Inhibition of Zinc-Dependent Histone Deacetylases with a Chemically

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Inhibition of Zinc-Dependent Histone Deacetylases with a Chemically Triggered Electrophile Zarko V. Boskovic,†,‡,∇ Melissa M. Kemp,†,∇ Allyson M. Freedy,†,‡ Vasanthi S. Viswanathan,† Marius S. Pop,§,∥ Jason H. Fuller,† Nicole M. Martinez,† Samuel O. Figueroa Lazú,† Jiyoung A. Hong,†,§,⊥ Timothy A. Lewis,† Daniel Calarese,# James D. Love,# Amedeo Vetere,† Steven C. Almo,# Stuart L. Schreiber,†,‡ and Angela N. Koehler*,†,§,∥ †

Center for the Science of Therapeutics, Broad Institute, Cambridge, Massachusetts 02142, United States Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts 02138, United States § Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ∥ Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States ⊥ Division of Hematology/Oncology, Boston Children’s Hospital, Boston, Massachusetts 02116, United States # Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States ‡

S Supporting Information *

ABSTRACT: Unbiased binding assays involving small-molecule microarrays were used to identify compounds that display unique patterns of selectivity among members of the zinc-dependent histone deacetylase family of enzymes. A novel, hydroxyquinoline-containing compound, BRD4354, was shown to preferentially inhibit activity of HDAC5 and HDAC9 in vitro. Inhibition of deacetylase activity appears to be time-dependent and reversible. Mechanistic studies suggest that the compound undergoes zinc-catalyzed decomposition to an orthoquinone methide, which covalently modifies nucleophilic cysteines within the proteins. The covalent nature of the compound−enzyme interaction has been demonstrated in experiments with biotinylated probe compound and with electrospray ionization−mass spectrometry.

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roles that the specific family members play in these diseases, as well as in normal states, may impact novel therapeutic developments. Despite significant efforts, many of the biological functions, precise molecular mechanisms, identity of substrates, and binding partners of HDACs are unknown or are poorly understood.2 Chemical probes can aid in studying their specific functions and clarify the relevance of the classes or individual HDACs as therapeutic candidates for specific diseases. For example, it has been suggested that class IIa HDACs function in cells primarily as “readers” of acetyl marks (bromodomain-like) rather than “erasers” (deacetylases)10,11 and are often thought to be associated with nonhistone targets.12,13 Recently, Lobera et al. described a potent class IIa inhibitor that is currently being used to characterize the roles of class IIa HDACs in autoimmune disease.14 Small molecule binders that can discriminate between different members of the HDAC family have the potential both to enable studies focused on novel

he zinc-dependent family of histone deacetylases (HDACs) regulates diverse and essential cellular activities by deacetylation of lysine residues that govern protein function for a broad spectrum of protein substrates.1 There are 11 family members of the zinc-dependent HDACs, which are divided into four subclasses based on sequence homology: class I (HDAC 1, 2, 3, and 8), class IIa (HDAC 4, 5, 7, and 9), class IIb (HDAC 6 and 10), and class IV (HDAC 11).1 They are widely thought to regulate transcriptional expression by altering chromatin structure through deacetylation of the N-ε-terminus of lysines on histone tails, promoting more compact packing of chromatin structure, and leading to repression of transcriptional activities.2,3 Evidence also suggests other important roles of HDACs outside of chromatin regulation. For example, these enzymes assist in protein stability, translocation, and recognition of other proteins as they often interact with other, nonhistone proteins, to form multicomponent complexes.4 HDACs are involved in many different biological processes, including development, differentiation, and proliferation. They have been implicated in many different diseases, including cancers,5 psychiatric disorders,6,7 metabolic disorders,8 and inflammatory diseases.9 Understanding the precise biological © XXXX American Chemical Society

Received: January 6, 2016 Accepted: April 11, 2016

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DOI: 10.1021/acschembio.6b00012 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. Small-molecule microarray screening. (A) Distribution of composite Z scores (x axis) for printed features (dark blue) and mock treatment (light green) grouped across all assays. Inlay: distribution of composite Z scores >5. (B) Binding signatures for top 200 hits from each HDACbinding assay across eight HDACs shown as a heatmap of compound ranks with respect to its composite Z score within different HDAC binding assays (1139 out of 17 164 total printed features excluding “empty” features). (C) Structures of representative positives identified through the screen.

9 in the biochemical assay. Further investigation of structural analogs of this compound revealed a similar pattern of selectivity. Unique structural features of these compounds prompted us to investigate the mechanism of inhibition in depth. We determined that the compound is a zinc-chelating, reversible inhibitor that covalently labels the enzyme’s cysteine residues. To our knowledge, there have thus far been no other characterized covalent modifiers of HDACs with this unique reversible mechanism or inhibitory profile.

biological activities of this protein class (e.g., proteinprotein interactions) and aid in evaluating the precise roles of deacetylase activity in a cellular context. Many small-molecule HDAC inhibitors fall into four main chemical classes: hydroxamic acids, ortho-amino anilides, aliphatic carboxylic acids, and cyclic peptides.15−19 These compounds often have similar inhibition profiles and preferentially target class I HDACs, although their molecular mechanisms of action can differ substantially.20 There is a growing interest in the development of small molecules that modulate protein activity through novel mechanisms that do not necessarily involve enzymatic activity, such as allosteric regulation. Efforts are ongoing to use new screening technologies and medicinal chemistry to discover and develop novel small molecules that possess unique selectivity, mechanism of action, or novel structure in order to improve upon the current HDAC probe set.14,21 Reported here is an unbiased binding assay enabled by smallmolecule microarrays (SMMs) to profile interactions of HDACs 1−11 to a structurally diverse collection of small molecules.21,22 In total, the screen probed over 220 000 theoretical protein−small molecule interactions. Approximately 530 SMM assay positives, spanning the full set of enzymes, were then evaluated in a secondary trypsin-coupled enzyme activity assay10,23 involving HDACs 1−5 and 7−9 in an effort to annotate compounds that were capable of inhibiting deacetylase enzymatic activity.24 We present our findings of a compound that exhibits preferential inhibition of HDACs 5 and

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RESULTS AND DISCUSSION

SMMs were used to identify novel candidate HDAC binders with unique specificity profiles using protocols described previously.25 Putative binders to recombinant poly-His or GST-tagged HDACs 1−11 were identified by screening arrays containing a total of 21 600 printed features, including various controls and DMSO-only spots as described previously.21,22,25 Briefly, individual HDACs were incubated with arrays in TBST (Tris-buffered saline with Tween 20) buffer for 30 min at room temperature, followed by incubation with either anti-GST or anti-His secondary antibodies conjugated with Alexa Fluor 647 fluorescent dye. A fluorescent signal was measured using a Genepix 4000B slide scanner. All assays were conducted in triplicate, and Z scores were computed as described previously, indicating how many standard deviations the signal was removed from the mean of signals within a particular assay.26,27 An average of three Z scores was used to generate B


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Figure 2. Activity of BRD4354. (A) Hypothesized MoA through Zn(II)-promoted retro-Mannich reaction revealing a thiol-reactive ortho-quinone methide. (B) Inhibition of HDACs 1−5 and 7−9 by BRD4354. (C) Heatmap of IC50 values of an assembled compound collection. (D) Graphical summary of structure−activity relationships around BRD4354. (E) Some representative structures from panel C. See the Supporting Information for complete compound collection.

a composite Z score for each printed feature in each assay. The distribution of composite Z scores is depicted in Figure 1A. To compare compound performance across assays, compounds were ranked on the basis of composite Z scores for each of the individual HDACs screened. Profiles for the 200 top-ranked compounds from each HDAC-binding assay were compared across all assays, and the corresponding heat map of individual ranks was generated (1139 unique compounds total, Figure 1B). The compounds were clustered based on calculated Euclidean distances between their ranks, and the HDACs were clustered based on correlations of their binding patterns to the compounds in the array.28,29 This approach uncovered several compounds with novel signatures of binding selectivity among the evaluated HDACs. To examine the impact of binding of the SMM assay positives on the deacetylase activities of the HDACs, we conducted a follow-up evaluation of compounds prioritized based on their composite Z scores in a fluorometric trypsincoupled deacetylase activity assay against HDACs 1−9 (full length, HDAC 1, 2, 3, 8, and 6; catalytic domains, HDAC 4, 5, 7, and 9; SI Figure 3).23 Representative structures for compounds evaluated in the enzymatic assay are shown in Figure 1C, including compounds with a pan-HDAC binding signature (1 and 2) as well as compounds with novel patterns of selectivity for various HDACs (3 and 4).30 The assay was initially performed at a single, high concentration for the larger set of SMM assay positives and in a dose−response format for selected active compounds of interest, such as 4, that displayed

novel patterns of selectivity in the SMM HDAC binding and in single-dose HDAC inhibition. We observed that compound 4 and its close analog BRD4354 (Figure 2A) were moderately potent inhibitors of HDAC5 and HDAC9, with BRD4354 having half-maximum inhibitory concentrations (IC50) of 0.85 μM and 1.88 μM, respectively (Figure 2B, SI Figure 4). BRD4354 also inhibited HDACs 4, 6, 7, and 8 at higher concentrations (3.88−13.8 μM) but demonstrated less of an inhibitory effect on other class I HDACs 1, 2, and 3 (IC50 > 40 μM). We noted that the inhibition selectivity profile did not completely agree with the binding selectivity profile from the SMM screen. This lack of concordance is likely due to several factors, including the qualitative nature of the SMM assay, subtle differences in the conditions used in the binding and activity assays and the fact that binding of ligand to a protein does not always translate into modulation of a specific function, in this case enzymatic activity. In addition, binding kinetics may vary in the surface- and solution-based assay formats.31 Structurally, the presence of a chelating 8-hydroxyquinoline moiety within both compound 4 and BRD4354 (Figure 2A)32 prompted speculation that this could play a role in the binding of zinc ions present within these enzymes.1 Indeed, a stoichiometric mixture of BRD4354 and zinc triflate in aqueous solution yields rapidly a yellow precipitate of the complex. Moreover, we hypothesized that upon zinc binding, a retroMannich reaction may ensue, effectively fragmenting the compound and producing a reactive ortho-quinone methide intermediate (Figure 2A).33 Similar reactivity has previously C


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Figure 3. (A) Time dependence of BRD4354 inhibition profiles for HDACs 3, 5, and 9 at 0, 3, and 6 h. (B) Partial restoration of HDAC5 activity after 100× dilution subsequent to incubation with BRD4354. HDAC5 was incubated with 8 μM or 800 μM BRD4354 for 1 h at RT. The solutions were diluted 100×, and activity was measured. Final concentrations of BRD4354 after dilution were 0.08 μM and 8 μM. (C) Restoration of activity of HDAC5 (100 nM) incubated with BRD4354 (50 μM) after dialysis.

been observed with para-hydroxy benzylamines34 and 7anilinomethyl-8-hydroxyquinolines whose quinone methide adduct with a proline residue of a macrophage inhibitory factor (MIF) has been crystallographically characterized.35 The novel structure of BRD4354 motivated further characterization of the chemical features contributing to the activity and selectivity of this compound. To better understand structure−activity relationships, a compound collection was assembled through acquisition of commercially available compounds or through synthesis via Mannich or Petasis reactions (structures of investigated compounds, and chemical syntheses, are shown in the Supporting Information). The hierarchical clustering of these compounds based on Euclidean distances of IC50 values for inhibition of individual HDACs in the biochemical assay is graphically summarized in Figure 2C, indicating that a number of active analogs demonstrated selectivity for HDAC5 and HDAC9 over other HDACs. Conclusions drawn from preliminary structure−activity relationship analysis are summarized in Figure 2D.28,29 Methylating the phenol led to a complete loss of activity (compound 26; Figure 2E and Supporting Information).36 Excision of the amino moiety from the ortho-aminomethyl position ablated the activity (compound 29). Removal of the aryl group from the same “central” carbon atom (compound 12) also reduced activity. Replacing 8hydroxyquinoline with a simple ortho-substituted phenol generated inactive compounds (compounds 25, 27). The aryl group could be substituted in a variety of ways, and the substitution of the pyridine did not affect the activity substantially (compounds 13, 18, 19). The nature of benzylic amine also did not affect the activity of the tested compounds (compounds 22 and 24). The quinoline portion of the molecule could also be substituted around the perimeter without an apparent major change in activity (compound 6 vs 13). Despite the metal chelating ability of 8-hydroxyquinolines,32 a naphthol analog (compound 14) retained activity, with a slightly modified selectivity profile compared to other active compounds. A simple Michael acceptor, 28, analogous to

the hypothetically formed ortho-quinone methide was insufficiently electrophilic and showed no inhibitory activity in these assays. Consistent with the hypothesized MoA of these compounds, the resolved enantiomers of BRD4354 (SI Figure 1) had virtually identical IC50 values. We also investigated the time-dependent behavior of BRD4354 by preincubating the compound with HDACs 3, 5, and 9 for varying times prior to initiating the deacetylase activity assay (Figure 3A). We observed a shift to ∼40-fold lower IC50 values for HDACs 5 and 9 after long preincubation periods. The IC50 values for HDAC5 decreased from 11 μM at 0 h to 0.6 μM at 3 h and 0.3 μM at 6 h, while the IC50 for HDAC9, at the same time points, decreased from 36 μM to 2 μM and 0.8 μM. The potency toward HDAC3 also shifted slightly, with an approximately 2-fold reduction after 6 h of incubation. The hydroxamic-acid-containing compound trichostatin A (TSA), a known noncovalent, reversible inhibitor, did not exhibit a time-dependent change in potencies for HDAC 3, 5, and 9 over the same time periods (SI Figure 5). Although time-dependence is normally a feature of irreversible inhibitors, the following two experiments suggest that BRD4354 acts in a reversible manner. Initially, we evaluated the effect of dilution upon the inhibitory properties of BRD4354 (Figure 3B). Specifically, the compound was incubated with HDAC5 at 8 μM (10-fold the approximate IC50) for 1 h. The solution was then diluted 100-fold in buffer containing fluorogenic peptide substrate and trypsin, and activity was compared to DMSO control and to conditions of full enzyme inhibition (BRD4354 concentration after 100× dilution: 8 μM). The deacetylase activity was gradually restored, with ∼75% activity compared with DMSO at 5 h after the 100× dilution with buffer. This is consistent with the result obtained when BRD4354 was incubated with HDAC5 and then subjected to dialysis (Figure 3C). Samples were extracted at different time points (0, 4, and 24 h), and deacetylase activities were evaluated. After 24 h of dialysis, the HDAC activity was restored compared to samples taken before D


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Figure 4. BRD4354 covalently labels HDACs. (A) LC-MS identification and quantification of adduct formation between BRD4354-derived fragment and N-acetylcysteine with various Lewis acidic additives. (B) Western blot quantification of covalently modified HDAC5 by a biotinylated BRD4354 analog, compound 17. (C) Electrospray ionization mass spectrometry demonstration of incorporation of reactive fragment of BRD4354 into the structure of HDAC9. (D) HDACs 5 and 9 amino acid residues covalently modified by BRD4354.

with lesser but measurable amounts of adduct being formed. This experiment demonstrated the zinc-dependent reactivity of the compound with cysteine in a model system and may hint to the mechanism of interaction with HDACs. Covalent binding of the compound to enzyme was also investigated with the biotinylated reagent 17 (Figure 4B). An azide-containing biotin derivative, prepared from biotin chloride and an amino-PEG-azide, was coupled to 20, a BRD4354 analog, through copper hexafluorophosphate catalyzed [3 + 2] cycloaddition.37 HDAC5 was incubated with various concentrations of 17 for 1 h at RT. Upon full denaturation of protein (i.e., reducing conditions and heat), the mixture was electrophoretically separated and transferred to a PVDF membrane. The biotin moiety was “recognized” with streptavidin-horeseradish peroxidase (SA-HRP), and the chemiluminescent signal was visualized and quantified (Figure 4B). We observed dose-dependent labeling of HDAC5 with 17,

dialysis and at 4 h after dialysis. These results indicate that the activity is restored after dialysis and dilution, suggesting that the compound acts as a reversible inhibitor. A similar result was obtained with TSA, albeit with complete restoration of enzymatic activity upon dilution (SI Figure 6). In order to investigate further the hypothesis that the mechanism of inhibition involved participation of Lewis acidic zinc and transient formation of ortho-quinone methide, we monitored the reactivity of BRD4354 with N-acetyl-L-cysteine (NAC) in the presence or absence of Zn2+ ions (Figure 4A). The reaction was monitored over several hours by LC-MS with various salt additives. Adduct formation was observed between NAC and the compound with the addition of zinc, resulting in overall substitution of the piperazine portion of the molecule with NAC. Little to no adduct formation was observed without zinc triflate, or in the presence of triflic acid. This experiment was performed with other Lewis acidic ions (Li+ and Mg2+) E


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acetylation of p53 from HEK293 cells co-transfected with p53 and p300 via HDAC5 overexpressed in HEK293 cells and isolated through immunoprecipitation via a FLAG-octapeptide epitope (SI Figure 10). As there are no current gold-standard phenotypic assays to evaluate HDAC5 and 9 deacetylase activity or their adaptor function,40 we opted to explore fairly general phenotypic assays. We initially investigated BRD4354 in a cell-viability assay using approximately 70 cancer cell lines noted in the Cancer Therapeutics Response Portal (www.broadinstitute.org/ctrp).41 Contrary to the preconception that a thiol-reactive compound would be generally toxic to cells,42 the viability profile for BRD4354 indicated that the compound did not affect the viability of most cell lines at concentrations near the IC50 values determined in the enzymatic assay, and we noted only a handful of sensitive cell lines (Figure 5). Focusing on a small subset of these lines that were sensitive and resistant, the viability assay was repeated and the observed activity was verified (SI Figure 11).

whereas inactive quinoline-containing 30 showed no propensity to label the enzyme. As a control, bovine serum albumin (BSA) was incubated with the compound and showed virtually no labeling compared to HDAC5 at compound concentrations as high as 100 μM. This corroborated preliminary isothermal titration calorimetry (ITC) data, where we observed a strong temperature differential upon the addition of BRD4354 to HDAC5, but a negligible temperature shift with the addition of compound to BSA (SI Figure 8). Interaction between HDAC5 and BRD4354 was also investigated by circular dichroism (CD) to assess the protein’s thermal stability (SI Figure 7).38 By monitoring the amplitude of the peak at 222 nm over increasing temperatures, we concluded that the compound stabilized the conformation of HDAC5. Guided by previous conditions used to study HDAC4 and HDAC7, both of which have been successfully crystallized, we attempted crystallization of HDAC5 with and without BRD4354. To date, these efforts have not yielded diffraction-quality crystals. The binding of the biotinylated analog and pure HDAC5 was also evaluated in the presence of additional zinc or in the presence of zinc-chelating EDTA. Additional Zn2+ ions did not enhance labeling efficiency of these compounds, nor did EDTA diminish it (SI Figure 2). This suggests that Zn2+ ions already embedded within the enzyme’s structure (whether catalytic or structural) are sufficient to catalyze the retro-Mannich reaction and generate an electrophilic ortho-quinone methide species responsible for labeling of these enzymes. Electrospray ionization mass spectrometry (ESI/MS) experiments involving HDAC9 provided additional evidence to suggest that this enzyme forms a covalent bond with the compound fragment. Recombinant HDAC9 showed two major peaks (48.8 kDa and 50.8 kDa) in the ESI-MS spectrum. Upon treatment of HDAC9 with BRD4354, a clear incorporation of the reactive fragment was observed in both peaks. Interestingly, under the conditions used (relatively high protein concentration2 μMcompared to that used in the enzymatic activity assays), the introduction of multiple reactive fragments to HDAC9 was observed (Figure 4C). This prompted a more in-depth attempt to identify specific amino acid residues that were being modified in response to treatment with BRD4354. Tryptic digestion of compound-treated catalytic domains for HDAC5 and HDAC9 (2 μM enzyme, 20 μM compound) was performed, and several cysteine residues were detected as being modified using LC-MS/MS (Figure 4D). Analogous cysteines, C757 in HDAC9 and C807 in HDAC5 (C154 and C150 in catalytic domains shown), were modified in both proteins. Based on sequence identity of HDACs 1−9, these particular cysteines are conserved in HDAC4, 5, and 9. Weerapana and Pace recently demonstrated reduced nucleophilicity of metalbinding cysteines compared to free cysteines.39 Conceivably, the inertness of other cysteines in the catalytic domains toward reaction with electrophilic compounds, such as BRD4354, reflects their status as metal-chelating residues. While BRD4354 inhibits HDACs both reversibly and covalently, it is difficult to define the precise mechanism of inhibition in the absence of additional structural information. For example, it is possible that zinc-chelation is reversible, whereas labeling may be an irreversible step. Finally, we initiated experiments to evaluate BRD4354 in a cellular context. HeLa cells treated with BRD4354 did not show an increase in bulk acetylation levels of histones or tubulin, proteins considered targets of class I HDACs (SI Figure 9). In a more focused experiment, we observed a small increase in

Figure 5. Effect of BRD4354 on viability of 70 cell lines.

The impact of BRD4354 treatment on live cells could also be assessed through measurement of change in expression of 1000 “landmark genes” in response to compound treatment.43 A549 adenocarcinoma cells were treated with BRD4354 for 24 h at 10 μM, and the top 50 upregulated and top 50 down regulated genes were compared to other compound treatments involving drugs, bioactive compounds with established MoA, and novel synthetic compounds. Hydroxamic-acid-containing, matrix metalloprotein 3 (MMP-3) inhibitor UK 356618 and a selective acyl-coenzyme A:cholesterol acyltransferase inhibitor CI-976 produced the most similar signature in terms of up- and downregulation of genes (SI Figure 12). Additional experiments are needed to understand the mechanism of selective toxicity or gene expression patterns observed in cells treated with BRD4354, including unbiased experiments focused on the identification of other proteins that may engage the compound in a cellular setting. BRD4354 and its congeners represent novel, biocompatible, masked, thiol-reactive chemical entities that diversify the commonly used cysteine-reactive molecules, such as acrylamides and vinyl sulfones. 44 Covalent inhibitors have demonstrated importance as chemical probes and in therapeutic-driven research,45,46 with benefits ranging from increased selectivity and potency, to prolonged effects and pharmacodynamics.47 Many covalent inhibitors target the nucleophilic thiol group of cysteines. These residues are among the least abundant residues in proteins and are often involved in imparting structural stability, by forming disulfide bridges or interacting with cofactors, such as metals.48 Targeting noncatalytic cysteines with reactive inhibitors has F



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the potential to increase specificity within a protein family.49 Several kinases have successfully been targeted using this strategy despite concerns about possible off-target effects.50 Shokat and colleagues demonstrated that covalent compound− enzyme interaction can provide an avenue to mutant-selective (when the mutation gives rise to a cysteine residue) inhibitors of oncogenic K-Ras.51 Recently, Taunton and colleagues demonstrated the importance of targeting noncatalytic cysteine residues of RSK2 with covalent, reversible inhibitors, achieving the selectivity and prolonged effect of covalent modifiers, but with the benefit of reversibility over time.49,52 In conclusion, we have identified an HDAC inhibitor that demonstrates a novel selectivity pattern in vitro, with enhanced selectivity toward HDAC5 and HDAC9. Several model studies, time-course studies, and dilution experiments suggest a mechanism that involves reversible inhibition of the enzymatic activity, and the covalent labeling of nucleophilic cysteines, the contribution of which to the enzyme inhibition is currently unknown. Notwithstanding the many caveats pertaining to covalent modifiers and reactive electrophilic compounds, where nonselective reactivity is often perceived as a liability, the observed selectivity within this class of enzymes leads us to believe that this small molecule could be potentially used as a probe to further understand the roles of selected HDAC enzymes. Additionally, the compound may serve as an example for the development of other “triggered” bioactive electrophiles, i.e., compounds whose intrinsic reactivity is revealed only under precisely defined biological conditions.53

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00012. Statistical analysis and heatmaps, chemistry, covalent adduct formation, enzyme activity data and reversibility studies, circular dichroism of HDAC5 with BRD4354, isothermal titration calorimetry, and change in expression of 1000 landmark genes in response to treatment with BRD4354 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Author Contributions ∇

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by funds from the National Institutes of Health: R01-CA160860 (A.N.K), 2R01GM038627 (S.L.S.), and the New York Structural Genomics Research Consortium U54-GM094662 (S.C.A.). S.L.S. is an Investigator with the Howard Hughes Medical Institute. Z.V.B. is a postdoctoral research associate at the Howard Hughes Medical Institute. N.M.M. and S.O.F.L. were participants of Summer Research Program in Genomics at the Broad Institute. We thank Olivia McPherson for assistance in manufacture of SMMs. G


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