Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain

Abstract

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is a multidomain protein that plays a critical role in maintaining DNA methylation patterns through concurrent recognition of hemimethylated DNA and histone marks by various domains, and recruitment of DNA methyltransferase 1 (DNMT1). UHRF1 is overexpressed in various cancers, including breast cancer. The tandem tudor domain (TTD) of UHRF1 specifically and tightly binds to histone H3 di- or trimethylated at lysine 9 (H3K9me2 or H3K9me3, respectively), and this binding is essential for UHRF1 function. We developed an H3K9me3 peptide displacement assay, which was used to screen a library of 44,000 compounds for small molecules that disrupt the UHRF1-H3K9me3 interaction. This screen resulted in the identification of NV01, which bound to UHRF1-TTD with a K value of 5 μM. The structure of UHRF1-TTD in complex with NV01 confirmed binding to the H3K9me3-binding pocket. Limited structure-based optimization of NV01 led to the discovery of NV03 (K of 2.4 μM). These well-characterized small-molecule antagonists of the UHRF1-H3K9me2/3 interaction could be valuable starting chemical matter for developing more potent and cell-active probes toward further characterizing UHRF1 function, with possible applications as anticancer therapeutics.

RETRACTED: The antineoplastic drug metformin downregulates YAP by interfering with IRF-1 binding to the YAP promoter in NSCLC

“…we then used fluorescein isothiocyanate-labeled IRF-1 (1−120) peptides that included the DNA-binding domain to perform peptide-displacement assays (Supplementary Fig. 3k) [22,35]. After verifying IRF-1 peptide (1–120) binding to the YAP promoter (Supplementary Fig.…”

“…An excitation wavelength of 485 nm and an emission wavelength of 528 nm were used. The experiment process and parameters were described in Senisterra G. et al [22].…”

Defining UHRF1 Domains that Support Maintenance of Human Colon Cancer DNA Methylation and Oncogenic Properties

“…Only after 6 weeks does tumor growth resume after UHRF1 depletion (EV) or for H3 mut and SRA mutant (G448D) expressing groups when there is outgrowth of cells that may escape endogenous UHRF1 depletion. Although the TTD aromatic cage is a target of current drug discovery activity (Houliston et al, 2017;Senisterra et al, 2018), the TTD mutant (Y188A) demonstrates negligible impact on CRC cell growth both in vitro and in vivo, while the PHD mutant (DAEA) strongly decreases CRC cell proliferation and tumor burden to comparable levels as UHRF1 depletion (EV) and the H3 mut . These results support the maintenance of abnormal DNA methylation by the histone-and DNA-reading PHD and SRA domains, but not the methyllysine binding TTD or RING E3 ligase domain, underpins the oncogenic functions of UHRF1 for CRC proliferation.…”

Design and Construction of a Focused DNA-Encoded Library for Multivalent Chromatin Reader Proteins

“…Over the past few years, several labs, including ours, have published small molecule inhibitors of Kme readers [53][54][55][56][57][58][59][60][61] (Figure 1). We have been utilizing a target class platform for Kme reader proteins and have gained insight into the types of molecules that bind to Kme readers [53,56,[62][63][64].…”