A selective Jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. The study presents a structure-guided small-molecule and chemoproteomics approach to elucidate the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insights into the specificity determinants for cofactor, substrate, and inhibitor recognition by the KDM6 subfamily of demethylases. The researchers developed the first small-molecule catalytic site inhibitor selective for the H3K27me3-specific JMJ subfamily. This inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. The study resolves the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provides encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family. The study also shows that the H3K27me3 peptide binds to the H3K27-specific demethylase JMJD3 in the opposite orientation to KDM7A. Structural and sequence comparisons of JMJD3, UTX, and UTY suggest that these recognition determinants are preserved within the KDM6 subfamily of demethylases. The researchers exploited these structural insights to optimize a series of weakly active hits discovered from a screening of the GlaxoSmithKline corporate compound collection. The resultant lead, GSK-J1, had an IC50 of 60 nM in the JMJD3 AlphaScreen assay. A 1.8 Å co-crystal structure of GSK-J1 bound to human JMJD3 revealed the critical interactions within the active site. The propanoic acid of GSK-J1 mimics α-ketoglutarate binding by maintaining interactions with K1381, T1387, and N1480. The aromatic ring of the tetrahydrobenzazepine of GSK-J1 sits in a narrow cleft between R1246 and P1388, mimicking P30 of the histone peptide. GSK-J1 is competitive with α-ketoglutarate but non-competitive with the peptide substrate. This finding suggests that GSK-J1's mechanism does not follow a simple random model for bisubstrate inhibition. GSK-J1 is selective for the H3K27 demethylases JMJD3 and UTX and is inactive against a panel of demethylases of the JMJ family. The bidentate interaction between GSK-J1 and the catalytic metal is critical for enzyme inhibition. The pyA selective Jumonji H3K27 demethylase inhibitor modulates the proinflammatory macrophage response. The study presents a structure-guided small-molecule and chemoproteomics approach to elucidate the functional role of the H3K27me3-specific demethylase subfamily (KDM6 subfamily members JMJD3 and UTX). The liganded structures of human and mouse JMJD3 provide novel insights into the specificity determinants for cofactor, substrate, and inhibitor recognition by the KDM6 subfamily of demethylases. The researchers developed the first small-molecule catalytic site inhibitor selective for the H3K27me3-specific JMJ subfamily. This inhibitor binds in a novel manner and reduces lipopolysaccharide-induced proinflammatory cytokine production by human primary macrophages, a process that depends on both JMJD3 and UTX. The study resolves the ambiguity associated with the catalytic function of H3K27-specific JMJs in regulating disease-relevant inflammatory responses and provides encouragement for designing small-molecule inhibitors to allow selective pharmacological intervention across the JMJ family. The study also shows that the H3K27me3 peptide binds to the H3K27-specific demethylase JMJD3 in the opposite orientation to KDM7A. Structural and sequence comparisons of JMJD3, UTX, and UTY suggest that these recognition determinants are preserved within the KDM6 subfamily of demethylases. The researchers exploited these structural insights to optimize a series of weakly active hits discovered from a screening of the GlaxoSmithKline corporate compound collection. The resultant lead, GSK-J1, had an IC50 of 60 nM in the JMJD3 AlphaScreen assay. A 1.8 Å co-crystal structure of GSK-J1 bound to human JMJD3 revealed the critical interactions within the active site. The propanoic acid of GSK-J1 mimics α-ketoglutarate binding by maintaining interactions with K1381, T1387, and N1480. The aromatic ring of the tetrahydrobenzazepine of GSK-J1 sits in a narrow cleft between R1246 and P1388, mimicking P30 of the histone peptide. GSK-J1 is competitive with α-ketoglutarate but non-competitive with the peptide substrate. This finding suggests that GSK-J1's mechanism does not follow a simple random model for bisubstrate inhibition. GSK-J1 is selective for the H3K27 demethylases JMJD3 and UTX and is inactive against a panel of demethylases of the JMJ family. The bidentate interaction between GSK-J1 and the catalytic metal is critical for enzyme inhibition. The py