Novel targets for drug discovery in celiac disease.

Celiac disease is a lifelong, immunological disorder induced by dietary protein-gluten, in a genetically susceptible populations, resulting in different clinical manifestations, the release of antibodies, and damage to the intestinal mucosa. The only recommended therapy for the disease is to strictly follow a gluten-free diet (GFD), which is difficult to comply with. A GFD is found to be ineffective in some active Celiac disease cases. Therefore, there is an unmet need for an alternative nondietary therapeutic approach. The review focuses on the novel drug targets for Celiac disease. Copyright:

Introduction

Celiac disease is a chronic disease which involves the deregulation of the immune system along with inflammation of the small bowel on exposure to an environmental factor, i.e., dietary protein – gluten, in individuals carrying specific genetic background. Different studies depicted the prevalence of Celiac disease worldwide is 0.7%.[1] The literature reported a prevalence of 1.04% (1 in 96) along with seropositivity of anti-tissue transglutaminase antibody 1.44% (1 in 69) in North India.[2] A gluten-free diet (GFD) is the lone recommended therapy for Celiac disease. To comply with a GFD is challenging because of many reasons, for instance, GFD is not easily available, no proper consensus of available gluten in the GFD, low palatability, and the therapy is not found to be effective in refractory Celiac disease (RCD). Moreover, about 30% experience persistent symptoms with GFD. Therefore, there is an urgent need for an alternative nondietary therapeutic approach. A new understanding of the disease pathophysiology has opened new doors to develop novel therapies.[3]

Novel Drug Targets for Celiac Disease

Compromised gut permeability – A drug target

Compromised gut permeability is a vital characteristic of Celiac disease. Zonulin, an intestinal enzyme has a structural similarity with growth factors that regulate tight junction integrity. Once Zonulin interacts with specific gliadin peptide, it results in upregulated gut paracellular permeability.[4] Larazotide acetate (AT-1001) is a protein extracted from a cholera toxin found to inhibit the zonulin activity and thereby ameliorate gut permeability.[56] Currently, AT-1001 is in Phase 2 clinical trial [Table 1].

Table 1

Potential drugs for celiac disease under different phases of clinical trial

CXCR3, a chemokine receptor and its ligand (specifically CXCL10) overexpress in the disease state. The interaction of CXCR3 with the gliadin peptide regulates Zonulin production and increase gut permeability. MyD88, an adapter protein is also involved during the interaction of chemokine and gliadin. This chemokine could be used as a novel therapeutic target for Celiac disease.[78]

Secretory IgA presents in the apical region of intestinal epithelium forms complexes with gliadin peptide. These complexes, with the help of transferrin receptor CD71 moves into the basal region of lamina propria from the apical region. This way gliadin peptide bypasses the lysosomal degradation. Once these harmful peptides reach the lamina propria, it gets the license to destroy the intestinal cells.[9]

Undigested peptides – A drug target

The human gut lacks prolyl endopeptidase activity which is required to digest the long gliadin peptide. Once these undigested gliadin peptides crosses the tight junction and reaches to lamina propria, it activates different immunological cascades which leads to intestinal damage. One of the approaches is also to introduce enzymes which could break or digest proline and glutamine bond in the immunogenic peptides.[678] ALV003 is an enzyme supplement, the mixture of two recombinant gluten-specific proteases and is in Phase 2b clinical trial as an adjunct therapy with a GFD [Table 1].[10]

Gluten sensitization – A drug target

Apart from targeting a specific cascade which alters in Celiac disease, an alternative approach is vaccination, which aimed to provide gluten tolerance. NexVax 2 is a therapeutic or desensitization vaccine developed by ImmuSanT and is in the Phase 1 clinical trial. The vaccine employed three peptides of gluten to induce tolerogenic in Celiac patients [Table 1].[11]

By encapsulating a component of wheat within a nanoparticle, Cour pharmaceutical is developing a novel treatment that proposes to develop gluten tolerance in Celiac disease. Phase 1 clinical trial is being conducted to characterize the safety and tolerability of a drug called TIMP-GLIA to develop immune tolerance (NCT02679014) [Table 1].

Adaptive immune response – A drug target

When undigested gliadin peptide breaches the intestinal tight junction, it interacts with tissue transglutaminase enzymes (TG2), which deamidate these peptides. Now, these negatively charged and stable peptide interacts with major histocompatibility complex (MHC) Class-II specifically HLADQ2 or HLA-DQ8 which are present on antigen-presenting cells (APCs).[1213] APCs present deamidated peptide to CD4+ T-cells. Once CD4+ T-cells get activated, it releases Th1 cytokines like interferon-γ (IFN-γ), interleukin-18 (IL-18), IL-21. IFN-γ activates the Zonulin enzyme and further increased the tight junction permeability of the intestinal barrier.[13] Secreted IFN-γ also induces metalloproteases production by myofibroblasts lamina propria mononuclear cells, which leads to tissue remodeling.[14]

CD4+ T-cells through Th2 response also increases the population of B-Cells and increase in the release of antibodies for instance, tissue transglutaminase, anti-gliadin, deamidated gliadin peptide, and endomysial antibodies [Figure 1].[15]

Figure 1

Novel drug targets for Celiac disease: (1) Endopeptidases/glutenases break down immunogenic gliadin peptide; (2) CXCR3 inhibitors prevents receptor-gliadin complex formation thereby inhibit zonulin secretion; (3) probiotics ameliorate gliadin induced dysbiosis; (4) tight junction modulator inhibit zonulin production which prevents gliadin induced compromised gut permeability; (5) gluten tolerization provide gluten tolerance; (6) TG2 inhibitor prevents deamidation of gliadin peptide; (7) DQ2/DQ8 blocking peptide inhibit gliadin-HLA complex formation and activation of T-cells; (8) cathepsin S inhibitor inhibits the proteolyses of MHC Class II invariant chain (9) anti interferon-γ/anti tumor necrosis factor-alpha prevent inflammation; (10) anti interleukin-15 inhibits IEL production. CXCR3 = Chemokine receptor, IELs = Intraepithelial lymphocytes, TG2 = Tissue transglutaminase, HLA = Human leukocyte antigen, DC = Dendritic cells

Antibodies deposited on the extracellular surface leads to alteration in the enterocyte cytoskeletal and actin redistribution. This further leads to epithelial damage.[16]

These all cascades also generates Fas/Fas ligand or activation of the NKG2D-MIC and perforin granzyme signaling pathway through IL-15 which leads to the interaction of the natural killer (NK) receptor on CD8+ T-cells and MICA (MHC Class I polypeptide-related sequence A) present on epithelial cells and results in further epithelial damage.[17]

It seems to be a promising strategy to inhibit the human leukocyte antigen (HLA) binding grooves with the gliadin antagonist. The strategy can further inhibit the T-cell activation pathway, which is vital for the destructive signaling cascade.[18] Dimeric and azidoproline analogs of gluten peptide are some HLA blockers that are presently in the preclinical stage [Table 1].[19]

Cathepsin S is a cysteine protease which proteolysis MHC Class II invariant chain for the presentation of antigen. This makes it a redundant target for drug therapies in various disease conditions. The protease inhibitor, RG7625 (Cathepsin S inhibitor) is in the Phase 1 clinical trial since 2016 [Table 1]. The result of the trial is still awaited.[2021]

Under in vitro and ex vivo studies using Caco cells and organ culture simultaneously, TG2 inhibitors, i.e., R281 and R283 showed a protective effect against gliadin-induced toxicity.[22] Inhibition of TG2 using TG2 inhibitor cystamine reduced the T-cell population. The results depicted that the inhibition of TG2 can ameliorate the harmful response of gluten-specific T-cells in the intestine.[23]

The introduction of anti-IFN-γ antibodies in gliadin-specific T-cell lines showed to avert the intestinal damage from the secreted inflammatory cytokines.[24]

Innate immune response – A drug target

Immunogenic gliadin peptide activates the release of cytokines such as IL-15, epidermal growth factor and IFN-α, which leads to the proliferation of enterocytes, dendritic cells, and intraepithelial lymphocytes (IELs), important for the pathogenesis of the disease.[2526] Increase expression of IL-15 in active Celiac disease upregulates NK receptors, i.e., CD94 and NKG2D by CD3+ IEL, which finally leads to T-cell receptor-independent destruction.[27]

Tumor necrosis factor-alpha (TNF-α) along with IFN-γ is cytotoxic to IELs. Infliximab, antibodies against TNF-α, showed to be efficacious in RCD.[2829] In the transgenic mouse model, inhibition of IL-15 demonstrated the reduction of IELs by promoting IELs apoptosis. IL-15 inhibits the apoptosis of cytotoxic IELs, which regulates RCD via the JAK3/STAT5 signaling pathway.[30] AMG-714, a monoclonal antibody for IL-15 is a promising drug strategy and has completed the Phase 2 clinical trial in Celiac disease [Table 1]. The study found no significant difference in villous height and crypt depth ratio between Celiac patients and control.[31]

Alfa amylase/trypsin inhibitors used in Wheat crops for pest control increase the TLR4-MD2 cluster of differentiation complex activity. It also upregulates proinflammatory cytokines levels, which further leads to the activation of the innate immune system.[32]

Cell adhesion molecules such as mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and integrin (a4b7) are important for the recruitment of lymphocyte at the site of injury in Celiac patients. Hence, the inhibition of both these molecules found to be a beneficial strategy for celiac patients.[16] Moreover, Natalizumab, a monoclonal antibody of a4b7 observed to be efficacious in Crohn's disease.[33]

Toll-like receptor as a drug target

Previous studies suggested the involvement of toll-like receptors (TLRs) in an innate immune response. TLR's recognize the by-products of gut microbiota[3435] and in response activates the release of inflammatory cytokines such as IL-6, TNFα, and IFN-γ.[35363738] The inflammatory cascade activated results in characteristic pathological changes of the intestine.[2739] The alteration in the TLR-4 and TLR-9 in blood and biopsy of Celiac disease patients showed its important role in innate immunity. The previous study also depicted the role of an immunogenic peptide in activating TLR's.[740] Thus, it is a potent drug target for Celiac disease, which further requires validation using large data.

Micro RNA – A drug target

In general, micro RNA (miRNA) acts in cell differentiation and proliferation. miRNA found to alter epithelial cell function and differentiation, increased crypt apoptosis, and alter gut permeability. miRNA alteration found to affect NOTCH1 signaling activity, thereby affecting intestinal cell differentiation in Celiac disease. Studies showed that miRNA such as miR-124a, miR-189, miR-299-5p, miR-379, miR-449a, and miR-34 dysregulated in Celiac disease.[41424344]

By minute identification of these miRNAs and related targets and mechanisms, specific therapeutics can be developed for suppression of these pathophysiological pathways through miRNAs enhancement or inhibition.[4142]

Dysbiosis – A drug target

Previous studies depicted dysbiosis in active Celiac patients. The beneficial microbiota for instance Bifidobacteria level gets decreased, and harmful microbiota levels such as Gram-negative bacteria, such as Proteobacteria genera (Staphylococcus spp. And Bacteroides) increases in the disease state.[45464748]

Studies suggested that the imbalance in microbiota in active Celiac condition results in altered gut permeability, which could be deleterious for immune activation. Probiotic therapy, along with GFD could be useful to ameliorate the dysbiosis, which ultimately prevents gliadin toxicity and immune activation.

Genetic susceptibility – A drug target

Genome-wide association study (GWAS) and immunochip analyses studies found about 39 important regions which increase the susceptibility toward the disease.[4950] These targets are crucial for future drug therapy approaches.

Thymocyte-expressed molecule involved in selection (THEMIS), which leads to T-cell maturation and thymocyte maturation into CD4+ and CD8+ T-cells, leads to a decrease in T-cell receptor signaling.[51] Once THEMIS gets activated, it decreases TCR signaling through the recruitment of SHP1.[52] TCR signaling finally affects the selection of immunological tolerant T-cells or overly self-reactive T-cells. Expression of THEMIS increases in Celiac disease, thereby making it a novel target for drug therapy.

Protein tyrosine phosphatase, receptor type, kappa (PTPRK) gene codes for transmembrane protein tyrosine phosphatase. TGF-β induces expression of PTPRK and modulates T-cell development which involves the progression of Celiac disease.[53] The expression of PTPRK decreases in Celiac disease. In the PTPRK knockout animal, IgG decreases, which ultimately decreases the T-helper function.[54] PTPRK maintain cell-cell adhesion, thereby maintaining the membrane permeability.[55]

Α-1,2-fucosyltransferase (FUT2) catalyzes the transformation of the type-1 precursor into H-antigen type-1 through the addition of α-1,2-linked fucose.[56] Now H-antigen type-1 acts as a precursor of A, B-blood group antigen, which mainly expressed in mucus, gastrointestinal mucosa and served as an anchor for the microbes. It is associated with an increased susceptibility toward Celiac disease.[57]

Polymorphisms of B-cell specific transcriptional repressor (BACH 2) associates with allergies and autoimmune disease. BACH2 in T-cells are important for regulatory T-cells, and naïve T-cells state.[50585960] BACH2 helps in maintaining immune homeostasis, which is vital for T-cell differentiation.[61] It is interesting to validate its role in Celiac disease.

Previous studies have observed that SNPs in the RGS1 gene lead to an autoimmune condition such as Celiac disease, type-1 diabetes, and multiple sclerosis.[626364] RGS1 encodes for regulator G-protein signaling-1 which modulates chemokine-induced GPCR activity. It also regulates cell chemotaxis.[6566] It has been found that the mRNA expression of RGS1 gets increased more in the gut than in peripheral blood during inflammation.[67] In Celiac disease, the SNPs for RGS1 affect its transcriptional regulation.

Conclusion

There is a dearth of drug therapy in Celiac disease. The only recommended therapy for Celiac disease is a GFD throughout life which is difficult to follow. The ideal therapy for the disease would provide the opportunity to the Celiac disease patients to include gluten in their diet. There are many promising therapeutic strategies that are under preclinical and clinical phases

IL-15 inhibitor (AMG 714) has shown to be promising in the Phase 2 clinical trial. Zonulin antagonist (Larazotide Acetate) also proved to be potential therapy. Glutenases for instance ALV003 seem to be more beneficial and is under Phase 2 clinical trials. Many promising molecules are at early stages of research such as TG2 inhibitors, HLA blockers, and probiotics

Drug therapies targeting miRNA which alter in Celiac disease are promising while GWAS studies also paved the way to find novel drug targets. For validation these targets require further studies with large sample size

The lacuna in the development of drugs for Celiac disease is the unavailability of suitable animal model for the disease which can be used to evaluate therapeutic approaches. Hence, it is the need of the hour to establish and validate suitable animal model for the disease

With the increase in understanding of the mechanism of Celiac disease, numerous novel targets for the disease have identified, which provided a scope to develop new drug strategies for Celiac disease. The current and future therapies are targeting to provide an alternate of GFD instead of searching adjuvant therapy.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.