D r u G D I s C o v e r Y

Reader’s block Protein factors can regulate gene expression by binding to specifically modified DNA-associated proteins. Small molecules that selectively interfere with such interaction may be of therapeutic value. See Article p.1067 & Letter p.1119

s e a n D . t a v e r n a & P H I l I P a . C o l e

Protein factors are crucial for control-ling gene expression. One group of such factors affects gene activity by ‘reading’

epigenetic marks — reversible modifications such as the addition of phosphate, acetyl or methyl groups — on proteins after their trans-lation from RNA. The factors’ target proteins are histones, which associate with DNA to form chromatin. The reading ability of these protein factors is a result of specific, well-folded sub-domains, sometimes called readers, which can distinguish between the post-translationally modified state of their binding partner and its unmodified state. Two papers1,2 in this issue describe highly potent and selective inhibi-tor molecules that compete with acetylated histones for binding to a set of such readers.

These data have therapeutic implications. Post-translational modifications can

often influence transient protein–protein inter actions by creating or disrupting bind-ing surfaces on the molecules. Among such modifications, acetylation on lysine amino-acid residues has been centre stage: originally discovered3 more than 40 years ago as a regula-tor of chromatin structure, this modification has now been detected in thousands of other proteins4. Acetyl-lysine modifications facili-tate the interaction of the protein with proteins that contain bromodomains — evolutionarily conserved subdomains that can specifically bind, or read, the acetylated form of the lysine during regulatory processes5. Such interactions are thought to regulate transcription and to be involved in various diseases, including cancer.

Nucleusaccumbens

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Serotonin↓

HTR2Bmutation

Impulsive behaviours

Figure 1 | HTR2B and the regulation of impulsivity. Bevilacqua et al.1 find that, in a Finnish subpopulation, a mutation in the serotonin receptor HTR2B is linked to severe impulsivity. In the nucleus accumbens region (green) of the brain, projections of neurons that secrete serotonin (red) interact with those that secrete dopamine (blue). This region has been repeatedly shown to play a crucial part in choice and impulsivity. Mutations in HTR2B, which modulates the release of dopamine and serotonin in the nucleus accumbens, may reduce the release of these neurotransmitters, leading to increased impulsive behaviour.

of the 5-HT1A receptor, which may inhibit serotonin release, has been linked to impulsiv-ity in animal models8. Moreover, the levels of 5-hydroxyindoleacetic acid — a metabolite of serotonin — are reduced in the cerebrospinal fluid of people who are suicidal9. Further-more, individuals whose serotonin levels have been experimentally lowered by diet are more likely to make impulsive choices10. Nonethe-less, the role of serotonin is probably com-plex, not least because the serotonin system includes 14 different receptors with sometimes opposing actions.

The HTR2B receptor received little attention in earlier studies of impulsivity. So, to support their human data, Bevilacqua et al.1 examined mice that lack the Htr2b gene. They observed increased impulsive behaviour in these animals according to several measures. How exactly HTR2B deficiency leads to this effect remains unclear, although the authors find that both male mice lacking Htr2b and men carrying the HTR2B Q20* mutation have elevated levels of the hormone testosterone.

Previous work2 suggests that HTR2B may function by modulating both serotonin and dopamine in the nucleus accumbens — a brain region involved in impulsive behaviour (Fig. 1). For instance, the ‘club drug’ ecstasy has been shown11 to stimulate the release of both sero-tonin and dopamine in the nucleus accumbens by directly activating HTR2B. It could there-fore be that depletion of the HTR2B receptor results in increased impulsive behaviour by reducing the release of both serotonin and dopamine in the nucleus accumbens. However, much more work is required to elucidate how HTR2B regulates impulsive behaviour through

its modulation of the interaction between pathways involving serotonin and dopamine.

Bevilacqua and colleagues’ observation1 that the HTR2B Q20* mutation is unique to Finns serves as yet another reminder of the high level of heterogeneity likely to be seen in complex

genetic traits and the importance of population history. But although this specific mutation is absent in non-Finnish populations, different mutations in the HTR2B gene might operate in other populations.

Bevilacqua and colleagues’ paper also illus-trates the power of exon-based sequencing in founder populations, and suggests that exonic mutations of strong functional effect do play a part in complex behavioural traits. ■

John R. Kelsoe is in the Department of Psychiatry, University of California, San Diego, and the VA San Diego Healthcare System, La Jolla, California 92014, USA. e-mail: jkelsoe@ucsd.edu

1. Bevilacqua, L. et al. Nature 468, 1061–1066 (2010).

2. Cardinal, R. N. Neural Netw. 19, 1277–1301 (2006).3. Moeller, F. G., Barratt, E. S., Dougherty, D. M.,

Schmitz, J. M. & Swann, A. C. Am. J. Psychiatry 158, 1783–1793 (2001).

4. Swann, A. C., Lijffijt, M., Lane, S. D., Steinberg, J. L. & Moeller, F. G. Bipolar Disord. 11, 280–288 (2009).

5. Peltonen, L., Jalanko, A. & Varilo, T. Hum. Mol. Genet. 8, 1913–1923 (1999).

6. Robbins, T. W. Psychopharmacology 163, 362–380 (2002).

7. Pattij, T. & Vanderschuren, L. J. M. J. Trends Pharmacol. Sci. 29, 192–199 (2008).

8. Winstanley, C. A., Theobald, D. E., Dalley, J. W. & Robbins, T. W. Neuropsychopharmacology 30, 669–682 (2005).

9. Träskman, L., Åsberg, M., Bertilsson, L. & Sjüstrand, L. Arch. Gen. Psychiatry 38, 631–636 (1981).

10. Rogers, R. D. et al. Psychopharmacology 146, 482–491 (1999).

11. Doly, S. et al. J. Neurosci. 28, 2933–2940 (2008).

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Figure 1 | Targeting the interaction between bromodomains and acetyl-lysine moieties. a, Filippakopoulos et al.1 show that JQ1 — a small-molecule competitive inhibitor that blocks the interaction of bromodomains of BET proteins with acetylated lysines (Ac) — can inhibit the proliferation of tumour cells expressing the BRD4–NUT oncoprotein. b, Nicodeme et al.2 show that pretreatment of cells with another small-molecule competitive inhibitor, I-BET, which interferes with the interaction between the bromodomain of the BET protein BRD4 and Ac, can mute the transcription of genes that are induced during inflammatory responses.

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A range of acetyltransferase enzymes (writers) add acetyl groups to lysine residues, and two families of deacetylase enzymes (erasers) remove these groups6. Two recently approved anticancer drugs7 — SAHA and dep-sipeptide — work by blocking deacetylases, and have galvanized the pharmaceutical industry’s interest in targeting chromatin modifications. In fact, several start-up biotech companies have attempted to target erasers and writers of lysine acetylation. In general, however, even highly specific inhibitors of acetyltransferases and deacetylases that mediate post-transla-tional modification can have undesired side effects, because blocking these enzymes can affect many different protein substrates and biochemical pathways.

As for targeting protein–protein inter-actions, with several notable exceptions the use of small-molecule drugs has been considered extremely difficult8 because their binding regions frequently consist of wide, shallow surfaces. For example, despite decades of work, pharmacologically practical com-pounds that disrupt the binding of phosphor-ylated proteins to their SH2-domain-containing protein partners have remained elusive. There have also been a couple of attempts to use small molecules to inhibit the interactions between proteins containing bromodomains and those carrying acetyl-lysines, but focus on this line of research has generally been limited9.

Using very different approaches, Filippa-kopoulos et al. (page 1067) and Nicodeme et al. (page 1119) now converge on a closely related set of chemical scaffolds — the triazole-diazepine-fused ring compounds JQ1 and I-BET — that inhibit the acetyl-lysine-read-ing ability of a specific class of bromo domain. Both sets of compounds bind tightly to bromo-domains in proteins of the BET family by exploiting the unusual pockets characteristic of this protein family (Fig. 1).

The bromodomains of BET proteins show a strong preference for housing doubly modi-fied acetyl-lysine histone tails in their wide and highly structured hydrophobic pockets10. Because of their shape and electrical prop-erties, these pockets are also well suited for binding small molecules. Indeed, the present papers’ structural data1,2 confirm that JQ1 and I-BET fit snugly into the acetyl-lysine pockets in a stereo specific fashion. Thermodynamic measurements further establish that both of the bromodomain–inhibitor interactions are of high affinity (with dissociation constants below 100 nM) and, compared with their interaction with other non-BET types of bromodomain, show great selectivity (at least 100-fold).

The two teams also pursue distinct bio-medical applications for JQ-1 and I-BET. Filippakopoulos et al.1 examine whether JQ1 can antagonize the growth of a rare but aggressive form of cancer called midline carcinoma. This cancer is defined by a gene fusion that results in the unnatural linkage of BRD4 — a BET protein

containing two bromodomains — with another protein called NUT. The BRD4–NUT fusion protein mediates increased acetylation of certain chromatin domains that are normally transcrip-tionally inactive11, and so it was predicted that inhibitors of the BRD4 bromo domains would shut down tumour growth mediated by this mechanism. Filippakopoulos and colleagues confirm this hypothesis, showing that JQ1 could blunt the growth of midline carcinoma cells in culture, as well as in mice into which the tumour cells were introduced.

Nicodeme et al.2 investigate whether I-BET modulates genes mediating immunological and inflammatory responses. They find that it inhibits the expression of a subset of genes nor-mally induced in response to toxic injury, with histone acetylation being reduced in the chro-matin regions around these genes. In a practi-cal application of these findings, the authors demonstrate that treating mice with I-BET protects against the excessive inflammatory response to septic shock. Such results point to the clinical potential of BET-bromodomain inhibitors in immuno-modulation therapies.

The two papers1,2 provide credibility for the idea of extending the pharmacology of targeting chromatin modifications beyond enzymatic activities and into the challenging arena of protein–protein interactions. One appeal of this strategy is that it avoids the promiscuity of enzyme inhibitors.

The studies further raise the prospect of identifying inhibitors of other readers, such as those that bind proteins containing methyl-lysine modifications. Nonetheless,

antagonizing a reader of post-translational modifications might also prompt unknown and unwanted alterations in biological pathways — a complication that necessitates extensive follow-up studies before such agents can move into the clinic. Moreover, the possible uniqueness of BET-bromodomain structures makes it difficult to predict whether small- molecule inhibitors would be similarly effective in antagonizing other bromodomain forms and reader modules. Nevertheless, the new tools described by these studies will undoubtedly prove attractive to biologists interested in the dynamics of chromatin and gene expression in physiology and disease. ■

Sean D. Taverna and Philip A. Cole are in the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. e-mails: staverna@jhmi.edu; pcole@jhmi.edu

1. Filippakopoulos, P. et al. Nature 468, 1067–1073 (2010).

2. Nicodeme, E. et al. Nature 468, 1119–1123 (2010).3. Gershey, E. L., Vidali, G. & Allfrey, V. G. J. Biol. Chem.

243, 5018–5022 (1968).4. Choudhary, C. et al. Science 325, 834–840 (2009).5. Taverna, S. D., Li, H., Ruthenburg, A. J., Allis, C. D. &

Patel, D. J. Nature Struct. Mol. Biol. 14, 1025–1040 (2007).

6. Cole, P. A. Nature Chem. Biol. 4, 590–597 (2008).7. Lemoine, M. & Younes, A. Discov. Med. 10, 462–470

(2010).8. Wells, J. A. & McClendon, C. L. Nature 450,

1001–1009 (2007).9. Sachchidanand et al. Chem. Biol. 13, 81–90 (2006). 10. Morinière, J. et al. Nature 461, 664–668 (2009).11. Reynoird, N. et al. EMBO J. 29, 2943–2952 (2010).

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