-phycoerythrin/Cy5 conjugate

(antibody RA3-6B2), CCR3-phycoerythrin (antibody 83101, R&D Systems, Abingdon, Oxfordshire,

UK). FcγR blocking agent was antibody 2.4G2. Dendritic cells were enumerated as non-autoﬂuorescent

CD3/B220⁻, MHC II⁺, CD11c⁺. Eosinophils were SSC^high, CCR3⁺, moderate CD11c⁺, low-absent

MHC II, B220/CD3⁻.

4.20. Data Presentation and Statistical Analyses

Data are presented as mean values

±

s.e. from the indicated number of observations.

Signiﬁcance was determined using one-way analysis of variance with post hoc testing using the

Student–Newman–Keuls procedure in SigmaPlot vs. 12.0 unless stated otherwise. A probability value

of p < 0.05 was considered statistically signiﬁcant.

Author Contributions: Writing-Review & Editing, J.Z. (Jihui Zhang), J.C., J.Z. (Jie Zuo), C.K.N., M.R.S., T.R.P.,

D.R.G. and C.R.; Supervision, T.R.P., D.R.G. and C.R.; Project Administration, D.R.G., C.R.; Funding Acquisition,

C.R.

Funding: This work was supported by the Wellcome Trust (award 087650, to CR).

Acknowledgments: We would like to thank John Ayrton and John Dixon for stimulating discussions and support.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

Abz

aminobenzoyl

ACh

acetylcholine

ADI

allergen delivery inhibitor

Ado

adenosine

ADZ 50,059

(3S,6S,9S,12S,15S,18S)-1-(2-aminophenyl)-9-butyl-18-carbamoyl-15-(4-hydroxy-3-nitrobenzyl)-

12-(hydroxymethyl)-3-isopropyl-6-methyl-1,4,7,10,13,16-hexaoxo-2,5,8,11,14,17-hexaazaicosan-

20-oic acid

AEBSF

AMC

BAL

4-(2-aminoethyl)benzenesulphonyl ﬂuoride

7-amino-4-methylcoumarin

bronchoalveolar lavage

Bz

benzoyl

CH₂Ph

CLD1.1

DCM

Der f

Der p

DMF

DMSO

DNP

DTT

benzyl

extracellular domain 1 of claudin-1

dichloromethane

Dermatophagoides farinae

Dermatophagoides pteronyssinus

dimethylformamide

dimethyl sulphoxide

2,4-dinitrophenol

dithiothreitol

E-64

trans-epoxysucciny-L-leucyl-amido (4-guanidino) butane

ethylenediaminetetraacetic acid

minimum essential medium with Earle’s salts

granulocyte macrophage-colony stimulating factor

Hanks’ balanced salt solution

house dust mite

4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid

high performance liquid chromatography

high-throughput screening

intraperitoneal

EDTA

EMEM

GM-CSF

HBSS

HDM

HEPES

HPLC

HTS

i.p.

iPr

isopropyl

IL

interleukin

Article Doi

Allergen delivery inhibitors: Characterisation of potent and selective inhibitors of der p 1 and their attenuation of airway responses to house dust mite allergens

DOI: 10.3390/ijms19103166

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Article abstract of DOI:10.3390/ijms19103166

Full text of DOI:10.3390/ijms19103166

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