NOVEL PLATFORMS FOR IDENTIFYING AND TESTING SMALL MOLECULE DRUG BASED TREATMENTS FOR INFLAMMATORY BOWEL DISEASE.

BACKGROUND: Tofacitinib and upadacitinib are JAK inhibitors (JAKi) approved for Inflammatory Bowel Disease (IBD) treatment. Response rates to JAKi in IBD are varied, and there are concerns for serious side effects like venous thromboembolism, cancer and cardiovascular events.
PURPOSE: There is a need for a deeper understanding of the diverse effects of JAKi and for designing more targeted JAKi to alleviate some of the safety concerns. We used a novel AI platform to identify small molecules that bind JAK proteins in more specific binding modalities favoring JAK1 over JAK2, JAK3, and TYK2. We tested the functional immune response to these molecules compared to approved JAK inhibitors as measured by CyTOF. CyTOF is a highly dimensional, single cell technology that uses heavy metal labeled antibodies detected by a mass spectrometer to identify up to 50 proteins per cell.
METHODS: An AI-driven Molecular Fingerprint™ Platform identified a small molecule that bound to JAK1 like tofacitinib, IBIS003. Under IRB approved protocols, blood was collected from adults not on steroids: 12 healthy adults (HA), 4 with Ulcerative Colitis (UC) and 4 with Crohn’s Disease (CD). Blood was stimulated with IL-2+IL-6 and then treated with IBIS003, tofacitinib (JAK1/JAK3i), upadacitinib (JAK1i) or ritlecitinib (JAK3i).
CyTOF identified over 15 different major innate and adaptive immune cell types and their subsets. Additionally, 14 functional proteins in the STAT, MAPK, PI3K, and NFkB pathways were evaluated. Standard bioinformatic best-practice pathways identified differences.
RESULTS: IBD samples showed distinct responses to JAKi compared to HA. For example, JAKi suppression of phospho-STAT5 in CD4+ T-Cells was seen only in IBD. Moreover, there were divergent reactions to JAKi between UC and CD. For instance, phospho-STAT5 in CD8+ T-Cells was decreased in UC but not CD in response to JAKi. IBIS003 showed JAKi-like responses in a subset of features. For example, in monocytes, IBIS003 decreased phospho-ERK-1/2 levels like tofacitinib and upadacitinib. However, IBIS003 had more of a ritlecitinib-like response in monocyte STAT signaling; IBIS003 and ritlecitinib did not decrease phospho-STAT1 or 3 levels whereas tofacitinib and upadacitinib did decrease phospho-STAT1 and 3.
CONCLUSION: We used a novel AI platform to identify IBIS003, a small molecule modeled to bind JAK1 like known JAKi. We identified immune features of JAKi across multiple cell types and signaling pathways toward a deeper understanding of their function. There were differential response signatures of UC and CD to JAKi. Importantly, we identified immune responses to IBIS003, both resembling and differing from approved JAK inhibitors. This is an important step for tailoring small molecule drug design through more specific targeting of JAK proteins toward optimizing efficacy while minimizing off-target effects.