Challenges in IBD Research 2024: Preclinical Human IBD Mechanisms.

Preclinical human inflammatory bowel disease (IBD) mechanisms is one of 5 focus areas of the Challenges in IBD Research 2024 document, which also includes environmental triggers, novel technologies, precision medicine, and pragmatic clinical research. Herein, we provide a comprehensive overview of current gaps in inflammatory bowel diseases research that relate to preclinical research and deliver actionable approaches to address them with a focus on how these gaps can lead to advancements in IBD interception, remission, and restoration. The document is the result of multidisciplinary input from scientists, clinicians, patients, and funders and represents a valuable resource for patient-centric research prioritization. This preclinical human IBD mechanisms section identifies major research gaps whose investigation will elucidate pathways and mechanisms that can be targeted to address unmet medical needs in IBD. Research gaps were identified in the following areas: genetics, risk alleles, and epigenetics; the microbiome; cell states and interactions; barrier function; IBD complications (specifically fibrosis and stricturing); and extraintestinal manifestations. To address these gaps, we share specific opportunities for investigation for basic and translational scientists and identify priority actions.

To address the unmet medical needs of patients with inflammatory bowel diseases (IBD) and move toward cures, preclinical human-relevant research must center on mechanistic questions pertinent to patients with IBD in the 3 areas of disease interception, remission, and restoration.

Profiling drugs for rheumatoid arthritis that inhibit synovial fibroblast activation.

Activation of synovial fibroblasts (SFs) contributes to rheumatoid arthritis (RA) by damaging synovial membranes and generating inflammatory cytokines that recruit immune cells to the joint. In this paper we profile cytokine secretion by primary human SFs from healthy tissues and from donors with RA and show that SF activation by TNF, IL-1α, and polyinosinic-polycytidylic acid (Poly(I:C)) cause secretion of multiple cytokines found at high levels in RA synovial fluids. We used interaction multiple linear regression to quantify therapeutic and countertherapeutic drug effects across activators and donors and found that the ability of drugs to block SF activation was strongly dependent on the identity of the activating cytokine. (5z)-7-oxozeaenol (5ZO), a preclinical drug that targets transforming growth factor-ß-activated kinase 1 (TAK1), was more effective at blocking SF activation across all contexts than the approved drug tofacitinib, which supports the development of molecules similar to 5ZO for use as RA therapeutics.

BI 1002494, a Novel Potent and Selective Oral Spleen Tyrosine Kinase Inhibitor, Displays Differential Potency in Human Basophils and B Cells.

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor-mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor-mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.

A High-Throughput Genetic Complementation Assay in Yeast Cells Identified Selective Inhibitors of Sphingosine Kinase 1 Not Found Using a Cell-Free Enzyme Assay.

Sphingosine kinase 1 (SphK1) is a lipid kinase that phosphorylates sphingosine to produce the bioactive sphingolipid, sphingosine-1-phosphate (S1P), and therefore represents a potential drug target for a variety of pathological processes such as fibrosis, inflammation, and cancer. We developed two assays compatible with high-throughput screening to identify small-molecule inhibitors of SphK1: a purified component enzyme assay and a genetic complementation assay in yeast cells. The biochemical enzyme assay measures the phosphorylation of sphingosine-fluorescein to S1P-fluorescein by recombinant human full-length SphK1 using an immobilized metal affinity for phosphochemicals (IMAP) time-resolved fluorescence resonance energy transfer format. The yeast assay employs an engineered strain of Saccharomyces cerevisiae, in which the human gene encoding SphK1 replaced the yeast ortholog and quantitates cell viability by measuring intracellular adenosine 5'-triphosphate (ATP) using a luciferase-based luminescent readout. In this assay, expression of human SphK1 was toxic, and the resulting yeast cell death was prevented by SphK1 inhibitors. We optimized both assays in a 384-well format and screened ∼10(6) compounds selected from the Boehringer Ingelheim library. The biochemical IMAP high-throughput screen identified 5,561 concentration-responsive hits, most of which were ATP competitive and not selective over sphingosine kinase 2 (SphK2). The yeast screen identified 205 concentration-responsive hits, including several distinct compound series that were selective against SphK2 and were not ATP competitive.