REVIEWS Lysine demethylases.First-generation mechanism- oinding sit lyine-pef ofsclective and Structure-act es(HKMTs)has been rest poc Ited in more potent BX-thefirst or of a PKMT s of demethylas tinc umonji domain- its use to cell-ba assays tion inhibi oline coreasakey structural modific ion,showe otency and and bind to the ytic ir n in the active xygena cture-based studies which have been insightful ntidigthcdcsigofnod ies the peptide groove(as previo n with esothat the comp und binds directly to theron inth d tha ng P cy,as seer -mole re selective for the jumonii domair containing demeth e inhib d to the pepti es over other 2-o prope ing prote compound may limit bio vo new onji d ontaining ple SID85736331)and 2.2bipyridines (for examp ds that bind to the SAM bindi mpound G.51,are po Z00477 er and more comp activity ag or fur The ue (it ret otency and 51 i Other comp nds that b ases h now been id ntifi d,the next cha be bette hat targeting the ofactor binding site of protein methyl- containing family of pro ins represents an importan vithin the SAM binding acid enes from huma fruitflies and veast'The humar odomain-cont and PRMT4 have also been identificd mains ence ssing a totalof61 unique human bromo omains" romod omains a conmonl d in proter y NATURE REVIEWSIDRUG DISCOVERY VOLUME 11 MAY 2012 395molecules can bind and inhibit enzyme function. Indeed, both the peptide substrate channel and the binding site for the cofactor SAM have been exploited to produce potent inhibitors of protein methyltransferases135,136. Currently, the successful identification of selective and cell-active inhibitors of histone lysine methyltrans￾ferases (HKMTs) has been restricted to those targeting the closely related enzymes G9A and GLP1, as well as  DOT1L. BIX-01294 was the first selective inhibitor of a PKMT. BIX-01294 binds at the protein substrate channel of G9A and GLP1, but its modest affinity and cytotoxicity limit its use to cell-based assays137. Second-generation inhibi￾tors such as E72 (REF. 138) and UNC321 (REF. 139), both of which incorporate a 7-alkoxyamine tethered to the quinazoline core as a key structural modification, showed significantly improved enzyme affinity. UNC638 (REF. 140) is a potent and selective inhibitor of G9A and GLP1, and was further optimized for improved cellular potency and low toxicity. UNC638 also retains the 7-alkoxyamine group, indicating that the incorporation of this group may represent a viable strategy for designing compounds that target this HKMT family. Structure-based studies — which have been insightful in aiding the design of novel compounds — have shown that the conserved quinazoline core of UNC638 (REF. 140) occupies the peptide groove (as previously seen with BIX-01294)141 and that the new alkoxyamine substituents bind inside the lysine channel in a similar manner to the lysine of the histone substrate140 (FIG. 4). Other small-mole￾cule inhibitors of HKMTs that bind to the peptide-binding groove include AZ505, which is a potent and selective inhibitor of SET and MYND domain-containing protein 2 (SMYD2)142. The oncogenic protein SMYD2 represses the functional activities of p53 and retinoblastoma protein, making it an attractive drug target for the development of small-molecule inhibitors. Compounds that bind to the SAM binding site include the DOT1L inhibitor EPZ004777, which has activity against mixed lineage leukaemia fusions that cause aberrant localization of DOT1L34. Although EPZ004777 was designed as a SAM analogue (it retains the nucleoside core), it displays remarkable selectivity (>1,000-fold) for inhibition of DOT1L over other his￾tone methyltransferases. Other compounds that bind to the SAM binding site include: the fungal metabo￾lite chaetocin, which is an inhibitor of SUV39H1 and G9A143; and sinefungin, which is a promiscuous natural product and an analogue of SAM144. Thus, in analogy to kinase inhibitors that bind at the ATP site, it appears that targeting the cofactor binding site of protein methyl￾transferases could be a general strategy for this target class. This is supported by computational analysis of the structural diversity observed within the SAM binding site across all human SAM-dependent methyltrans￾ferases, indicating that selectivity should be achievable145. Potent inhibitors of histone arginine methyltransferases such as protein arginine methyltransferase 1 (PRMT1) and PRMT4 have also been identified146–148, providing further evidence that protein methyltransferases can be inhibited by small molecules. Lysine demethylases. First-generation mechanism￾based inhibitors of the flavin-dependent lysine-specific demethylases LSD1 and LSD2, such as tranylcypromine, lacked potency and selectivity over their historical tar￾gets — the monoamine oxidases149,150. Structure–activity relationships subsequently demonstrated that extension of the chemical structure further into the lysine substrate pocket resulted in more potent and selective inhibi￾tors151–153 (for example, compound 10; FIG. 5)154. The LSD class of demethylases are structurally and mechanisti￾cally distinct from the Jumonji domain-containing his￾tone demethylases and appear to primarily target H3K4. Thus, LSDs may offer the possibility of developing selec￾tive H3K4 demethylase antagonists more readily than by selectively targeting the subset of Jumonji domain￾containing H3K4 demethylases. All current inhibitors of Jumonji domain-containing lysine demethlyases compete with the cofactor 2-oxoglut￾arate and bind to the catalytic iron in the active site. The highly polar compound 2,4-pyridine-dicarboxylate inhib￾its the Jumonji domain-containing demethylases as well as other 2-oxoglutarate-dependent oxygenases such as HIF prolyl hydroxylase 1 (HPH1; also known as EGLN2) and HPH2 (also known as EGLN1)155. As observed with the LSD1 inhibitors, extending the chemical structure of the Jumonji domain-containing demethylase inhibitor tem￾plate so that the compound binds directly to the iron in the substrate binding pocket increases potency, as seen with metal-chelating hydroxamic acids156. These compounds are selective for the Jumonji domain-containing demeth￾ylases over other 2-oxoglutarate-dependent oxygenases, but the molecular and physicochemical properties of the compound may limit bioavailability156. Two new series of Jumonji domain-containing demethylase inhibitors, 8-hydroxyquinolines (for exam￾ple, SID 85736331) and 2,2′-bipyridines (for example, compound 15c) (FIG. 5), are potent inhibitors with sub￾type selectivity and more drug-like properties157,158. These new lead compounds have smaller and more compact chemical structures, and represent good lead compounds for further optimization. The compounds gain their potency and selectivity through favourable inhibitor– protein interactions in the active site closer to the metal centre. Thus, as potent and selective inhibitors of histone demethylases have now been identified, the next chal￾lenge will be to identify compounds that have improved cell permeability, which will be better suited to investigate activity in whole-cell assays. Bromodomain-containing proteins. The bromodomain￾containing family of proteins represents an important class of histone modification reader proteins that recog￾nize acetylated lysine residues. The bromodomain was first described in 1992 as a domain of ~110 amino acids that was conserved in several transcriptionally important genes from humans, fruitflies and yeast16. The human genome encodes 42 bromodomain-containing proteins, each of which contains between one and six bromodo￾mains, encompassing a total of 61 unique human bromo￾domains15. Interestingly, bromodomains are commonly found in proteins that also contain enzymatic domains REVIEWS NATURE REVIEWS | DRUG DISCOVERY VOLUME 11 | MAY 2012 | 395 © 2012 Macmillan Publishers Limited. All rights reserved