Epigenetic regulations are involved in numerous physiological and pathogenic processes. Among the key regulators that orchestrate epigenetic signaling are over 50 human protein lysine methyltransferases (PKMTs). Interrogating the functions of individual PKMTs can be facilitated by target-specific PKMT inhibitors. Given the emerging need of such small molecules, we envision an approach to identify target-specific methyltransferase inhibitors by screening privileged small-molecule scaffolds against diverse methyltransferases. Here we demonstrate such feasibility by identifying the inhibitors of SETD2. N-propyl sinefungin (Pr-SNF) was shown to preferentially interact with SETD2 by matching the distinct transition-state features of SETD2’s catalytically-active conformer. With Pr-SNF as a structure probe, we further revealed the dual roles of SETD2’s post-SET loop on regulating substrate access through a distinct topological reconfiguration. Privileged sinefungin scaffolds are expected to have broad use as structure and chemical probes of methyltransferases.

SETD8/SET8/Pr-SET7/KMT5A is the sole protein lysine methyltransferase (PKMT) known to monomethylate lysine 20 of histone H4 in vivo. SETD8’s methyltransferase activity has been implicated in many essential cellular processes including DNA replication, DNA damage response, transcription modulation, and cell cycle regulation. Developing SETD8 inhibitors with cellular activity is a key step toward elucidating the diverse roles of SETD8 via convenient pharmacological perturbation. From the hits of a prior high throughput screen (HTS), SPS8I1–3 (NSC663284, BVT948, and ryuvidine) were validated as potent SETD8 inhibitors. These compounds contain different structural motifs and inhibit SETD8 via distinct modes. More importantly, these compounds show cellular activity by suppressing the H4K20me1 mark of SETD8 and recapitulate characteristic S/G2/M-phase cell cycle defects as observed for RNAi-mediated SETD8 knockdown. The commonality of SPS8I1–3 against SETD8, together with their distinct structures and mechanisms for SETD8 inhibition, argues for the collective application of these compounds as SETD8 inhibitors.

Protein methyltransferases (PMTs) catalyze arginine and lysine methylation of diverse histone and nonhistone targets. These posttranslational modifications play essential roles in regulating multiple cellular events in an epigenetic manner. In the recent process of defining PMT targets, S-adenosyl-L-methionine (SAM) analogues have emerged as powerful small molecule probes to label and profile PMT targets. To examine efficiently the reactivity of PMTs and their variants on SAM analogues, we transformed a fluorogenic PMT assay into a ready high throughput screening (HTS) format. The reformulated fluorogenic assay is featured by its uncoupled but more robust character with the first step of accumulation of the commonly-shared reaction byproduct S-adenosyl-L-homocysteine (SAH), followed by SAH-hydrolyase-mediated fluorogenic quantification. The HTS readiness and robustness of the assay were demonstrated by its excellent Z′ values of 0.83–0.95 for the so-far-examined 8 human PMTs with SAM as a cofactor (PRMT1, PRMT3, CARM1, SUV39H2, SET7/9, SET8, G9a and GLP1). The fluorogenic assay was further implemented to screen the PMTs against five SAM analogues (allyl-SAM, propargyl-SAM, (E)-pent-2-en-4-ynyl-SAM (EnYn-SAM), (E)-hex-2-en-5-ynyl-SAM (Hey-SAM) and 4-propargyloxy-but-2-enyl-SAM (Pob-SAM)). Among the examined 8×5 pairs of PMTs and SAM analogues, native SUV39H2, G9a and GLP1 showed promiscuous activity on allyl-SAM. In contrast, the bulky SAM analogues, such as EnYn-SAM, Hey-SAM and Pob-SAM are inert toward the panel of human PMTs. These findings therefore provide the useful structure-activity guidance to further evolve PMTs and SAM analogues for substrate labeling. The current assay format is ready to screen methyltransferase variants on structurally-diverse SAM analogues.

No abstract

CARM1 is a cancer-relevant protein arginine methyltransferase that regulates many aspects of transcription. Its pharmacological inhibition is a promising anti-cancer strategy. Here SKI-73 (6a in this work) is presented as a CARM1 chemical probe with pro-drug properties. SKI-73 (6a) can rapidly penetrate cell membranes and then be processed into active inhibitors, which are retained intracellularly with 10-fold enrichment for several days. These compounds were characterized for their potency, selectivity, modes of action, and on-target engagement. SKI-73 (6a) recapitulates the effect of CARM1 knockout against breast cancer cell invasion. Single-cell RNA-seq analysis revealed that the SKI-73(6a)-associated reduction of invasiveness acts by altering epigenetic plasticity and suppressing the invasion-prone subpopulation. Interestingly, SKI-73 (6a) and CARM1 knockout alter the epigenetic plasticity with remarkable difference, suggesting distinct modes of action for small-molecule and genetic perturbations. We therefore discovered a CARM1-addiction mechanism of cancer metastasis and developed a chemical probe to target this process.