Improved Glucocorticoid Receptor Ligands: Fantastic Beasts, but How to Find Them?

PERSPECTIVE

published: 24 September 2020

doi: 10.3389/fendo.2020.559673

Frontiers in Endocrinology | www.frontiersin.org 1 September 2020 | Volume 11 | Article 559673

Edited by:

Ines Pineda-Torra,

University College London,

United Kingdom

Reviewed by:

Andrew Alt,

University of Michigan, United States

Andrew C. B. Cato,

Karlsruhe Institute of Technology

(KIT), Germany

*Correspondence:

Karolien De Bosscher

[email protected]

Specialty section:

This article was submitted to

Molecular and Structural

Endocrinology,

a section of the journal

Frontiers in Endocrinology

Received: 06 May 2020

Accepted: 26 August 2020

Published: 24 September 2020

Citation:

Van Moortel L, Gevaert K and

De Bosscher K (2020) Improved

Glucocorticoid Receptor Ligands:

Fantastic Beasts, but How to Find

Them? Front. Endocrinol. 11:559673.

doi: 10.3389/fendo.2020.559673

Improved Glucocorticoid Receptor

Ligands: Fantastic Beasts, but How

to Find Them?

Laura Van Moortel

1,2,3

, Kris Gevaert

2,3

and Karolien De Bosscher

1,2,3

Translational Nuclear Receptor Research (TNRR) Laboratory, VIB, Ghent, Belgium,

VIB Center for Medical Biotechnology,

VIB, Ghent, Belgium,

Department of Biomolecular Medicine, Ghent University, Ghent, Belgium

Exogenous glucocorticoids are widely used in the clinic for the treatment of inﬂammatory

disorders and hematological cancers. Unfortunately, their use is associated wit h

debilitating side effects, including hyperglycemia, osteoporosis, mood swings, and

weight gain. Despite the continued efforts of pharma as well as academia, the search

for so-called selective glucocorticoid receptor modulators (SEGRMs), compounds with

strong anti-i nﬂammatory or anti-cancer properties but a reduced number or level of side

effects, has had limited success so far. Although monoclonal antibody therapies have

been successfully introduced for the treatment of certain disorders (such as anti- TNF for

rheumatoid arthritis), glucocorticoids remain the ﬁrst-in-line option for many other chronic

diseases including asthma, multiple sclerosis, and multiple myeloma. This perspective

offers our opinion on why a continued search for SEGRMs remains highly relevant in an

era where small molecules are sometimes unrightfully considered old-fashioned. Besides

a discussion on which bottlenecks and pitfalls might have been overlooked in the past, we

elaborate on potential solutions and recent developments t hat may push future research

in the right direction.

Keywords: glucocorticoids, glucocorticoid receptor, selective GR modulators, drug discovery, inﬂammation, assay

development, GR, SEGRM

INTRODUCTION

Glucocorticoids (GCs) are endogenous steroidal hormones involved in metabolism, stress,

development, and immunity (

1). They exert their eﬀects by binding the glucocorticoid receptor

(GR), a nuclear receptor (NR) consisting of an intrinsically disordered N-terminal domain (NTD),

a central DNA binding domain (DBD), a hinge region (HR), and a C-terminal ligand-binding

domain (LBD) (2). Upon ligand binding, GR typically translocates from the cytoplasm to

the nucleus where it acts as a genuine transcription factor to regulate target gene expression

via multiple mechanisms (Figure 1A), which are discussed in detail in (3). The discovery of

the anti-inﬂammatory eﬀects of endogenous GCs preceded the development of synthetic GCs,

which are used to treat, among others, inﬂammatory disorders, and hematological cancers (4).

Unfortunately, the therapeutic eﬃcacy of such exogenous GCs is, particularly for systemic use,

overshadowed by an unacceptably high number of undesired side eﬀects such as hyperglycemia,

osteoporosis, mood swings, and weight gain (

5).

Van Moortel et al. Improved Glucocorticoid Receptor Ligands

FIGURE 1 | Overview of glucocorticoid receptor activity with classic glucocorticoids and selective GR modulators. (A) General action mechanism of the glucocorticoid

receptor (GR). Glucocorticoids (GCs) diffuse through the cellular membrane and bind GR. The latter dissociates from its chaperone complex and migrates to the

nucleus. There, it dimerizes and binds glucocorticoid response elements (GREs) to upregulate downstream target genes. Monomeric GR also undergoes

protein-protein interactions with DNA-bound pro-inﬂammatory transcription factors (TFs) to downregulate their activity, or it binds directly to the TF response elements

(TF-RE). (B) Distinct actions of classic GCs and selective GR modulators (SEGRMs). In contrast to classic GCs, SEGRMs are hypothesized to reduce GR’s capacity to

dimerize and therefore reduce GRE-mediated transcription. Interference with TF activity is driven via monomeric GR and therefore maintained with SEGRMs.

Frontiers in Endocrinology | www.frontiersin.org 2 September 2020 | Volume 11 | Article 559673

Van Moortel et al. Improved Glucocorticoid Receptor Ligands

Some of these side eﬀects stem from direct binding of

homodimeric GR to pseudopalindromic glucocorticoid

reponse elements (GREs) in the promoter regions of genes

controlling key metabolic pathways (Figure 1). The resulting

GRE-driven upregulation of tyrosine aminotransferase (TAT),

glucose 6-phosphatase (G6P) and phosphoenolpyruvate

carboxykinase (PEPCK) for instance leads to hyperglycemia

(

6). The suppression of nuclea r factor (NF)-κB- and activator

protein (AP)-1 activity on the other hand, is typically explained

via protein-protein interactions with monomeric GR (called

tethering) (7). Despite the controversies on the actual underlying

mechanism (see further), the targeting of activities of pro-

inﬂammatory transcription factors undoubtedly contributes

substantially to the anti-inﬂammatory actions of GCs.

The discrepancy between monomer- and dimer-driven eﬀects

of GR was ﬁrst suggested in 1994 with the demonstration that GR

with a dimerization-disrupting mutation in the DBD (GRdim) is

still able to repress AP-1-driven genes, while no longer able to

induce GRE-mediated activation (

8). Four years later, Reichardt

et al. established that mice carrying this homozygous mutation

were viable and healthy, in contrast to GR full knock-out mice

(9), arguing for an equally viable mechanistic basis to separate

beneﬁcial from undesired eﬀects. This was the starting point of

the sea rch for so-called dissociative or selective GR modulators

(SEGRMs), GR ligands that can still repress inﬂammation via

monomer-driven NF-κB, and AP-1 inhibition, while no longer

inducing GRE-driven side eﬀects (Figure 1B).

In the meantime, a few shades of gray have been added

to the original black-and-white monomer-dimer paradigm.

First of all, dimer-mediated gene activation also contributes

to the anti-inﬂammatory eﬀects of GCs via the upregulation

of anti-inﬂammatory genes such as glucocorticoid-induced

leucine zipper (GILZ) and dual speciﬁcity phosphatase (DUSP1)

(

10). This helps explaining why GRdim mice show increased

sensitivity to acute inﬂammation such as septic shock (11).

Secondly, it was shown in cellulo that Dex still promotes

dimerization of the GRdim mutant (12). However, introducing

an extra point mutation in th e GR LBD almost completely

disrupted dimerization and abrogated GRE-driven activity, but

preser ved the inhibition of NF-κB activity. Thirdly, monomeric

GR was shown to bind directly to genomic NF-κB and AP-1

response elements, without the need for the transcription factor

itself (

13, 14). This ﬁnding challenges the original tethering

hypothesis but still supports the notion that suppression of NF-

κB and AP-1 activities does not require GR dimerization. Taken

together, given the bodies of evidence on a large contribution of

dimeric GR to particular side eﬀects vs. the role of GR monomers

to support anti-inﬂammatory actions in a chronic setting, the

notion that compounds that favor signaling via monomeric GR

can hold a therapeutic beneﬁt against persistent inﬂammation,

still stands.

The development of successful SEGRMs has proven to be a

long and extremely bumpy road. Many compounds that showed

promising initial results (listed in Table 1) never got past the

pre-clinical stage or failed la ter on in clinical trials. It is well-

known that the success rate for the development of any kind of

small molecule drug from bench to clinic is very low, typically

starting from 10,000 compounds to end up with one market-

approved drug (44, 45). In addition, we believe that in the case of

GR, multiple technical, and biological f actors have been reducing

the prospect to success even further. Fortunately, molecules

are still being developed, trying to meet the hope of many

patients wh o would beneﬁt from GR modulators. For instance,

AZD7495 (asthma, NCT03622112) and AZD9567 (rheumatoid

arthritis, NCT03368235) are currently under evaluation in

clinical trials.

This perspective oﬀers our opinion as molecular biologists

on the rationale why a continued search for SEGRMs still

makes sense and bears signiﬁcant relevance. We oﬀer our

view on a number of bottlenecks and pitfalls that might

have hampered research progress in the past and elaborate on

which new developments and insights could help overcome

these issues.

SEGRMs: THE UNMET MEDICAL NEED

The need for more selective GR ligands remains highly

relevant. Although more targeted therapies have successfully

been introduced, such as anti-tumor necrosis factor (TNF)

for arthritic disorders and inﬂammatory bowel diseases (IBD),

these therapies are not without limitations. For one, anti-

TNF t herapy has been associated with a 250% increase in the

occurrence of tuberculosis (

46). Furthermore, these therapies

have been reported to trigger multiple sclerosis (MS) and other

demyelinating conditions (

47–50). This is in line with the

reported disease worsening in patients with pre-existing MS in

clinical trials for Lenercept and cA2, two types of anti-TNF

therapy (51, 52). Beside such side eﬀects, monoclonal therapies

are generally very expensive, laying a huge burden on hea lth

care systems, which will only increase with aging populations

in western countries. Their price also makes them unaﬀordable

for most people in low income countries, which is particularly

a problem for asthma, for which 80% of disease-related deaths

occur in low to low-medium income countries (53).

GCs on the other hand are generally much cheaper and

are still the ﬁrst-line treatment for asthma, multiple sclerosis,

and multiple myeloma among others (

54–56). However, their

side eﬀects are a well-known problem and not necessarily

limited to patients receiving oral or intravenous GCs. While

topical skin treatments, especially with t he newest generation

glucocorticoids, impose a very low risk for systemic side eﬀects

(57–59), topic al eye treatments, and inhaled GCs (IGCs) have

both been associated with adrenal suppression (60, 61). This can

lead to growth retardation in infants and children, who form a

large cohort of t h e asthma patient population. The long-term use

of high doses IGCs has also been associated with decreased bone

mineral density in both children and adults (62–64). Although

the beneﬁts of ocular and IGCs usually outweigh the risks,

patients would still beneﬁt from GCs with lower risks for syst emic

side eﬀe c ts.

Taken together, the need for more selective GCs reaches

further than systemic treatments and is also high for ocular and

inhalation therapies.

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Van Moortel et al. Improved Glucocorticoid Receptor Ligands

TABLE 1 | Available pre-clinical data of SEGRMs.

Compound In vitro assays and in cellulo

overexpre ssion a ssays

In cellulo assays for endogenous

anti-inﬂammatory and/or side effect

targets

Inﬂammatory animal models Status and latest

progress

References

LGD-5552 Ligand-binding assays GR, AR, MR,

MMTV-luciferase in CV-1 cells

(overexpressed GR)

E-selectin-luciferase in CV-1 and

HepG2 cells (overexpressed GR)

IL-6-luciferase in HepG2 cells

(overexpressed GR)

Cofactor binding assays

PEPCK and PDK4 mRNA in H4IIE cells

COX2 and APOCIII mRNA in H4IIE cells

POMC mRNA in ATT20 cells

Collagen-induced arthritis in

mice

Freund’s complete

adjuvant-induced arthritis in rats

Experimental autoimmune

encephalomyelitis in rats

Discontinued (preclinical) (

15, 16)

AL-438 Ligand-binding assays GR, PR

RSV-LTR-GRE-luciferase in CV-1 cells

(overexpressed GR)

TAT-luciferase in HepG2 cells

(overexpressed GR)

E-selectin-luciferase in HepG2 cells

(overexpressed GR)

Cofactor binding assays

Eosinophil counts in BAL

Human PBMC cell and rat splenocyte T-cell

proliferation assays

Osteocalcin protein in MG-63 cells

Aromatase activity in hDSF cells

IL-6 release in HSKF1501 cells

Carrageen-induced paw edema

in rats

Freund’s complete

adjuvant-induced arthritis in rats

Discontinued (preclinical) (

17–19)

MK-5932 MMTV-luciferase in A549 cells

MMTV-luciferase in HeLa cells

TNFα-β-lactamase in U937 cells

TNFα, IFNγ, IL-1β, IL-6 secretion in human

whole blood

TNFα, IL-6 secretion in rat whole blood

Oxazolone-induced contact

dermatitis in rats

Discontinued (preclinical) (

20, 21)

GW870086 Functional selectivity MR, AR, PR, ER

on MMTV-luciferase in CV-1 cells

MMTV-LTR-luciferase in A549 and

MG-63 cells

E-selectin-κB-RE-alkaline

phosphatase in A549 cells

Lymphotoxin-β, COX-2, Cyp24a1, MAP-7,

GPR64, GILZ, DUSP1, MICAL2, FKBP5,

CDKN1C, RGS2, SGK mRNA in A549 cells

Delayed-type oxazolone-induced

contact hypersensitivity in mice

Ovalbumin-induced airway

inﬂammation in mice

Discontinued

Phase II for asthma: no

difference with placebo

(NCT00945932)

Phase II for atopic

dermatitis: weaker effects

than ﬂuticasone propionate

standard (NCT01299610)

(

22–24)

(Continued)

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Van Moortel et al. Improved Glucocorticoid Receptor Ligands

TABLE 1 | Continued

Compound In vitro assays and in cellulo

overexpre ssion a ssays

In cellulo assays for endogenous

anti-inﬂammatory and/or side effect

targets

Inﬂammatory animal models Status and latest

progress

References

BI653048 Ligand-binding assays GR, PR

MMTV-luciferase in HeLa cells

IL-6 release in CCD-1112Sk cells

Osteocalcin levels in MG-63 cells

Human ERG potassium channel inhibition in

Hek293T cells

Canine low dose endotoxemia

model

Discontinued

Phase I: no improvement on

side effect proﬁle compared

to prednisolone

(NCT02217631,

NCT02224105, NCT02217644)

(

25–27)

Mapracorat Ligand-binding assays GR, PR, AR, MR

MMTV-luciferase in HeLa cells

Collagenase-luciferase in HeLa cells

κB-RE-luciferase in SV-40 transformed

hCEpiC cells

TPA-RE-luciferase in SV-40 transformed

hCEpiC cells

TAT activity in HepG2 cells

IL-12p40, IFNγ secretion in PBMC cells

Eotaxin-1 (+/– GR siRNA), −3, CCL5 (+/– GR

siRNA), G-CSF, IL-6, IL-8, MCP-1 release in

hConF cells

Eotaxin-3, CCL5 (+/– GR siRNA), CCL27,

ICAM-1 (+/– GR siRNA), IL-6, IL-8, MCP-1,

TNFα release in hCEpiC cells

IL-6, MCP-1 release and (p)p38, (p)JNK protein

in SV-40 transformed hCEpiC cells

IL-6, IL-8 release in hONA cells

IL-1β, ICAM-1 release in hREC cells

IL-6, IL-12p40, MCP-1 release in THP-1 cells

(p)JNK, (p)p65, (p)p38, IκBα levels in hCEpiC

cells

MYOC levels in mkTM cells

Migration, apoptosis, IL-8 release, annexin-1,

and CXCR4 expression in human eosinophils

IL-6, IL-8, CCL5, TNFα release in hMC-1 cells

GM-CSF, TNFα, PGE2 production and COX-2,

(p)p38, (p)MK2, DUSP1 protein in Raw 264.7

cells

IL-6, IL-8, MCP-1, PGE2 release, COX-2, RelB,

(p)IκBα protein, RelA and RelB DNA binding in

human keratinocytes

Croton oil-induced irritant

contact dermatitis in mice and

rats

Dinitroﬂuorobenzene

(DNFB)-induced allergic contact

dermatitis in mice and rats Dry

eye model in rabbits

Paracentesis model in rabbits

Ovalbumin-induced allergic

conjunctivitis in guinea pigs

Compound 48/80-induced

wheal and erythema skin

inﬂammation in beagles

Discontinued

Phase III for cataract

surgery, no results reported

(NCT01591655)

Phase I for psoriasis, no

results reported

(NCT03399526)

Phase I to assess corneal

endothelial cell changes, no

results

reported (NCT01736462)

(

28–36)

(Continued)

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Van Moortel et al. Improved Glucocorticoid Receptor Ligands

TABLE 1 | Continued

Compound In vitro assays and in cellulo

overexpre ssion a ssays

In cellulo assays for endogenous

anti-inﬂammatory and/or side effect

targets

Inﬂammatory animal models Status and latest

progress

References

(Fos)dagrocorat Gal4-RE-luciferase with

Gal4-DBD-LBD in Huh7 cells

Cofactor binding assays

IL-6 release in A549 cells

IFNγ in human whole blood assays

Human pre-adipocyte differentiation

FABP4 mRNA in adipocytes

TAT, PEPCK in human primary adipocytes

Osteocalcin levels in human

primary osteoblasts

Murine LPS-induced

endotoxemia model

Discontinued

Phase II for rheumatoid

arthritis: no improved

beneﬁt-risk ratio compared

prednisone (NCT01393639)

(

37, 38)

AZD5423 Ligand-binding assays GR, MR, PR,

AR, ERα, ERβ

TPA-RE-β-galactosidase stable in

ChaGoK1 cells

TNFα-release in hPBMC cells Sephadex-induced airway

inﬂammation in rats

Discontinued

Phase II for asthma

(NCT01225549)

Phase II for

COPD (NCT01555099)

(

39–41)

AZD7594 Ligand-binding assays GR, MR, PR,

AR, ERα, ERβ

TPA-RE-β-galactosidase stable in

ChaGoK1 cells

TNFα-release in hPBMC cells Sephadex-induced airway

inﬂammation in rats

Ongoing

Second phase II for asthma

completed 11/2019

(NCT03622112)

Phase I in adolescents

ongoing (NCT03976869)

(

39, 42)

AZD9567 Ligand-binding assays GR, MR, PR,

AR, ERα, ERβ

MMTV-β-galactosidase stable in

ChaGoK1 cells

TPA-RE-β-galactosidase stable in

ChaGoK1 cells

Cofactor binding assays

TAT in primary hepatocytes

Osteoprotegerine in human fetal osteoblasts

Streptococcal cell wall

reactivation arthritis model in rats

Ongoing

Phase II for rheumatoid

arthritis completed

11/2019 (NCT03368235)

(

43)

Information on clinical trials was retrieved from clinicaltrials.gov. A549, human lung epithelial carcinoma cell line; APOCIII, apolipoprotein C18; AR, androgen receptor; ATT20, mouse pituitary tumor cell line; BAL, bronchoalveolar

lavage; C C D-1112Sk, human foreskin ﬁbroblast cell line; CCL (5), C-C motif chemokine (5); CDKN1C, cyclin-dependent kinase inhibitor 1C; hCEpiC, human corneal epithelial cells; ChaGoK1, human bronchogenic carcinoma cell line;

hConF, human conjunctival ﬁbroblasts; COX-2, cyclo-oxygenase 2; CV-1, African green monkey kidney cell line; Cyp24a1, vitamin D (3) 24-hydroxylase; CXCR4, C-X-C chemokine receptor type 4; hDSF, human dermal skin ﬁbroblasts;

ER, estrogen receptor; hERG, human ether-a-go-go potassium channel; FKBP5, 51 kDa FK506-binding protein; G(M)-CSF, granulocyte (macrophage) colony-stimulating factor; GPR-64, G-protein coupled receptor 64; H4IIE, rat

hepatocellular carcinoma cell line; Hek293T, human embryonic kidney cell line; HeLa, human cervical adenocarcinoma cell line; HepG2, human hepatocellular carcinoma cell line; HSKF1501, human foreskin ﬁbroblast cell line; Huh7,

human hepatocellular carcinoma cell line; ICAM-1, intracellular adhesion molecule 1; IFNγ , interferon γ ; Iκ Bα, NF-kappa-B inhibitor α; IL(-12p40), interleukin (12 subunit p40); (p)JNK, (phospho-)c-Jun N-terminal kinase; κ B-RE, NF-κ B

response element; LTR, long terminal repeat; MAP-7, microtubule-associated protein 7; hMC-1, human mast cell line; MCP-1, monocyte chemotactic protein 1; MG-63, human osteosarcoma cell line; MICAL2, molecule interacting

with CasL protein 2; (p)MK-2, (phospho-)mitogen-activated protein kinase-activated protein kinase 2; MMTV, mouse mammary tumor virus; MR, mineralocorticoid receptor; MYOC, myocillin; hONA, human optic nerve astrocytes;

(p)p38, (phospho-)mitogen activated protein kinase p38; (p)p65, (phospho-)nuclear factor kappa B subunit p65; PBMC, peripheral blood mononuclear cells; PDK4, pyruvate dehydrogenase kinase 4; PGE2, prostaglandin E2; POMC,

pro-opiomelanocortin; PR, progesterone receptor; Raw264.7, mouse leukemia macrophage cell line; hREC, human retinal endothelial cells; RGS2, regulator of G-protein signaling 2; RSV, Rous sarcoma virus; SGK, serum/glucocorticoid

regulated kinase; THP-1, human leukemic monocyte cell line; mkTM, monkey trabecular meshwork cells; TPA-RE, 12-O-Tetradecanoylphorbol-13-acetate response element; U937, human histiocytic lymphoma cell line.

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Van Moortel et al. Improved Glucocorticoid Receptor Ligands

BOTTLENECKS AND PITFALLS

OBSERVED IN THE PAST

Current tools for screening potential SEGRMs suﬀer from

shortcomings and do not always capture the complexity of

GR signaling. First of all, the lack of three-dimensional

structures of full-length GR highly restricts our knowledge of

GR’s structure-activity relationship and decreases the predictive

power of molecular modeling and docking studies. Additionally,

all existing crystal structures of ligands in complex with

GR’s LBD were obtained upon the introduction of one or

more mutations in this LBD. Although these mutations were

predicted not to inﬂuence the LBD structure, this can never

be claimed with absolute certainty. F602S for instance, one

of the most commonly used GR mutations allowing growth

of LBD crystals, causes chemical shift perturbations in LBD

nuclear magnetic resonance spectra compared to wild-type

LBD (

65). Furthermore, GR is allosterically regulated through

interactions with its corresponding response elements and

cofactors (

66), and more general also for other NR members,

conformational changes in one NR domain can allosterically

alter the conformation of another domain within the same NR

molecule (67). Thus, most probably the conformation of the LBD

studied in isolation is an incorrect reﬂection of this domain’s

conformation in the full-length protein.

Further, while high aﬃnity and selectivity for GR can be

captured using in vitro ligand-binding assa ys, conﬁrmations in

a cellular or in vivo context are sometimes lacking. This harbors

an inherent risk to miss out on oﬀ-target eﬀects of the compound

in question. Therefore, the conﬁrmation of GR dependency in

a cellular and an in vivo context is still an important valida tion

to make, for instance by testing compounds in wild-type vs. GR

knock-out models.

Table 1 provides an overview of the assays typically carried

out to characterize GR-mediated actions of a set of well-

known SEGRMs. To our opinion, a lack of predictive power

is one of the problems most diﬃcult to solve, especially

when moving from simpliﬁed assays to more complex biology.

Direct GRE-driven activity, potentially leading to side eﬀects,

is almost universally monitored via reporters driven by a

mouse mammary tumor virus (MMTV) promoter. Although

a fast and straightforward and thus defendable method for

initial compound characterization, a GRE-driven reporter assay

can be a poor predictor for regulation of endogenous GRE-

driven genes, as was also observed for MMTV (

15, 22).

The eﬀects of GCs are highly gene-speciﬁc and GRE-driven

activity can diﬀer depending on the sequence of the GRE

and the surrounding chromatin environment (68–70). The use

of overexpressed GR should also be avoided in such assays,

as this may lead to compound potencies and eﬃcacies t hat

are not necessarily representative for an endogenous context.

Additionally, not all side eﬀects are dimer-driven and are

therefore not predict able v ia GRE-driven reporters. Mimicking

the right gene- and context-speciﬁcity of GR activity remains one

of the greatest challenges. Making a switch from reporters driven

by minimal recombinant promoters to more physiologically

relevant promoters could already oﬀer some beneﬁt. These

promoters would ideally belong to genes that are conﬁrmed

mediators of underlying therapeutic - or side eﬀects. Validation

on a well-representative set of relevant endogenous target genes is

even more important (see below, section Potential Solutions: The

Way Forward).

Cell- and tissue-speciﬁcity of GC actions is another variable

parameter. The MMTV-driven reporter for instance showed

stronger upregulation by GW870086 in bone osteosarcoma

cells compared to lung epithelial carcinoma cells (

22). It thus

remains essential to screen compounds in cell types that are the

best proxies for the underlying therapeutic and/or side eﬀects

in vivo, for instance the use of hepatocy tes to study eﬀects

on glucose and lipid metabolism, or th e use of osteoblast or

osteoclast cell lines for drugs that would be used in arthritis

patients. Further, although characterization of compound activity

in cellulo is essential, this will always be an oversimpliﬁcation of

the situation in a living organism. Therefore, validation of an

improved therapeutic beneﬁt depends on representative animal

models. While this is readily implemented for anti-inﬂammatory

eﬀect scoring, concomitant testing of side eﬀect parameters

(such as glucose tolerance, insulin tolerance, cortisol levels,

bone mineral density) presents a bottleneck, because a longer

treatment protocol may be needed to surpass the thresholds of

measurable results for these parameters or because of species

diﬀerences (see below) (

22, 43).

Lack of translatability from animal models to human patients

is yet another hurdle to overcome. Diﬀerences in ligand activity

between species can be an underlying cause, as observed for AL-

438 and MK-5932, which both had stronger anti-inﬂammatory

eﬀects in rat vs. human blood (

17, 20). While it would be

recommended to perform initial cellular tests in human cells

as much as possible, in vivo interspecies diﬀerences remain a

hurdle in the entire ﬁeld of drug discovery and are currently

diﬃcult to overcome. Another concern is when animal models

used to study a particular disease insuﬃciently mimic the

pathology observed in humans. A careful design and set-up

of animal models remains key to study anti-inﬂammatory as

well as side eﬀects. If a well-known side eﬀect (marker) of

a classic GC in man is not observed in the animal model

used, this model will obviously have no predictive power on

(markers of) this particular side eﬀect in patients and will

therefore be unsuited to evaluate the improved beneﬁt-risk

ratio of SEGRMs over classic GCs. For instance, in a canine

model of low dose endotoxemia used to investigate the anti-

inﬂammatory and bone-sparing eﬀects of BI653048, neither

BI653048 nor prednisolone treatment aﬀected osteocalcin levels

(25). However, prednisolone does reduce bone mineral density

in dogs and decreases bone formation markers in humans after

1 day (71, 72). Indeed, in a phase I clinical trial, BI653048,

and prednisolone both caused decreased serum osteocalcin

levels (26). Studies with other SEGRMs also concluded that

osteocalcin levels in cellulo do not always reﬂect in vivo decreases

in bone density (18, 26, 27, 37, 38), casting doubts on the

value of osteocalcin as proxy for the in vivo reduction of bone

mineral density.

Lastly, notwithstanding the notion that dissociating GCs may

improve the beneﬁt-risk ratio in chronic inﬂammatory disorders,

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Van Moortel et al. Improved Glucocorticoid Receptor Ligands

a portion of the anti-inﬂammatory eﬀects of GR does remain

dimer-driven (73). Hence, the likelihood decreases for truly

dissociating compounds to match the therapeutic eﬃcacy of

the strongest classic GCs. Taking into account that some side

eﬀects, such as osteoporosis, are at least partially mediated by

monomeric GR (

18), makes the quest to ﬁnd a SEGRM that

scores better on multiple side eﬀects even more challenging.

POTENTIAL SOLUTIONS: THE WAY

FORWARD

Although pre-clinical characterization of compounds will

never suﬃce to accurately predict their eﬀects in patients,

particular improvements on current screenings could increase

the predictive power. First of all, reporter genes driven by

physiological promoters relevant for the clinical context of the

tested SEGRM should be preferred over artiﬁcial promoters. An

example could be the use of the G6P- or PEPCK-promoters in

liver cells to monitor hyperglycemia (

74, 75), or a Runt-related

transcription factor (Runx)2-driven promoter in osteoblasts

or Smad-driven promoters in osteoclasts as markers for GC-

induced osteoporosis (76, 77). A consistent and thorough

screening of endogenous targets in a relevant human cellular

context adds to importance. While monitoring GR activity in

every targeted pathway for every compound is impossible to

achieve, identiﬁcation of reliable in cellulo biomarkers with a

higher predictive power for species-independent in vivo anti-

inﬂammatory and/or side eﬀects would be a tremendous help.

This requires a full understanding of the molecular mechanisms

driving both anti-inﬂammatory and side eﬀects in human tissues

as well as in animal models. This is, particularly for side

eﬀects, not always the case. Continued eﬀorts to unravel the

underlying molecular mechanisms driving particular GC side

eﬀects are therefore crucial. However, some important side eﬀect

mediators have already been identiﬁed and could be suitable

markers. Examples are muscle ring ﬁnger (MuRF)1, atrogin-

1, and Krüppel-like factor (KLF)15 in muscle atrophy (

78),

regulated in development and DNA damage response (REDD)1

in skin (and muscle) atrophy (79, 80), and G6P and PEPCK in

liver (

74, 75). In bone, the upregulation of cleaved caspase 3

and −9 or the reduction of bone morphogenetic protein (BMP)2

and Runx2 activity are important predictors for reduction in

osteoblast numbers (81, 82), while upregulation of receptor

activator of nucle ar factor-κB ligand (RANKL)-RANK signaling

and cathepsin K activity are important markers for increased

osteoclast diﬀerentiation and activity (respectively) (76, 83).

Reduction of publication bias toward “negative results” and

joining forces between pharmaceutical companies and academic

groups should push the current boundaries and drive research

forward. At times, underlying reasons for discontinuation

of (pre-)clinical research remain enigmatic. As one concrete

example of many other examples that can be brought forward,

results from t hree completed phase III clinical trials on the

use of Mapracorat for post-operative treatment of cataract

surgery (NCT01230125, NCT01591161, and NCT01591655)

await publication, leaving fundamental scientists on the sideline

wondering why Mapracorat was never market approved. More

insights on where exactly discontinued SEGRMs failed, if those

reasons are on the scientiﬁc level, will encourage academic

labs with the right expertise to dig deeper into the underlying

causes, and create feedback-knowledge that may ﬂow back to

industrial programs.

Even though fully dissociating SEGRMs might never reach the

therapeutic eﬃcacy of the most potent classic GCs, they can still

oﬀer relevant therapeutic beneﬁt. Many inﬂammatory disorders

are characterized by a disease course that alternates between

periods of remission and exacerbation or relapse. SEGRMs might

not trigger the full-on anti-inﬂammatory casc ade that is required

to suppress an exacerbation, but might be ideal for maintenance

therapy. To maintain disease control, lower GC doses often

suﬃce. SEGRMs could match the anti-inﬂammatory eﬃcacy of

the lower dose classic GCs while still showing a reduced side

eﬀect burden. Combination of classic GCs with SEGRMs or

other therapeutic agents is another strategy to increase beneﬁt-

risk ratios. Combination of Dex with CpdA was for instance

shown to increase anti-inﬂammatory eﬀects while reducing

GRE-driven signaling in cellulo (84). Finally, the development

of compounds that do not bind the classic ligand-binding

pocket but instead target the dimerization interface might be an

interesting alternative strategy to disrupt GRE-driven signaling.

The intrinsically disordered nature of the GR NTD (85), has

so far prohibited resolving a crystal structure of full-length GR.

However, some smaller (however technically still challenging)

advances could already lead to important new insights. Crystal

structures of wild-type LBD in absence of stabilizing muta tions

would already give more conﬁdence in the reliability of current

docking approaches. Secondly, crystal structures of the DBD-

hinge-LBD portion would not only lead to a better understanding

of the structure-activity relationship of GR, but might pose extra

advantages for molecular modeling or docking studies. Since

eﬃcient GR dimerization seems to require both DBD and LBD

(

12), crystal structures of at least the DBD-hinge-LBD portion of

GR should improve predictions on those molecular entities that

are truly dimer-disrupting.

Another important emerging strategy to ﬁnd more eﬃcacious

GCs is to minimize exposure to non-inﬂamed tissues. IGCs can

for instance be optimized to undergo rapid elimination once

they enter the systemic circulation, a strategy that was applied

for the development of AZD7594 (

39). For systemic GCs, the

use of liposomal formulations is showing very promising results.

While these not only improve distribution to tissues that are

anatomically diﬃcult to reach (86–88), they can lower the side

eﬀects of systemic GCs by maximizing concentrations at the

inﬂamed tissues while minimizing distribution to other tissues

(89, 90).

While it may be utopia to try and develop compounds that

alleviate all side eﬀects, improved proﬁles for particular side

eﬀects may be achievable. Skin thinning and ocular hypertension

are for instance among the most problematic side eﬀects for

topical and ocular GC treatments, respectively (5). For systemic

treatments, liposomal formulations in combination with selective

improvement of particular side eﬀects may be a viable way

forward. Liposomal SEGRMs that do not aﬀect bone metabolism

Frontiers in Endocrinology | www.frontiersin.org 8 September 2020 | Volume 11 | Article 559673

Van Moortel et al. Improved Glucocorticoid Receptor Ligands

might for instance have an increased beneﬁt-risk ratio over classic

GCs for the treatment of arthritic disorders.

CONCLUSION

While there still seems a long road ahead toward SEGRMs

with a real improved beneﬁt-risk ratio, there is light at the

end of the tunnel. The pipeline of SEGRM compounds under

clinical evaluation is not empty and new insights from ongoing

(or future) research is expected to lead to optimized screening

tools with maximized predictive power. Additionally, strategies

to limit exposure to oﬀ-targets tissues, such as liposomal

formulations for systemic treatments, show promising results

(

86–90). Combination of these approaches with the identiﬁcation

of reliable markers to predict on-target side eﬀects, (e.g., ocular

hypertension in ocular tre atment , osteoporosis in rheumatoid

arthritis, skin thinning in topic applications) may be an eﬀective

and achievable leap forward.

DATA AVAILABILITY STATEMENT

The original contributions presented in the study are included

in the article/supplementary material, further inquiries can be

directed to t h e corresponding author/s.

AUTHOR CONTRIBUTIONS

LVM wrote the manuscript with contributions from KG and

KDB. LVM made the artwork with contributions from KG and

KDB. All authors approved the ﬁnal version.

FUNDING

LVM was supported by a strategic basic research fellowship

of the Research Foundation-Flanders (FWO-Vlaanderen), grant

number 1S14720N.

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Frontiers in Endocrinology | www.frontiersin.org 11 September 2020 | Volume 11 | Article 559673