Pharmacological inhibition of PRMT7 links arginine monomethylation to the cellular stress response

Abstract

Protein arginine methyltransferases (PRMTs) regulate diverse biological processes and are increasingly being recognized for their potential as drug targets. Here we report the discovery of a potent, selective, and cell-active chemical probe for PRMT7. SGC3027 is a cell permeable prodrug, which in cells is converted to SGC8158, a potent, SAM-competitive PRMT7 inhibitor. Inhibition or knockout of cellular PRMT7 results in drastically reduced levels of arginine monomethylated HSP70 family stress-associated proteins. Structural and biochemical analyses reveal that PRMT7-driven in vitro methylation of HSP70 at R469 requires an ATP-bound, open conformation of HSP70. In cells, SGC3027 inhibits methylation of both constitutive and inducible forms of HSP70, and leads to decreased tolerance for perturbations of proteostasis including heat shock and proteasome inhibitors. These results demonstrate a role for PRMT7 and arginine methylation in stress response.

Fig. 1. SGC8158 is a potent and selective PRMT7 inhibitor in vitro.

a Structures of HTS hit compound SGC0911, potent compound SGC8172, active component of the chemical probe SGC8158 and its negative control SGC8158N. b SGC8158 inhibits PRMT7 in vitro with IC₅₀ of <2.5 nM, whereas negative control compound SGC8158N has IC₅₀ of 14 ± 2 μM, (n = 3 biological replicates, mean ± SEM). c SGC8158 is selective against a panel of 35 proteins, DNA, and RNA methyltransferases. IC₅₀ values are represented by colored circles indicated top left of the panel. d Crystal structure of MmPRMT7 in complex with SGC8158. MmPRMT7 is shown in cartoon representation in cyan, hydrophobic pocket residues are shown in cyan sticks, and SGC8158 is in orange. The THW motif loop region is highlighted in red with dashed lines representing the unmodeled H313 and W314 residues. e For comparison, the crystal structure of MmPRMT7_SAH (PDB ID: 4C4A) is shown in cartoon representation in yellow, hydrophobic pocket residues are shown in yellow sticks, and SAH is in pink. The THW motif loop region is highlighted in green. f Comparison of the THW motif loop region of MmPRMT7_SGC8158 (in cyan) with that of PRMT5 (PDB ID: 5GQB) (in magenta), and PRMT1, 2, 3, 4, 6, and 8 (in gray) (PDB IDs: 1OR8, 5FUL, 2FYT, 2V74, 4Y30, and 5DST, respectively). SGC8158 is shown in orange sticks and SAH in gray sticks. Source data are provided as a Source Data file.

Fig. 2. Identification of PRMT7 substrates.

a Localization of exogenous FLAG-tagged PRMT7 as analyzed by immunofluorescence. Scale bar—25 µm. Green—FLAG, Blue—Hoechst dye (nucleus). The experiment repeated independently three times with similar results. b Cellular fractionation of endogenous PRMT7 in several cell lines. C cytoplasmic fraction, N nuclear fraction. Tubulin indicates β-Tubulin control. The experiment was repeated independently twice for HEK293T cells with similar results. c Volcano plot showing log2 heavy/light ratio of SILAC-labeled monomethyl arginine peptides from WT (L, unlabeled) relative to PRMT7 KO (H, heavy RK labeled) HCT116 cells. Dashed lines represent significance cut-offs of H/L ratio < −1 and adjusted p-value < 0.01 (n = 4). Labeled points, further highlighted in red, correspond to reported Rme1 sites found in the PhosphoSitePlus® v6.5.8. p-values from four independent replicates calculated by empirical Bayes moderated t-tests and adjusted using the Benjamini–Hochberg procedure as implemented in the Bioconductor package limma (v3.38.3). d Cellular component gene ontology terms associated with 27 significantly depleted arginine methylation events in PRMT7 KO relative to WT cells identified in c. e HSP family sequence alignment showing HSPA8 R469 resides in a highly conserved region where R469 is boxed in red. Source data are provided as a Source Data file.

Fig. 3. HSP70 R469 is methylated by PRMT7 in cells.

a PRMT7 (P7) knockout (KO) or knockdown (KD) reduces HSP70 methylation in various cell lines. 94A, 21B—HCT116 CRISPR PRMT7 KO clones; P parental C2C12; C-C2C12 expressing control guide RNA; 32 C2C12 CRISPR clone expressing PRMT7 catalytic mutant (delY35,A35S); 4,57,74—C2C12 CRISPR Prmt7 KO clones. PRMT7 was knocked down in HEK293T and MCF7 cells using siRNA, Con control, P7KD PRMT7 knockdown. b Monomethylation of inducible and constitutive HSP70 is PRMT7-dependent. MDA-MB-231 cells were transfected with PRMT7 siRNA for 3 days, heat-shocked for 1 h at 42 °C and analyzed 24 h after heat shock. c Only wild-type PRMT7 is able to rescue the HSP70 arginine monomethylation in HCT116 PRMT7 KO cells. Cells were transfected with GFP-tagged PRMT7 WT or catalytic mutant (R44A). d HSP70 R469A mutation blocks PRMT7-mediated methylation of HSPA8 and HSPA1. HCT116 PRMT7 KO cells were co-transfected with FLAG-tagged PRMT7 WT or R44A mutant and GFP-tagged HSPA8 or HSPA1 WT or R469A mutant. HSPA1/8-GFP was immunoprecipitated and analyzed for arginine monomethylation levels. The HSP70 methylation in MCF7, HCT116, and HEK293T cells was analyzed in cytoplasmic fraction to avoid unspecific band overlap. The experiments in a–d were repeated independently at least three times with similar results. Source data are provided as a Source Data file.

Fig. 4. PRMT7 monomethylation of HSP70 depends on the open (ATP-bound) form of HSP70.

a–c HSP70 structures in closed and open confirmations reveal differential accessibility of the conserved R469-containing sequence (HSPA8) monomethylated by PRMT7. Structures are color coded for domains (orange—ATP binding, blue—substrate binding, and green—lid domains). The HSP70 substrate-binding domain loop containing PRMT7 methylated arginine is colored red. a, b Closely related homolog HSPA5 structures (65% overall sequence identity to HSPA8) were analyzed to investigate the position of the arginine methylation site in the different conformations. In the ADP-bound state, the lid of the substrate-binding domain is closed (PDB 5E85), limiting accessibility of the R492 (analogous to R469 in HSPA8) residue for methylation by PRMT7. In the ATP-bound form (PDB 5E84), the arginine residue is accessible therefore permitting access by the PRMT7 enzyme. c The structure of the more closely HSPA8 related HSPA1A (86% overall sequence identity and 82% sequence identity for aa. 386–646 in the substrate-binding domain, PDB 4PO2) in the closed conformation in which R469 is occluded by the lid subdomain. d–f Kinetic analysis of HSPA8 methylation by PRMT7 in vitro. Kinetic parameters were determined for HSPA8 methylation in the presence and absence of ATP. PRMT7 had no activity in the absence of ATP. d Kinetic analysis at fixed 10 µM HSPA8 (SAM K_m = 1.6 ± 0.1 µM). e Kinetic analysis at fixed 20 µM of SAM (HSPA8 K_m = 10.6 ± 0.1 µM and k_cat of 2.2 ± 0.1 h⁻¹). f HSPA8–R469K mutant is not methylated by PRMT7 in vitro. The results are mean ± SEM of three technical replicates. Source data are provided as a Source Data file.

Fig. 5. SGC3027 inhibits HSP70 methylation in cells and its active component SGC8158 methylation in vitro.

a SGC8158 inhibits PRMT7 methylation of HSPA8 in vitro. SGC8158 IC₅₀ = 294 ± 26 nM (2 biological replicates, each with technical n = 3, mean ± SEM), SGC8158N IC₅₀ > 100 µM (n = three technical replicates). The methylation assay was performed in the presence of ATP (n = 3 technical replicates). b SGC3027 is a prodrug cellular inhibitor of PRMT7 as illustrated by the prodrug conversion to the active component in cells. c SGC3027 inhibits PRMT7-dependent HSP70 monomethylation in C2C12 cells. Cells were treated with the compound for 2 days. The experiment was repeated four times with similar results. d Quantification of SGC3027 and SGC3027N effects on HSP70 monomethylation in C2C12 cells. The graphs represent non-linear fits of Rme1 signal intensities normalized to intensities of HSP70. SGC3027: n = 11, four separate experiments, IC₅₀ = 2.4 ± 0.1 µM; SGC3027N: n = 4 technical replicates, IC₅₀ > 40 µM (mean ± SEM). e A representative blot for SGC3027N effects on HSP70 methylation. Rme1—arginine monomethylation. The experiment with 3 and 10 µM SGC3027N concentration was repeated three times with similar results. Source data are provided as a Source Data file.

Fig. 6. PRMT7 knockout/inhibition affects cell survival after heat shock or proteasomal stress.

a, b Loss of PRMT7 decreases cell survival and increases apoptosis levels after heat shock. MEF cells were heat-shocked for 20 min at 44 °C. Apoptosis was monitored immediately after the heat shock and cell number was determined 24 h later. The results shown are mean ± SEM of two biological replicates, each technical triplicate (a) and three technical replicates (b). Statistical significance was determined with unpaired Student t-test (two-tailed). c SGC3027 inhibition of PRMT7 activity decreases cell survival and increases apoptosis levels after heat shock. Cells were pretreated with 3 µM compounds for 2 days before heat shock. Cell number was determined as in a. The results shown are mean ± SEM of two biological replicates, each technical n = 5. Statistical significance was determined with one-way ANOVA with Tukey’s post-hoc test. d Loss of PRMT7 decreases cell survival after bortezomib (BTZ) treatment. BTZ was removed after 4 h and the cell confluence was monitored 4 h after BTZ treatment. The results are mean ± SEM of 3–6 technical replicates. e, f SGC3027 decreases cell survival after BTZ treatment (30 nM) in Prmt7 WT MEF (e) but not in Prmt7 KO MEF (f). Cells were pretreated with 4 µM compounds for 2 days before BTZ treatment. After 20 h, BTZ was removed and SGC3027 or SGC3027N were replaced. The results are mean ± SD of 6–8 technical replicates. Red arrow indicates the time BTZ was removed. Confluency is a measure of cell number. g Overexpression of WT HSPA1 (HSP70/HSP72) inhibits the induction of G3BP-mcherry and TIAR-positive stress granules in response to proteasome inhibition. Scale bar is 14 μm, arrow indicates stress granules. h Quantitation of G3BP-mcherry and TIAR stress granules in GFP-positive cells overexpressing WT GFP-HSPA1 (with/without 3 µM SGC3027) or the catalytic mutant GFP-HSPA1 R469K and non-transfected cells (NT). The results are mean ± SD of three biological replicates. Statistical significance was determined with one-way ANOVA with Tukey’s post-hoc test. Source data are provided as a Source Data file.