REVIEWS rial re tion ofmultinle histone marks To nium moietyThe try of the aromatic cage a he structures of 23 of the 61 human bromodomains nd configuration of a c harg ates one ing pockets,the deepna wclefts that bind mono-and and onmaincouldatas antaeonist.UNC6g,wasrceTntydeiopcdforthcM teins Based on these findings,Zhou ues D) he sam eG9A)to mim he may t oey e orodomain nging to target al design of cy icpeptide m ulat s of the ides for the to demo strate that epig netic modifiers have a clea ighe er than ofh ine-acetylated histo and tur 53.Th evaluatin targets.owing to rthat of the linear ys382-c ed p53 ide.This Th first wave of r the main o have been bene reatment of cutaneou sriptional protein ted the f andem bromodomain-containing family of tran ip HDAC inhibitors for n n as th ron mental endoc e disruptor nt restriction r range nent an I sex of the into the nultiple generations ecause mo P300/CBP-associated factor epigenome has th中 have been obs (or on germ cells be ore conception or embryonic d hstineea s.As is the case with bromodc will need to be elope tonnteoctod nd may incu de too ppears to be possible.A com on theme among all 396 MAY 2012 VOLUME 11 www.nature.com/reviews/drugdisc 2012 Macmillan Publishers Limited.All riehts reserved P300/CBP-associated factor A trancriptional co-activator protein containing a bromodomain and a histone acetyltransferase domain. π electron interactions A non-covalent interaction between the π-electron cloud of aromatic rings and the cationic charge of, for example, methylated lysine. (for example, HATs) or other reader domains (for exam￾ple, PHDs)159 in configurations that contribute to specific combinatorial recognition of multiple histone marks160. To date, the structures of 23 of the 61 human bromodomains have been experimentally determined, demonstrating a conserved hydrophobic pocket that accommodates one (and sometimes two) acetyl-lysine side chains15,161. Bromodomains adopt a left-handed, four-helix bun￾dle comprising amphipathic helices known as alphaZ, alphaA, alphaB and alphaC. At one end of the helical bundle, the amino- and carboxyl termini come together, emphasizing the modular architecture of this domain and underscoring the idea that the bromodomain could act as an independent functional unit that interacts with other proteins. Based on these findings, Zhou and colleagues162 conducted a nuclear magnetic resonance (NMR)-based chemical screen to identify compounds that bound to the bromodomain of the HAT P300/CBP-associated factor with an affinity comparable to that of the Tat peptide acetylated on Lys50 (IC50 (half-maximal inhibitory con￾centration) ~5μM). The lead compound did not bind to the structurally related bromodomains of CREBBP and TIF1β (transcriptional intermediary factor 1β), indicat￾ing that it is possible to identify small-molecule inhibitors that have specificity within the bromodomain family162. Building on this observation, Zhou et al.163 described the rational design of cyclic peptide modulators of the bromo￾domain-containing transcriptional co-activator CREBBP. The affinity of the cyclic peptides for the CREBBP bromo￾domain was significantly higher than the affinity of the bromodomain for its biological ligands, which included lysine-acetylated histones and tumour suppressor p53. The best cyclic peptide exhibited a Kd (dissociation constant) of 8.0μM, representing a 24-fold improvement in affinity over that of the linear Lys382-acetylated p53 peptide. This lead peptide was highly selective for the bromodomain of CREBBP compared with bromodomains from other tran￾scriptional proteins163. Recently, two independent groups reported the first selective inhibitors with low nanomolar affinity for the tandem bromodomain-containing family of transcrip￾tional regulators known as the BET proteins (BRD2, BRD3, BRD4 and BRDT)36,76,164. The compounds JQ1 and IBET represent novel chemical templates that are distinct from the previously reported simple acetyl-containing templates, and they have a clear mode of action. These studies demonstrate that it is feasible to produce inhibi￾tors that have a high affinity (in the nanomolar range), specificity and cell permeability (FIG. 3). The development of these inhibitors has revealed novel insights into the physiological role and therapeutic potential of inhibiting BET function. Indeed, beneficial effects of these inhibi￾tors have been observed in several cancers and systemic inflammatory conditions36,40,41,76. Methyl-lysine readers. As is the case with bromodomains, the ability to inhibit the interaction between a methyl￾lysine reader domain and its target methyl-lysine mark appears to be possible. A common theme among all methyl-lysine reader domains is the presence of a con￾served ‘aromatic cage’ that comprises the binding cleft for the methyl-lysine side chain and provides π electron interactions with the positively charged methylammo￾nium moiety160. The geometry of the aromatic cage as well as the presence and configuration of a countercharge or hydrogen-bond acceptor determines the degree of methylation that is optimal for binding. These structural features are attractive for drug discovery. Of all the known structures of methyl-lysine bind￾ing pockets, the deep narrow clefts that bind mono- and di-methyl-lysine (such as those found in MBT domains), may be the most attractive targets for the design of small molecules. The first example of such a small-molecule antagonist, UNC669, was recently developed for the MBT domain-containing protein L3MBTL1 (lethal 3 MBT￾like protein 1), using a structure-guided approach165. Interestingly, UNC669 uses the same pyrrolidine moiety as the protein methyltransferase inhibitor UNC638 (which is an inhibitor of the dimethylase G9A) to mimic dimethyl-lysine, suggesting that pyrrolidine may be used as a universal ‘warhead’ in compounds that target dimethyl￾lysine binding pockets in proteins. At present, there are no reported antagonists of trimethyl-lysine readers. Because some trimethyl-lysine binding pockets tend to be more open and shallow compared with those of MBT domains, they may be more challenging to target. Safety of drugging epigenetic modifiers As with all new potential drug targets, there will be a need to demonstrate that epigenetic modifiers have a clear benefit in the treatment of diseases that can be achieved with an acceptable safety and tolerability profile. This is especially important when evaluating epigenetic protein targets, owing to their fundamental role as general fac￾tors in the regulation of global gene expression patterns. The first wave of epigenetic drugs — HDAC inhibi￾tors — have been beneficial in the treatment of cutaneous T cell lymphoma, with acceptable adverse event profiles; additional clinical studies are underway to determine their utility in treating other cancers166. There are ongoing studies to determine the potential therapeutic utility of HDAC inhibitors for non-oncology indications in which the adverse event profile requirements may be more stringent167. For non-oncology indications, key safety issues include the long-term effects of the drug on stem cells and germ cells, especially potential transgenerational effects168. For example, embryonic exposure to environ￾mental endocrine disruptors169 or nutrient restriction170 during gonadal development and sex determination is capable of inducing adult-onset disease states that can be perpetuated across multiple generations. Because modulation of the epigenome has the poten￾tial to reprogramme all cells, adverse effects on stem cells (or on germ cells before conception or embryonic devel￾opment) may only become apparent over longer periods of time. Strategies to investigate and avoid such effects will need to be developed, and may include tools such as epigenomic profiling of histone and DNA marks in the appropriate cell types. Identification of inhibitors that are truly subtype- or target-selective for their epigenetic protein will also help to tease apart the balance between efficacy and safety for a given target. REVIEWS 396 | MAY 2012 | VOLUME 11 www.nature.com/reviews/drugdisc © 2012 Macmillan Publishers Limited. All rights reserved