Tumor necrosis factor α (TNF-α) is a central driver of inflammation in autoimmune conditions such as Crohn's disease and rheumatoid arthritis (RA). Targeting epigenetic regulators involved in cytokine expression holds therapeutic promise, yet the precise role of the CBP/EP300 bromodomains (BRDs) in modulating immune responses remains poorly understood. Here, we introduce a distinct class of selective CBP/EP300-BRD inhibitors based on a unique 3-methylcinnoline acetyl-lysine mimic, identified through high-throughput fragment docking. These inhibitors significantly reduce TNF-α-driven cytokine expression in vitro by blocking NFκB signaling in immune cells. In vivo, BRD inhibition led to a robust anti-inflammatory effect, decreasing cytokine secretion (including IL-1β, MCP-1, IL-1α, and IL-6) and preventing immune cell migration to inflamed lymph nodes in a TNF-α-stimulated murine model. Our findings highlight CBP/EP300-BRDs as promising targets for autoimmune therapy, with these non-cytotoxic inhibitors offering a potential complementary approach for RA and other TNF-α-mediated inflammatory conditions.
© 2025 The Authors. Published by American Chemical Society.

The complex of methyltransferase-like proteins 3 and 14 (METTL3-14) is the major enzyme that deposits N6-methyladenosine (m6A) modifications on messenger RNA (mRNA) in humans. METTL3-14 plays key roles in various biological processes through its methyltransferase (MTase) activity. However, little is known about its substrate recognition and methyl transfer mechanism from its cofactor and methyl donor S-adenosylmethionine (SAM). Here, we study the MTase mechanism of METTL3-14 by a combined experimental and multiscale simulation approach using bisubstrate analogues (BAs), conjugates of a SAM-like moiety connected to the N6-atom of adenosine. Molecular dynamics simulations based on crystal structures of METTL3-14 with BAs suggest that the Y406 side chain of METTL3 is involved in the recruitment of adenosine and release of m6A. A crystal structure with a BA representing the transition state of methyl transfer shows a direct involvement of the METTL3 side chains E481 and K513 in adenosine binding which is supported by mutational analysis. Quantum mechanics/molecular mechanics (QM/MM) free energy calculations indicate that methyl transfer occurs without prior deprotonation of adenosine-N6. Furthermore, the QM/MM calculations provide further support for the role of electrostatic contributions of E481 and K513 to catalysis. The multidisciplinary approach used here sheds light on the (co)substrate binding mechanism, catalytic step, and (co)product release, and suggests that the latter step is rate-limiting for METTL3. The atomistic information on the substrate binding and methyl transfer reaction of METTL3 can be useful for understanding the mechanisms of other RNA MTases and for the design of transition state analogues as their inhibitors.
© 2023, Corbeski et al.

Methylation of adenine N6 (m6A) is the most frequent RNA modification. On mRNA, it is catalyzed by the METTL3-14 heterodimer complex, which plays a key role in acute myeloid leukemia (AML) and other types of blood cancers and solid tumors. Here, we disclose the first proteolysis targeting chimeras (PROTACs) for an epitranscriptomics protein. For designing the PROTACs, we made use of the crystal structure of the complex of METTL3-14 with a potent and selective small-molecule inhibitor (called UZH2). The optimization of the linker started from a desfluoro precursor of UZH2 whose synthesis is more efficient than that of UZH2. The first nine PROTAC molecules featured PEG- or alkyl-based linkers, but only the latter showed cell penetration. With this information in hand, we synthesized 26 PROTACs based on UZH2 and alkyl linkers of different lengths and rigidity. The formation of the ternary complex was validated by a FRET-based biochemical assay and an in vitro ubiquitination assay. The PROTACs 14, 20, 22, 24, and 30, featuring different linker types and lengths, showed 50% or higher degradation of METTL3 and/or METTL14 measured by Western blot in MOLM-13 cells. They also showed substantial degradation on three other AML cell lines and prostate cancer cell line PC3.
© 2024 The Authors. Published by American Chemical Society.

The small GTPase Cdc42 is an integral component of the cytoskeleton, and its dysregulation leads to pathophysiological conditions, such as cancer. Binding of Cdc42 to the scaffold protein IQGAP1 stabilizes Cdc42 in its active form. The interaction between Cdc42 and IQGAP1 enhances migration and invasion of cancer cells. Disrupting this association could impair neoplastic progression and metastasis; however, no effective means to achieve this has been described. Here, we screened 78,500 compounds using a homogeneous time resolved fluorescence-based assay to identify small molecules that disrupt the binding of Cdc42 to IQGAP1. From the combined results of the validation assay and counter-screens, we selected 44 potent compounds for cell-based experiments. Immunoprecipitation and cell viability analysis rendered four lead compounds, namely NCGC00131308, NCGC00098561, MLS000332963 and NCGC00138812, three of which inhibited proliferation and migration of breast carcinoma cells. Microscale thermophoresis revealed that two compounds bind directly to Cdc42. One compound reduced the amount of active Cdc42 in cells and effectively impaired filopodia formation. Docking analysis provided plausible models of the compounds binding to the hydrophobic pocket adjacent to the GTP binding site of Cdc42. In conclusion, we identified small molecules that inhibit binding between Cdc42 and IQGAP1, which could potentially yield chemotherapeutic agents.
© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.

The cAMP-responsive element binding protein (CREB) binding protein (CBP) and adenoviral E1A-binding protein (P300) are two closely related multifunctional transcriptional coactivators. Both proteins contain a bromodomain (BrD) adjacent to the histone acetyl transferase (HAT) catalytic domain, which serves as a promising drug target for cancers and immune system disorders. Several potent and selective small-molecule inhibitors targeting CBP BrD have been reported, but thus far small-molecule inhibitors targeting BrD outside of the BrD and extraterminal domain (BET) family are especially lacking. Here, we established and optimized a TR-FRET-based high-throughput screening platform for the CBP BrD and acetylated H4 peptide. Through an HTS assay against an in-house chemical library containing 20 000 compounds, compound DC_CP20 was discovered as a novel CBP BrD inhibitor with an IC50 value of 744.3 nM. This compound bound to CBP BrD with a KD value of 4.01 μM in the surface plasmon resonance assay. Molecular modeling revealed that DC_CP20 occupied the Kac-binding region firmly through hydrogen bonding with the conserved residue N1168. At the celluslar level, DC_CP20 dose-dependently inhibited the proliferation of human leukemia MV4-11 cells with an IC50 value of 19.2 μM and markedly downregulated the expression of the c-Myc in the cells. Taken together, the discovery of CBP BrD inhibitor DC_CP20 provides a novel chemical scaffold for further medicinal chemistry optimization and a potential chemical probe for CBP-related biological function research. In addition, this inhibitor may serve as a promising therapeutic strategy for MLL leukemia by targeting CBP BrD protein.