Fenebrutinib, a Bruton's tyrosine kinase inhibitor, blocks distinct human microglial signaling pathways

Bruton's tyrosine kinase (BTK) is an intracellular signaling enzyme that regulates B cell and myeloid cell functions. Due to its involvement in both innate and adaptive immune compartments, inhibitors of BTK have emerged as a therapeutic option in autoimmune disorders such as multiple sclerosis (MS). Brain penetrant small molecule BTK inhibitors may also help to address compartmentalized neuroinflammation which is proposed to underlie MS disease progression. BTK is expressed by microglia, the resident innate immune cells of the brain, however the precise roles of microglial BTK and the impact of BTK inhibitors on microglial functions is still being elucidated. Much research to date has also focused on the effects of BTK inhibitors using rodent disease models. Here we characterize the pharmacological and functional properties of fenebrutinib (FEN), a potent, highly selective, noncovalent, reversible BTK inhibitor, in human microglia and complex human brain cell systems including brain organoids. We find that FEN blocks the effects of microglial Fc?R activation including cytokine and chemokine release, microglial clustering and neurite damage in diverse human brain cell systems. Gene expression analyses identified pathways linked to inflammation, matrix metalloproteinase production and cholesterol metabolism that were modulated by FEN treatment. In contrast, FEN had no significant impact on human microglial pathways linked to TLR4 or NLRP3 signaling nor myelin phagocytosis. Our study increases the understanding of BTK functions in human microglial signaling relevant to MS pathogenesis and suggests that FEN could attenuate detrimental microglial activity associated with Fc?R activation in MS patients. Overall design: Bulk RNA sequencing of iPSC-derived brain tricultures (neurons, astrocytes, microglia) and immunocompetent human brain organoids