Introduction
GLPG3970, a potent and selective small molecule salt-inducible kinase (SIK) 2/3 inhibitor, exhibits a dual mechanism modulating both pro-inflammatory and immunoregulatory pathways that holds potential as a novel therapeutic option for inflammatory bowel disease.1,2 Oral administration of GLPG3970 in colitis mouse models reduced disease activity scores, inhibited pro-inflammatory cytokine release and improved histological endpoints including protection of goblet cell health and MUC2 expression.2,3 Mechanistic investigations demonstrated a broad immunomodulatory activity on colonic immune cell types. It remains unclear whether SIK2/SIK3 inhibition exerts a direct therapeutic activity on intestinal epithelial cell types or if these effects result indirectly from modulating pro-inflammatory and immunoregulatory mechanisms.3
Aims & Methods
We aimed to unravel the activity of SIK2/SIK3 inhibition using GLPG3970 in ulcerative colitis (UC) patient-derived epithelial cells focusing on SIK target engagement markers, inflammation and mucus production. Differentiated intestinal organoids from patients with UC were exposed to vehicle control, GLPG3970 (3 µM and 20 µM) or PGE2 (20 µM; positive control) for 24 hours (gene expression) or 48 hours (immunohistochemistry). Gene expression was evaluated in organoid cultures derived from five patients with UC, with or without 24 hours of pro-inflammatory stimulation (using a mix of 100 ng/mL TNFα, 20 ng/mL IL-1β and 1 µg/mL flagellin)4 prior to 24 hours of exposure in combination with the compound, and measured by RT-qPCR compared with vehicle control (Friedman, Dunn’s multiple comparison). Immunohistochemistry was performed for MUC2, DAPI, CK14 (n = 3 patients, minimum 10 images/condition, 3 replicates). Randomized images were analysed by Imaris Software by sectioning regions of interest (two-way ANOVA, Dunnett’s multiple comparison).
Results
Upon GLPG3970 exposure, UC patient-derived organoids showed transcriptional upregulation of SIK target engagement markers such as the CREB1 activation genes NR4A1, NR4A2 and NR4A3. There was no effect with GLPG3970 on gene expression of inflammatory markers in colon epithelial cells, in the presence or absence of inflammatory stimulation, except for IL-1β and IL-8 upregulation after 24 hours of GLPG3970 exposure. Genes related to mucus production were upregulated in both noninflamed (MUC5AC, 20 µM, p < 0.01; AGR2, 20 µM, p < 0.05) and inflamed conditions (MUC2, 20 µM, p < 0.01; MUC5AC, 3 µM and 20 µM, p < 0.01) at the highest GLPG3970 concentration tested. Immunohistochemistry confirmed increased MUC2 secretion (secreted MUC2 surface × secreted MUC2 intensity) after 48 hours of GLPG3970 stimulation (3 µM, p < 0.05; 20 µM, p < 0.01); however, no significant increase was measured in absolute goblet cell number or goblet surface intensity compared with vehicle control.
Conclusion
SIK2/SIK3 inhibition by GLPG3970 showed a significant upregulation of target engagement markers as measured by the CREB activation genes NR4A1/2/3 in UC patient-derived colon epithelial cells, whereas no direct downregulation of inflammatory markers was observed. Exposure of UC patient-derived colonic organoids to GLPG3970 20 µM resulted in a signature of enhanced mucus secretion, both in the presence and absence of inflammation. Overall, these data indicate a direct impact of SIK2/SIK3 inhibition in intestinal epithelial cells with potential benefit in promoting barrier reinforcement via goblet cell mucus production in patients with active UC.
References
1. Peixoto C et al. J Med Chem 2024;67:5233–58.
2. Pereira-Fernandes A et al. [abstract] United European Gastroenterol J 2021;9:S8.
3. Lavazais S et al. [abstract] United European Gastroenterol J 2021;9:S8.
4. Arnauts K et al. Gastroenterology 2020;159:1564–67.
Disclosure
This study was funded by Galapagos NV (Mechelen, Belgium).
Editorial and publications management support was provided by PharmaGenesis London, London, UK, and was funded by Galapagos NV.
LG and KA are joint first authors.
LG and KA report no conflict of interest.
DM was an employee of Galapagos at the time of the work, and is now an employee of NovAliX.
BV reports research support from AbbVie, Biora Therapeutics (formerly Progenity), Landos Biopharma, Pfizer, Sosei Heptares and Takeda; speaker fees from Abbvie, Biogen, Bristol Myers Squibb, Celltrion, Chiesi, Falk, Ferring Pharmaceuticals, Galapagos, Janssen Pharmaceuticals, Lily, MSD, Pfizer, R-Biopharm, Sandoz, Takeda, Tillots Pharma, Truvion Healthcare and Viatris; consultancy fees from Abbvie, Alfasigma, Alimentiv, Applied Strategic, AstraZeneca, Atheneum, BenevolentAI, Biora Therapeutics (formerly Progenity), Boxer Capital, Bristol Myers Squibb, Galapagos, Guidepont, Landos, Lily, Mylan, Inotrem, Ipsos, Janssen Pharmaceuticals, Pfizer, Sandoz, Sanofi, Santa Ana Bio, Sapphire Therapeutics, Sosei Heptares, Takeda, Tillots Pharma and Viatris; and stock options from Vagustim.
SV reports speaker and/or consultancy fees from AbbVie, Abivax, AbolerIS Pharma, AgomAb Therapeutics, Alimentiv, Arena Pharmaceuticals, AstraZeneca, Biora Therapeutics (formerly Progenity), Boehringer Ingelheim, Bristol Myers Squibb, Celgene, Cytoki Pharma, Dr Falk Pharma, Ferring Pharmaceuticals, Galapagos, Genentech/Roche, Gilead Sciences, GSK, Hospira, IMIDomics, Janssen Pharmaceuticals, Johnson & Johnson, Lilly, Materia Prima, Mestag Therapeutics, MiroBio, Morphic Therapeutic, MRM Health, MSD, Mundipharma, Pfizer, ProDigest, Prometheus Biosciences, Robarts Clinical Trials, Surrozen, Takeda, Theravance Biopharma, Tillotts Pharma, VectivBio, Ventyx Biosciences and Zealand Pharma; grants from AbbVie, Galapagos, Johnson & Johnson, Pfizer and Takeda; and participation on a data safety monitoring board for Sanofi.
SDV is an employee of Galapagos.