In 2005, Greiner and co-workers screened a library of ca. 3,000 compounds using a standard radioactive filter-binding assay [94Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9 Nat Chem Biol 2005; 1: 143-5.] and identified chaetocin (Fig. 2), a fungal mycotoxin, as the first small-molecule inhibitor of recombinant Drosophila Su(var)3-9 (IC₅₀ = 0.6 µM). Chaetocin was also found to inhibit H3K9 PKMT SUV39H1 (IC₅₀ = 0.8 µM), the human orthologue of dSu(var)3-9. While chaetocin inhibited other H3K9 PKMTs, including Neurospora DIM5 (IC₅₀ = 3.0 µM) and mouse G9a (IC₅₀ = 2.5 µM), it was selective over non-H3K9 PKMTs, such as H3K27 PKMT dE(z) complex, H3K4 PKMT SET7/9, and H4K20 PKMT SETD8 [94Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9 Nat Chem Biol 2005; 1: 143-5.] (IC₅₀ dE(z) complex > 90 µM; SET7/9 and SETD8 >180 µM). Furthermore, mechanistic studies characterized chaetocin as a SAM-competitive inhibitor, which remained active even after the disulfide bonds of chaetocin were reduced in the presence of increasing amounts of dithiothreitol (DTT) [94Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9 Nat Chem Biol 2005; 1: 143-5.]. Interestingly, a total synthesis report found both natural (+)- and synthetic (–)-chaetocin to be equipotent against G9a (IC₅₀ = 2.4 and 1.7 µM, respectively) while the sulfur-deficient analogs were inactive (IC₅₀ > 50 µM, Fig. 2) [95Iwasa E, Hamashima Y, Fujishiro S, et al. Total synthesis of (+)-chaetocin and its analogues: their histone methyltransferase G9a inhibitory activity J Am Chem Soc 2010; 132: 4078-9.]. Like other members of the epidithiodiketopiperazine (ETP) class [96Gardiner DM, Waring P, Howlett BJ. The epipolythiodioxopiperazine (ETP) class of fungal toxins: distribution, mode of action, functions and biosynthesis Microbiology 2005; 151: 1021-32.], chaetocin is cytotoxic, although dependent on initial cell density. Chaetocin-treated Drosophila SL-2 cells at an inhibitor concentration of 0.5 µM showed marked cellular reduction of di- and trimethylation levels of H3K9 with no apparent changes in the degree of methylation of other lysines (H3K27, H3K36, H3K79, and H3K4) [94Greiner D, Bonaldi T, Eskeland R, Roemer E, Imhof A. Identification of a specific inhibitor of the histone methyltransferase SU(VAR)3-9 Nat Chem Biol 2005; 1: 143-5.].

Fig. (2) Lysine methyltransferase inhibitors (IC50 values in parentheses with corresponding enzyme).

A high throughput screen of ca. 125,000 compounds, preselected from the Boehringer Ingelheim (BI) compound collection, revealed BIX01294 (Fig. 2) as the first selective small-molecule inhibitor of G9a and GLP with low micromolar potency in vitro over other H3K9 PKMTs (SUV39H1 and SETDB1), H3K4 PKMT SET7/9, and arginine methyltransferase PRMT1, which all showed no inhibition at concentrations of 45 µ M [93Kubicek S, O'Sullivan RJ, August EM, et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase Mol Cell 2007; 25: 473-81.]. Under linear assay conditions, BIX01294 inhibited G9a and GLP with IC₅₀ values of 1.9 µ M and 0.7 µ M, respectively [84Chang Y, Zhang X, Horton JR, et al. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294 Nat Struct Mol Biol 2009; 16: 312-7.]. In cellular assays, BIX01294 was toxic at high concentrations (> 4.1 µ M). However, when cells were treated at an inhibitor concentration of 4.1 µ M, BIX01294 reduced H3K9me2 levels of bulk histones, while methylation levels of other known sites, including H3K27, H3K36, and H4K20, remained largely unchanged. Mechanistically, unlike chaetocin, BIX01294 did not inhibit G9a in a SAM-competitive manner but rather occupied the histone peptide binding pocket, as confirmed by the X-ray crystal structure of BIX01294 and GLP in the presence of SAH (Fig. 3A, PDB: 3FPD) [84Chang Y, Zhang X, Horton JR, et al. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294 Nat Struct Mol Biol 2009; 16: 312-7., 93Kubicek S, O'Sullivan RJ, August EM, et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase Mol Cell 2007; 25: 473-81.]. Interestingly, the X-ray structure revealed that while BIX01294 did not bind in the SAM-binding site, it also did not interact with the lysine binding channel [84Chang Y, Zhang X, Horton JR, et al. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294 Nat Struct Mol Biol 2009; 16: 312-7.]. Through the same high-throughput screen as mentioned above, non-selective lysine and arginine methyltransferase inhibitors, such as BIX01338, were also discovered (Fig. 2) [93Kubicek S, O'Sullivan RJ, August EM, et al. Reversal of H3K9me2 by a small-molecule inhibitor for the G9a histone methyltransferase Mol Cell 2007; 25: 473-81.].

Fig. (3) A) GLP-BIX01294 complex (PDB: 3FPD, BIX01294 is shown in orange) with SAH (cyan) superimposed with GLP-H3 co-crystal structure (PDB: 2RFI, H3 backbone (ribbon) and H3K9me2 are shown in green). B) G9a-UNC0224 complex (PDB: 3K5K, UNC0224 is shown in grey) with SAH (cyan) superimposed with GLP-H3 co-crystal structure (H3K9me2 is shown in green).

Structure-activity relationships (SAR) of the quinazoline scaffold exemplified by BIX01294 were investigated based on the reported X-ray structure of the GLP-BIX01294 complex (Fig. 3A). Tractable SAR were demonstrated for the 2- and 4-amino moieties [85Liu F, Chen X, Allali-Hassani A, et al. Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a J Med Chem 2009; 52: 7950-3., 97Liu F, Chen X, Allali-Hassani A, et al. Protein lysine methyltransferase G9a inhibitors: Design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines J Med Chem 2010; 53: 5844-7.]. To improve potency, the 7-methoxy moiety of the quinazoline template was explored in an attempt to design analogs that would interact with the lysine binding channel. These efforts resulted in the discovery of UNC0224 (Fig. 4) as a seven times more potent G9a inhibitor (IC₅₀ = 15 nM) when compared to BIX01294 (IC₅₀ = 106 nM) in the G9a ThioGlo assay [85Liu F, Chen X, Allali-Hassani A, et al. Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a J Med Chem 2009; 52: 7950-3., 98Collazo E, Couture JF, Bulfer S, Trievel RC. A coupled fluorescent assay for histone methyltransferases Anal Biochem 2005; 342: 86-92.]. The higher potency of UNC0224 was confirmed by isothermal titration calorimetry (ITC) (K_d = 23 nM; BIX01294 K_d = 130 nM) [85Liu F, Chen X, Allali-Hassani A, et al. Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a J Med Chem 2009; 52: 7950-3.]. Although UNC0224 was equipotent against GLP with an IC₅₀ of 20 nM, it was more than 1,000-fold selective for G9a over other PKMTs, including SET7/9 and SETD8. In addition, UNC0224 was clean against a broad panel of G-protein coupled receptors, ion channels, and transporters [85Liu F, Chen X, Allali-Hassani A, et al. Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a J Med Chem 2009; 52: 7950-3.]. A high resolution (1.7 Å) X-ray co-crystal structure of G9a and UNC0224 (PDB: 3K5K) was obtained, providing the first crystal structure of G9a in complex with a small molecule inhibitor (Fig. 3B). Indeed, the structure showed the 7-dimethylamino propoxy side chain of UNC0224 occupying the lysine binding channel of G9a, validating the design rationale for UNC0224 and providing an explanation for its higher potency [84Chang Y, Zhang X, Horton JR, et al. Structural basis for G9a-like protein lysine methyltransferase inhibition by BIX-01294 Nat Struct Mol Biol 2009; 16: 312-7., 85Liu F, Chen X, Allali-Hassani A, et al. Discovery of a 2,4-diamino-7-aminoalkoxyquinazoline as a potent and selective inhibitor of histone lysine methyltransferase G9a J Med Chem 2009; 52: 7950-3.]. However, the 7-alkoxy side chain did not completely occupy the lysine binding channel, and space remained for the channel to accommodate a longer side chain or larger amino-capping group. Therefore, additional SAR of the 7-alkoxy side chain of UNC0224 was investigated. These side chain optimization efforts led to the discovery of UNC0321 (Fig. 4), the most potent G9a inhibitor to date (IC₅₀ = 6 nM, AlphaScreen; 9 nM, ThioGlo) [97Liu F, Chen X, Allali-Hassani A, et al. Protein lysine methyltransferase G9a inhibitors: Design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines J Med Chem 2010; 53: 5844-7.]. Because UNC0321 likely reached the detection limits of the biochemical assays, Morrison K_i’s were determined using an endoproteinase-coupled microfluidic capillary electrophoresis (MCE) assay [99Wigle TJ, Provencher LM, Norris JL. Accessing protein methyltransferase and demethylase enzymology using microfluidic capillary electrophoresis Chem Biol 2010; 17: 695-704.]. UNC0321 (Morrison K_i = 63 pM) was about 40-fold more potent than UNC0224 (Morrison K_i = 2.6 nM) and 250-fold more potent than BIX01294 (Morrison K_i = 16 nM) [97Liu F, Chen X, Allali-Hassani A, et al. Protein lysine methyltransferase G9a inhibitors: Design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines J Med Chem 2010; 53: 5844-7.]. While UNC0224 was equipotent against G9a and GLP, UNC0321 showed some selectivity for G9a (IC₅₀ = 6 nM, AlphaScreen) over GLP (IC₅₀ = 23 nM, AlphaScreen). In addition, UNC0321 was inactive (IC₅₀ > 40 µM, ThioGlo) against other PKMTs, SET7/9 and SETD8, as well as PRMT3 [97Liu F, Chen X, Allali-Hassani A, et al. Protein lysine methyltransferase G9a inhibitors: Design, synthesis, and structure activity relationships of 2,4-diamino-7-aminoalkoxy-quinazolines J Med Chem 2010; 53: 5844-7.]. While highly potent in biochemical assays, UNC0321 was less potent in cellular assays compared to BIX01294.

Fig. (4) Lysine methyltransferase inhibitors (Morrison Ki or Kd values in parentheses with corresponding enzyme); inset, E72 (green) super-imposed with BIX01294 (orange) and UNC0224 (grey).

To improve cellular potency of this series, new analogs aimed at increasing lipophilicity, thus cell membrane permeability, while maintaining high in vitro potency were designed and synthesized. Among the newly synthesized compounds, UNC0638 (Fig. 4) had excellent in vitro potency (Morrison K_i G9a = 3.7 nM; K_i = 3.0 nM) and was > 100-fold selective over a wide range of epigenetic and non-epigenetic targets [100Vedadi M, Barsyte-Lovejoy D, Liu F, et al. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells Nat Chem Biol 2011; 7: 566-74.]. Michaelis-Menten kinetics found UNC0638 was indeed competitive with the peptide substrate instead of the SAM cofactor. This mechanism of action was confirmed by X-ray crystal structure of the G9a-UNC0638-SAH complex (2.56 Å resolution, PDB: 3RJW) which showed UNC0638 occupying the substrate binding groove and lysine binding channel, not the SAM binding pocket – the same binding mode that was previously observed for UNC0224 (see Fig. 3B). More importantly, UNC0638, which possesses balanced in vitro potency and physicochemical properties aiding cell penetration, had excellent potency in cellular assays and low cell toxicity. UNC0638 treatment of a variety of cell lines resulted in reduction of global H3K9me2 levels equivalent to that observed for shRNA knockdown of G9a/GLP. It significantly reduced the H3K9me2 mark at the promoter of known G9a-regulated endogenous genes and did not reduce the H3K9me2 mark at the promoter of a non-G9a-regulated gene. In addition, UNC0638 significantly reduced the clonogenicity of MCF7 cells and disproportionately affected a number of genomic loci encoding microRNAs. In mouse embryonic stem (mES) cells, UNC0638 reactivated a retroviral reporter gene and G9a-silenced endogenous genes in a concentration dependent manner without promoting differentiation. Furthermore, UNC0638 significantly reduced H3K9me2 levels at the promoter of the retroviral long terminal repeat and G9a-regulated genes and indirectly induced DNA hypomethylation in mES cells [100Vedadi M, Barsyte-Lovejoy D, Liu F, et al. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells Nat Chem Biol 2011; 7: 566-74.]. The combination of high potency, excellent selectivity, and robust on-target activities in cells makes UNC0638 a valuable tool for further investigating the biological function of G9a and GLP and their role in health and disease [101For the latest unpublished progress in this area, see: http://www.thesgc.org/chemical_probes/UNC0638/#overview ].

Chang and co-workers also designed and synthesized quinazoline analogs based on the previously reported GLP-BIX01294 X-ray co-crystal structure and discovered compound E72 (Fig. 4) as a potent G9a and GLP inhibitor (IC₅₀ GLP = 100 nM) with binding affinities determined by ITC (K_d GLP = 136 nM; G9a = 164 nM) [102Chang Y, Ganesh T, Horton JR, et al. Adding a lysine mimic in the design of potent inhibitors of histone lysine methyltransferases J Mol Biol 2010; 400: 1-7.]. A brief selectivity study showed E72 was inactive against H3K9 PKMT Suv39H2 with no inhibition at 5 µ M [102Chang Y, Ganesh T, Horton JR, et al. Adding a lysine mimic in the design of potent inhibitors of histone lysine methyltransferases J Mol Biol 2010; 400: 1-7.]. The X-ray co-crystal structure of the GLP-E72 complex in the presence of SAH (2.19 Å, PDB: 3MO5) showed that E72 occupied both the surface of the peptide binding groove and the lysine binding channel, in an analogous manner to UNC0224 with G9a (see Fig. 4 inset for an overlay of E72 with BIX01294 and UNC0224) [102Chang Y, Ganesh T, Horton JR, et al. Adding a lysine mimic in the design of potent inhibitors of histone lysine methyltransferases J Mol Biol 2010; 400: 1-7.]. In three separate cell types, E72 was significantly less toxic than BIX01294 at compound concentrations of 10 µ M and was able to reactivate K-ras-mediated epigenetic silencing of the Fas gene in NIH 3T3 cells [102Chang Y, Ganesh T, Horton JR, et al. Adding a lysine mimic in the design of potent inhibitors of histone lysine methyltransferases J Mol Biol 2010; 400: 1-7.].

In 2004, Bedford and co-workers discovered the first non-nucleoside specific inhibitors of PRMTs using a random screening approach [114Cheng DH, Yadav N, King RW, Swanson MS, Weinstein EJ, Bedford MT. Small molecule regulators of protein arginine methyltransferases J Biol Chem 2004; 279: 23892-9.]. A diverse library of 9,000 compounds from ChemBridge was screened and nine of these compounds were identified as low micromolar inhibitors (0.15–6.9 µM) of in vitro methylation of the RNA binding protein, Npl3p, by the yeast Hmt1p arginine methyltransferase. These hits, named arginine methyltransferase inhibitors (AMIs), also inhibited human PRMT1, a mammalian orthologue of Hmt1p, with potency ranging from 0.19 to 16.3 µM (Fig. 5) [114Cheng DH, Yadav N, King RW, Swanson MS, Weinstein EJ, Bedford MT. Small molecule regulators of protein arginine methyltransferases J Biol Chem 2004; 279: 23892-9.].

Fig. (5) Select examples of the first non-nucleoside arginine methyltransferase inhibitors (AMIs) identified from random screening (IC50 values in parentheses with corresponding enzyme).

To determine specificities, the AMIs were screened against a panel of other type I PRMTs (PRMT3, 4, and 6). Although each of the nine AMIs initially inhibited all of the PRMTs tested, displaying no specificity for individual type I PRMTs [114Cheng DH, Yadav N, King RW, Swanson MS, Weinstein EJ, Bedford MT. Small molecule regulators of protein arginine methyltransferases J Biol Chem 2004; 279: 23892-9.], a subsequent report found AMI-1 was selective over type II PRMT5 [115Bonham K, Hemmers S, Lim YH, Hill DM, Finn MG, Mowen KA. Effects of a novel arginine methyltransferase inhibitor on T-helper cell cytokine production FEBS J 2010; 277: 2096-108.]. Furthermore, AMI-1 and -6 were selective in vitro against several lysine methyltransferases including Suv39H1, Suv39H2, SET7/9, and DOT1. Cellular activity studies in HeLa cells showed AMI-1, but not -6, was able to inhibit hPRMT1, decreasing methylation levels of the GFP-Npl3 fusion [114Cheng DH, Yadav N, King RW, Swanson MS, Weinstein EJ, Bedford MT. Small molecule regulators of protein arginine methyltransferases J Biol Chem 2004; 279: 23892-9.]. AMI-1 was also screened against sirtuin, a class III histone deacetylase (HDAC), due to the minor similarity of the binaphthylurea motif of AMI-1 to the known sirtuin inhibitor, suramin. AMI-1 inhibited sirtuin, albeit with decreased potency relative to suramin (IC₅₀ on SIRT1: AMI-1, 32 µ M; suramin, 300 nM) [116Trapp J, Meier R, Hongwiset D, Kassack MU, Sippl W, Jung M. Structure-activity studies on suramin analogues as inhibitors of NAD(+)-dependent histone deacetylases (sirtuins) ChemMedChem 2007; 2: 1419-31.].

Several groups have since used the AMIs as leads for PRMT drug discovery. Using the dye-like core of AMI-5 and -6 as a lead scaffold, a number of analogs were synthesized [117Ragno R, Simeoni S, Castellano S, et al. Small molecule inhibitors of histone arginine methyltransferases: Homology modeling, molecular docking, binding mode analysis, and biological evaluations J Med Chem 2007; 50: 1241-53.]. The most potent of these analogs was compound 1 (IC₅₀ hPRMT1 = 4.8 µ M), which was less potent than AMI-5 (Fig. 6). Cellular activity data was not reported [117Ragno R, Simeoni S, Castellano S, et al. Small molecule inhibitors of histone arginine methyltransferases: Homology modeling, molecular docking, binding mode analysis, and biological evaluations J Med Chem 2007; 50: 1241-53.]. The AMI-5 (eosin) scaffold was subsequently simplified to curcumin-like structures [118Balasubramanyam K, Varier RA, Altaf M, et al. Curcumin, a novel p300/CREB-binding protein-specific inhibitor of acetyltransferase, represses the acetylation of histone/nonhistone proteins and histone acetyltransferase-dependent chromatin transcription J Biol Chem 2004; 279: 51163-71.] containing bromo- and dibromophenol moieties [119Mai A, Cheng D, Bedford MT, et al. Epigenetic multiple ligands: Mixed histone/protein methyltransferase, acetyltransferase, and class III deacetylase (sirtuin) inhibitors J Med Chem 2008; 51: 2279-90.]. These analogs were screened against a panel of methyltransferases. Several compounds, including 2 and 3 (Fig. 6), were selective for PRMT4 (CARM1) over PRMT1 and SET7/9 at concentrations of 100 µ M [119Mai A, Cheng D, Bedford MT, et al. Epigenetic multiple ligands: Mixed histone/protein methyltransferase, acetyltransferase, and class III deacetylase (sirtuin) inhibitors J Med Chem 2008; 51: 2279-90.].

Fig. (6) Synthetic analogs of the original AMI series identified as PRMT inhibitors (IC50 values in parentheses with corresponding enzyme).

In addition, Bonham and co-workers reported their efforts to generate a less polar version of AMI-1 while still maintaining PRMT potency [115Bonham K, Hemmers S, Lim YH, Hill DM, Finn MG, Mowen KA. Effects of a novel arginine methyltransferase inhibitor on T-helper cell cytokine production FEBS J 2010; 277: 2096-108.]. Using the aminonaphthol sulfonate scaffold of AMI-1 as the core, analogs were synthesized by adding structural attributes of AMI-6 and -9. The most potent hybrid, compound 4 (IC₅₀ hPRMT1 = 4.2 µM; hPRMT4 = 2.6 µ M), contained both the dichlorotriazine group of AMI-6 and the azo moiety of AMI-9 (Fig. 6). Selectivity studies showed compound 4 inhibited both type I and type II PRMTs but was mostly inactive against lysine methyltransferase SET7/9. Since PRMTs have been shown to regulate T-helper cell activation and cytokine secretion [120Mowen KA, Schurter BT, Fathman JW, David M, Glimcher LH. Arginine methylation of NIP45 modulates cytokine gene expression in effector T lymphocytes Mol Cell 2004; 15: 559-71.-122Blanchet F, Cardona A, Letimier FA, Hershfield MS, Acuto O. CD28 costimulatory signal induces protein arginine methylation in T cells J Exp Med 2005; 202: 371-7.], the effect of compound 4 on cytokine expression was also examined. Indeed, compound 4 enhanced T-helper cell proliferation without affecting cell viability and decreased IFN-γ and IL-4 production of type I and type II T-helper cells, respectively, thereby interfering with IL-4 promoter activity and impairing the interaction between PRMT1 and NIP45 [115Bonham K, Hemmers S, Lim YH, Hill DM, Finn MG, Mowen KA. Effects of a novel arginine methyltransferase inhibitor on T-helper cell cytokine production FEBS J 2010; 277: 2096-108.].

A high throughput screening effort by Purandare and coworkers led to the identification of pyrazole amide 5 as an initial hit (Fig. 7), which after preliminary optimization gave compound 6 (IC₅₀ hPRMT4 = 80 nM) as a potent and selective inhibitor of PRMT4 (CARM1) [123Purandare AV, Chen Z, Huynh T, et al. Pyrazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2008; 18: 4438-1.], albeit poor permeability [parallel artificial membrane permeability assay (PAMPA)] and pharmacokinetic (PK) properties [124Huynh T, Chen Z, Pang S, et al. Optimization of pyrazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2009; 19: 2924-7., 125Therrien E, Larouche G, Manku S, et al. 1,2-Diamines as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2009; 19: 6725-32.]. Further hit to lead optimization of this class by separate groups led to two potent derivatives: 7 and 8 (Fig. 7) with IC₅₀’s < 100 nM, although the compounds were either not active in cellular assays or the data were not reported [124Huynh T, Chen Z, Pang S, et al. Optimization of pyrazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2009; 19: 2924-7., 126Allan M, Manku S, Therrien E, et al. N-Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2009; 19: 1218-23.]. However, replacing the amide functionality with the 1,3,4-oxadiazole moiety in 7 improved membrane permeability (PAMPA) [124Huynh T, Chen Z, Pang S, et al. Optimization of pyrazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2009; 19: 2924-7.]. Compounds 6 and 7 were found to be significantly less potent against other type I PRMTs (PRMT1 and 3, IC₅₀ > 25 µM) [123Purandare AV, Chen Z, Huynh T, et al. Pyrazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2008; 18: 4438-1., 124Huynh T, Chen Z, Pang S, et al. Optimization of pyrazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2009; 19: 2924-7.] and 8 was selective against both PRMT1 and lysine methyltransferase SET7/9 (IC₅₀ > 100 µ M) [126Allan M, Manku S, Therrien E, et al. N-Benzyl-1-heteroaryl-3-(trifluoromethyl)-1H-pyrazole-5-carboxamides as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2009; 19: 1218-23.]. A subsequent report identified compound 9 as a potent pyrazole derivative (IC₅₀ mPRMT4 = 0.20 µ M, Fig. 7) with an improved PK profile in rats, although it did not show cellular activity when tested in the MTT cell viability assay [125Therrien E, Larouche G, Manku S, et al. 1,2-Diamines as inhibitors of co-activator associated arginine methyltransferase 1 (CARM1) Bioorg Med Chem Lett 2009; 19: 6725-32.]. Concurrently, an additional screening effort by Wan and co-workers identified benzo[d]imidazole related analogs as hits against PRMT4 (CARM1) with low micromolar potency [127Wan HH, Huynh T, Pang SH, et al. Benzo[d]imidazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1)-hit to lead studies Bioorg Med Chem Lett 2009; 19: 5063-6.]. Hit to lead optimization and SAR exploration led to the most potent analog in this series, compound 10 (IC₅₀ hPRMT4 = 70 nM, Fig. 7 inset), which was significantly less active against other type I PRMTs (PRMT1 and 3, IC₅₀ > 25 µ M) [127Wan HH, Huynh T, Pang SH, et al. Benzo[d]imidazole inhibitors of coactivator associated arginine methyltransferase 1 (CARM1)-hit to lead studies Bioorg Med Chem Lett 2009; 19: 5063-6.].

Fig. (7) PRMT inhibitors designed from the high throughput screening hit, pyrazole amide 5; inset, Benzo[d]imidazole related analog identified as a PRMT inhibitor (IC50 values in parentheses with corresponding enzyme).

The first target based virtual screening study for PRMTs was reported by Spannhoff and co-workers in 2007 [128Spannhoff A, Heinke R, Bauer I, et al. Target-based approach to inhibitors of histone arginine methyltransferases J Med Chem 2007; 50: 2319-5.-130Jung M. Epigenetic targets in drug discovery In: Sippl W, Jung M, Eds. Weinheim Wiley-VCH 2009; 251-68.]. In this study, a combination of molecular docking and pharamacophore-based filtering was used to virtually screen hPRMT1 and fungal RmtA, a PRMT1 homologue, against the NCI diversity subset consisting of 1990 compounds. Homology models of both enzymes were constructed from the known rat PRMT3 X-ray crystal structure (PDB: 1F3L). In order to target the histone binding pocket, SAH was included as part of the protein during docking. Compounds that were successfully docked were then tested in vitro against RmtA and recombinant hPRMT1. In this study, seven of the 36 virtual hits were able to inhibit RmtA and hPRMT1 with micromolar potency (IC₅₀ hPRMT1 = 2–90 µ M) [128Spannhoff A, Heinke R, Bauer I, et al. Target-based approach to inhibitors of histone arginine methyltransferases J Med Chem 2007; 50: 2319-5.]. Hit validation was conducted in cancer cells using antibody-mediated detection of histone hypomethylation caused by inhibition of PRMT1. Two of the hits, allantodapsone and stilbamidine (Fig. 8), inhibited methylation at PRMT1 target H4R3 in a dose-dependent manner while having only a marginal effect on methylation levels of PKMT target H3K4. In a reporter gene functional assay with MCF7a cells, both inhibitors showed a dose-dependent reduction of estrogen receptor activation by estradiol. In addition, kinetic assays showed allantodapsone and stilbamidine did not inhibit RmtA in a SAM-competitive manner but were rather competitive with regard to the histone substrate, as expected [128Spannhoff A, Heinke R, Bauer I, et al. Target-based approach to inhibitors of histone arginine methyltransferases J Med Chem 2007; 50: 2319-5.].

Fig. (8) PRMT inhibitors identified from virtual screens (IC50 values in parentheses with corresponding enzyme).

Based on the success of the structure-based virtual screen to discover potent and cell-penetrant PRMT inhibitors, a larger virtual screening effort was conducted using the ChemBridge compound collection containing 328,000 compounds [129Bissinger EM, Heinke R, Sippl W, Jung M. Targeting epigenetic modifiers: Inhibitors of histone methyltransferases Med Chem Commun 2010; 1: 114-24.-131Heinke R, Spannhoff A, Meier R, et al. Virtual screening and biological characterization of novel histone arginine methyltransferase PRMT1 inhibitors ChemMedChem 2009; 4: 69-77.]. The database was first filtered using a simple pharmacophore search resulting in an abbreviated library of ca. 6200 compounds. These compounds were then docked into the substrate binding pocket of PRMT1, resulting in nine inhibitors of hPRMT1 with IC₅₀ values ranging from 13 to 37 μM [131Heinke R, Spannhoff A, Meier R, et al. Virtual screening and biological characterization of novel histone arginine methyltransferase PRMT1 inhibitors ChemMedChem 2009; 4: 69-77.]. The three most potent compounds, 11–13, are represented in Fig. (8).

As an alternative approach for identifying new PRMT inhibitors, Spannhoff and co-workers performed a fragment-based virtual screen of ca. 900 fragment-like leads with molecular weights below 200 g/mol and identified an α-methylthioglycolic amide compound as a micromolar inhibitor of RmtA [129Bissinger EM, Heinke R, Sippl W, Jung M. Targeting epigenetic modifiers: Inhibitors of histone methyltransferases Med Chem Commun 2010; 1: 114-24., 130Jung M. Epigenetic targets in drug discovery In: Sippl W, Jung M, Eds. Weinheim Wiley-VCH 2009; 251-68., 132Spannhoff A, Machmur R, Heinke R, et al. A novel arginine methyltransferase inhibitor with cellular activity Bioorg Med Chem Lett 2007; 17: 4150-3.]. A subsequent structure similarity search led to the discovery of RM65 (Fig. 8) as an equipotent inhibitor of RmtA and hPRMT1 in vitro (IC₅₀ hPRMT1 = 55 µ M). A cursory selectivity study found RM65 was not active against PKMT SET7/9 at a concentration of 50 µ M. In cancer cells, histone hypomethylation was observed at PRMT1 target H4R3. Additionally, while the virtual screen was conducted with fragments binding to the substrate binding pocket, docking studies have suggested a bisubstrate binding mode for RM65, targeting both the substrate and cofactor binding sites [132Spannhoff A, Machmur R, Heinke R, et al. A novel arginine methyltransferase inhibitor with cellular activity Bioorg Med Chem Lett 2007; 17: 4150-3.].

An alternative mechanism of inhibiting PRMT-mediated arginine methylation was recently reported. Rather than developing inhibitors of the enzyme active site, Feng and co-workers developed small molecules that target the substrate, blocking PTMs on H4 by H4-modifying enzymes [133Feng Y, Li MY, Wang BH, Zheng YG. Discovery and mechanistic study of a class of protein arginine methylation inhibitors J Med Chem 2010; 53: 6028-39.]. The naphthalene-sulfonate (NS) analogs were discovered through a similarity structure search of a weak hit identified from a virtual screen of the ChemBridge compound collection. The most potent derivative, NS-1 (Fig. 9), was initially characterized as a substrate-competitive PRMT1 inhibitor with micromolar potency (IC₅₀ hPRMT1 = 13 µ M) [133Feng Y, Li MY, Wang BH, Zheng YG. Discovery and mechanistic study of a class of protein arginine methylation inhibitors J Med Chem 2010; 53: 6028-39.]. However, after a variety of kinetic and biophysical studies, NS-1 and similar structural analogs including AMI-1 were found to interact directly with the substrate, not the enzyme, blocking PRMT1-mediated arginine methylation. In a selectivity study, NS-1 was found to be significantly less potent against PRMT4 (CARM1) (IC₅₀ mPRMT4 ~ 2 mM) [133Feng Y, Li MY, Wang BH, Zheng YG. Discovery and mechanistic study of a class of protein arginine methylation inhibitors J Med Chem 2010; 53: 6028-39.]. Unlike PRMT1, PRMT4 (CARM1) targets H3 and does not methylate GAR sequences. In addition, Selvi and co-workers isolated the small molecule TBBD (ellagic acid) from pomegranate extract and found it inhibited the methyltransferase activity of PRMT4 (CARM1) in a substrate-targeting manner, selectively blocking methylation of H3R17 (Fig. 9) [134Selvi BR, Batta K, Kishore AH, et al. Identification of a novel inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1)-mediated methylation of histone H3 Arg-17 J Biol Chem 2010; 285: 7143-52.]. These and other substrate sequence-specific inhibitors may emerge as useful tools for mechanistic study of arginine methylation and other epigenetic modifications.

Fig. (9) Substrate-targeting PRMT inhibitors (IC50 values in parentheses with corresponding enzyme and substrate).