GNE-064: A Potent, Selective, and Orally Bioavailable Chemical Probe for the Bromodomains of SMARCA2 and SMARCA4 and the Fifth Bromodomain of PBRM1.

Bromodomains are acetyllysine recognition domains present in a variety of human proteins. Bromodomains also bind small molecules that compete with acetyllysine, and therefore bromodomains have been targets for drug discovery efforts. Highly potent and selective ligands with good cellular permeability have been proposed as chemical probes for use in exploring the functions of many of the bromodomain proteins. We report here the discovery of a class of such inhibitors targeting the family VIII bromodomains of SMARCA2 (BRM) and SMARCA4 (BRG1), and PBRM1 (polybromo-1) bromodomain 5. We propose one example from this series, GNE-064, as a chemical probe for the bromodomains SMARCA2, SMARCA4, and PBRM1(5) with the potential for in vivo use.

Discovery of CPI-1612: A Potent, Selective, and Orally Bioavailable EP300/CBP Histone Acetyltransferase Inhibitor.

The histone acetyltransferases, CREB binding protein (CBP) and EP300, are master transcriptional co-regulators that have been implicated in numerous diseases, such as cancer, inflammatory disorders, and neurodegeneration. A novel, highly potent, orally bioavailable EP300/CBP histone acetyltransferase (HAT) inhibitor, CPI-1612 or 17, was developed from the lead compound 3. Replacement of the indole scaffold of 3 with the aminopyridine scaffold of 17 led to improvements in potency, solubility, and bioavailability. These characteristics resulted in a 20-fold lower efficacious dose for 17 relative to lead 3 in a JEKO-1 tumor mouse xenograft study.

Early Drug-Discovery Efforts towards the Identification of EP300/CBP Histone Acetyltransferase (HAT) Inhibitors.

EP300 and CBP (KAT3A/3B) are two highly homologous, multidomain, epigenetic coregulators that play central roles in transcription through the acetylation of lysine residues on histones and other proteins. Both enzymes have been implicated in human diseases, especially cancer. From a high-throughput screen of 191 000 compounds searching for EP300/CBP histone acetyltransferase (HAT) inhibitors, 18 compounds were characterized by a suite of biochemical enzymatic assays and biophysical methods, including X-ray crystallography and native mass spectrometry. This work resulted in the discovery of three distinct mechanistic classes of EP300/CBP HAT inhibitors, including two classes not previously described. The profiles of an example of each class of inhibitor are described in detail. A subsequent medicinal chemistry effort led to the development of a novel class of orally bioavailable AcCoA-competitive EP300/CBP HAT inhibitors with in vivo activity. We believe that this work will prove to be a useful guide for other groups interested in the development of HAT inhibitors.

Make the right measurement: Discovery of an allosteric inhibition site for p300-HAT.

Histone acetyltransferases (HATs) and histone deacetylases (HDACs) catalyze the dynamic and reversible acetylation of proteins, an epigenetic regulatory mechanism associated with multiple cancers. Indeed, HDAC inhibitors are already approved in the clinic. The HAT paralogs p300 and CREB-binding protein (CBP) have been implicated in human pathological conditions including several hematological malignancies and androgen receptor-positive prostate cancer. Others have reported CoA-competitive inhibitors of p300 and CBP with cell-based activity. Here, we describe 2 compounds, CPI-076 and CPI-090, discovered through p300-HAT high throughput screening screening, which inhibit p300-HAT via binding at an allosteric site. We present the high resolution (1.7 and 2.3 Å) co-crystal structures of these molecules bound to a previously undescribed allosteric site of p300-HAT. Derivatization yielded actionable structure-activity relationships, but the full-length enzymatic assay demonstrated that this allosteric HAT inhibitor series was artifactual, inhibiting only the HAT domain of p300 with no effect on the full-length enzyme.

GNE-371, a Potent and Selective Chemical Probe for the Second Bromodomains of Human Transcription-Initiation-Factor TFIID Subunit 1 and Transcription-Initiation-Factor TFIID Subunit 1-like.

The biological functions of the dual bromodomains of human transcription-initiation-factor TFIID subunit 1 (TAF1(1,2)) remain unknown, although TAF1 has been identified as a potential target for oncology research. Here, we describe the discovery of a potent and selective in vitro tool compound for TAF1(2), starting from a previously reported lead. A cocrystal structure of lead compound 2 bound to TAF1(2) enabled structure-based design and structure-activity-relationship studies that ultimately led to our in vitro tool compound, 27 (GNE-371). Compound 27 binds TAF1(2) with an IC50 of 10 nM while maintaining excellent selectivity over other bromodomain-family members. Compound 27 is also active in a cellular-TAF1(2) target-engagement assay (IC50 = 38 nM) and exhibits antiproliferative synergy with the BET inhibitor JQ1, suggesting engagement of endogenous TAF1 by 27 and further supporting the use of 27 in mechanistic and target-validation studies.

Discovery of a Potent and Selective in Vivo Probe (GNE-272) for the Bromodomains of CBP/EP300.

The single bromodomain of the closely related transcriptional regulators CBP/EP300 is a target of much recent interest in cancer and immune system regulation. A co-crystal structure of a ligand-efficient screening hit and the CBP bromodomain guided initial design targeting the LPF shelf, ZA loop, and acetylated lysine binding regions. Structure-activity relationship studies allowed us to identify a more potent analogue. Optimization of permeability and microsomal stability and subsequent improvement of mouse hepatocyte stability afforded 59 (GNE-272, TR-FRET IC50 = 0.02 µM, BRET IC50 = 0.41 µM, BRD4(1) IC50 = 13 µM) that retained the best balance of cell potency, selectivity, and in vivo PK. Compound 59 showed a marked antiproliferative effect in hematologic cancer cell lines and modulates MYC expression in vivo that corresponds with antitumor activity in an AML tumor model.

Identification of potent, selective KDM5 inhibitors.

This communication describes the identification and optimization of a series of pan-KDM5 inhibitors derived from compound 1, a hit initially identified against KDM4C. Compound 1 was optimized to afford compound 20, a 10nM inhibitor of KDM5A. Compound 20 is highly selective for the KDM5 enzymes versus other histone lysine demethylases and demonstrates activity in a cellular assay measuring the increase in global histone 3 lysine 4 tri-methylation (H3K4me3). In addition compound 20 has good ADME properties, excellent mouse PK, and is a suitable starting point for further optimization.

S-adenosyl methionine is necessary for inhibition of the methyltransferase G9a by the lysine 9 to methionine mutation on histone H3.

Lysine to methionine (K-to-M) mutations in genes encoding histone H3 are thought to drive a subset of pediatric brain and bone cancers. These high-frequency K-to-M mutations occur at sites of methylation on histone H3, and tumors containing the mutant histones exhibit a global loss of specific histone methylation marks. Previous studies showed that K-to-M mutant histones, also known as oncohistones, are potent orthosteric inhibitors of specific Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain methyltransferases. However, the biochemical and biophysical details of the interaction between K-to-M mutant histones and the respective SET domain methyltransferases are currently unknown. Here, we use the histone H3K9-directed methyltransferase G9a as a model to explore the mechanism of inhibition by K-to-M oncohistones. X-ray cocrystal structures revealed that the K9M residue of histone H3 occupies the active site cavity of G9a, and kinetic analysis indicates competitive inhibition of G9a by histone H3K9M. Additionally, we find that the cofactor S-adenosyl methionine (SAM) is necessary for stable interaction between G9a and H3K9M histone. Consistent with the formation of a ternary complex, we find that the inhibitory peptide is uncompetitive with regard to SAM. These data and others indicate that K-to-M oncohistones promote global loss of specific lysine methylation through sequestration and inhibition of SAM-bound SET domain methyltransferases.

Diving into the Water: Inducible Binding Conformations for BRD4, TAF1(2), BRD9, and CECR2 Bromodomains.

The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2. The preference of distinct substituents for individual bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.

Regulatory T Cell Modulation by CBP/EP300 Bromodomain Inhibition.

Covalent modification of histones is a fundamental mechanism of regulated gene expression in eukaryotes, and interpretation of histone modifications is an essential feature of epigenetic control. Bromodomains are specialized binding modules that interact with acetylated histones, linking chromatin recognition to gene transcription. Because of their ability to function in a domain-specific fashion, selective disruption of bromodomain:acetylated histone interactions with chemical probes serves as a powerful means for understanding biological processes regulated by these chromatin adaptors. Here we describe the discovery and characterization of potent and selective small molecule inhibitors for the bromodomains of CREBBP/EP300 that engage their target in cellular assays. We use these tools to demonstrate a critical role for CREBBP/EP300 bromodomains in regulatory T cell biology. Because regulatory T cell recruitment to tumors is a major mechanism of immune evasion by cancer cells, our data highlight the importance of CREBBP/EP300 bromodomain inhibition as a novel, small molecule-based approach for cancer immunotherapy.

Discovery of Benzotriazolo[4,3-d][1,4]diazepines as Orally Active Inhibitors of BET Bromodomains.

Inhibition of the bromodomains of the BET family, of which BRD4 is a member, has been shown to decrease myc and interleukin (IL) 6 in vivo, markers that are of therapeutic relevance to cancer and inflammatory disease, respectively. Herein we report substituted benzo[b]isoxazolo[4,5-d]azepines and benzotriazolo[4,3-d][1,4]diazepines as fragment-derived novel inhibitors of the bromodomain of BRD4. Compounds from these series were potent and selective in cells, and subsequent optimization of microsomal stability yielded representatives that demonstrated dose- and time-dependent reduction of plasma IL-6 in mice.

Identification of a Benzoisoxazoloazepine Inhibitor (CPI-0610) of the Bromodomain and Extra-Terminal (BET) Family as a Candidate for Human Clinical Trials.

In recent years, inhibition of the interaction between the bromodomain and extra-terminal domain (BET) family of chromatin adaptors and acetyl-lysine residues on chromatin has emerged as a promising approach to regulate the expression of important disease-relevant genes, including MYC, BCL-2, and NF-κB. Here we describe the identification and characterization of a potent and selective benzoisoxazoloazepine BET bromodomain inhibitor that attenuates BET-dependent gene expression in vivo, demonstrates antitumor efficacy in an MV-4-11 mouse xenograft model, and is currently undergoing human clinical trials for hematological malignancies (CPI-0610).

A Subset of Human Bromodomains Recognizes Butyryllysine and Crotonyllysine Histone Peptide Modifications.

Bromodomains are epigenetic readers that are recruited to acetyllysine residues in histone tails. Recent studies have identified non-acetyl acyllysine modifications, raising the possibility that these might be read by bromodomains. Profiling the nearly complete human bromodomain family revealed that while most human bromodomains bind only the shorter acetyl and propionyl marks, the bromodomains of BRD9, CECR2, and the second bromodomain of TAF1 also recognize the longer butyryl mark. In addition, the TAF1 second bromodomain is capable of binding crotonyl marks. None of the human bromodomains tested binds succinyl marks. We characterized structurally and biochemically the binding to different acyl groups, identifying bromodomain residues and structural attributes that contribute to specificity. These studies demonstrate a surprising degree of plasticity in some human bromodomains but no single factor controlling specificity across the family. The identification of candidate butyryl- and crotonyllysine readers supports the idea that these marks could have specific physiological functions.

Development of methyl isoxazoleazepines as inhibitors of BET.

In this report we detail the evolution of our previously reported thiophene isoxazole BET inhibitor chemotype exemplified by CPI-3 to a novel bromodomain selective chemotype (the methyl isoxazoleazepine chemotype) exemplified by carboxamide 23. The methyl isoxazoleazepine chemotype provides potent inhibition of the bromodomains of the BET family, excellent in vivo PK across species, low unbound clearance, and target engagement in a MYC PK-PD model.

Discovery, Design, and Optimization of Isoxazole Azepine BET Inhibitors.

The identification of a novel series of small molecule BET inhibitors is described. Using crystallographic binding modes of an amino-isoxazole fragment and known BET inhibitors, a structure-based drug design effort lead to a novel isoxazole azepine scaffold. This scaffold showed good potency in biochemical and cellular assays and oral activity in an in vivo model of BET inhibition.

Crystal structure of a complex between amino and carboxy terminal fragments of mDia1: insights into autoinhibition of diaphanous-related formins.

Formin proteins direct the nucleation and assembly of linear actin filaments in a variety of cellular processes using their conserved formin homology 2 (FH2) domain. Diaphanous-related formins (DRFs) are effectors of Rho-family GTPases, and in the absence of Rho activation they are maintained in an inactive state by intramolecular interactions between their regulatory N-terminal region and a C-terminal segment referred to as the DAD domain. Although structures are available for the isolated DAD segment in complex with the interacting region in the N-terminus, it remains unclear how this leads to inhibition of actin assembly by the FH2 domain. Here we describe the crystal structure of the N-terminal regulatory region of formin mDia1 in complex with a C-terminal fragment containing both the FH2 and DAD domains. In the crystal structure and in solution, these fragments form a tetrameric complex composed of two interlocking N+C dimers. Formation of the tetramer is likely a consequence of the particular N-terminal construct employed, as we show that a nearly full-length mDia1 protein is dimeric, as are other autoinhibited N+C complexes containing longer N-terminal fragments. The structure provides the first view of the intact C-terminus of a DRF, revealing the relationship of the DAD to the FH2 domain. Delineation of alternative dimeric N+C interactions within the tetramer provides two general models for autoinhibition in intact formins. In both models, engagement of the DAD by the N-terminus is incompatible with actin filament formation on the FH2, and in one model the actin binding surfaces of the FH2 domain are directly blocked by the N-terminus.

The Skap-hom dimerization and PH domains comprise a 3'-phosphoinositide-gated molecular switch.

PH domains, by binding to phosphoinositides, often serve as membrane-targeting modules. Using crystallographic, biochemical, and cell biological approaches, we have uncovered a mechanism that the integrin-signaling adaptor Skap-hom uses to mediate cytoskeletal interactions. Skap-hom is a homodimer containing an N-terminal four-helix bundle dimerization domain, against which its two PH domains pack in a conformation incompatible with phosphoinositide binding. The isolated PH domains bind PI[3,4,5]P(3), and mutations targeting the dimerization domain or the PH domain's PI[3,4,5]P(3)-binding pocket prevent Skap-hom localization to ruffles. Targeting is retained when the PH domain is deleted or by combined mutation of the PI[3,4,5]P(3)-binding pocket and the PH/dimerization domain interface. Thus, the dimerization and PH domain form a PI[3,4,5]P(3)-responsive molecular switch that controls Skap-hom function.

Structure of the FH2 domain of Daam1: implications for formin regulation of actin assembly.

Daam1 (dishevelled-associated activator of morphogenesis-1) is a diaphanous-related formin first studied as a novel dishevelled binding protein and shown to be crucial for the planar cell polarity (PCP) pathway in Xenopus. Daam1, like other formins, directs nucleation and elongation of new actin filaments using its conserved formin-homology-2 (FH2) domain. Here we report the crystal structure of a large C-terminal fragment of human Daam1 containing the FH2 domain. The structure, determined at 2.25 A resolution using the single-wavelength anomalous diffraction (SAD) phasing method, reveals a "tethered dimer" architecture that is similar to that previously described for the FH2 domain of the yeast formin Bni1, which shares approximately 21% sequence identity with Daam1. Despite the overall similarity with the dimeric FH2 domain of Bni1 and with a truncated monomeric structure of mDia1, the Daam1 FH2 structure reveals a number of differences in secondary structure elements and in the "lasso/post" dimerization interface that may be functionally important. Most strikingly, the two halves of the crystallographic dimer pack together in a manner that occludes their actin binding surfaces. This "locked" conformation is stabilized by two novel, interacting beta-strands formed by the ends of the linkers that connect the two sides of the dimer. The Daam1 FH2 domain has weak actin assembly activity as compared with other mammalian formins, but mutations that disrupt the beta-strand lock increase activity about tenfold to a level comparable to other formins, suggesting that this occluded conformation may represent an auto-inhibited conformation of the Daam1 FH2 domain.

Structure of the autoinhibitory switch in formin mDia1.

Diaphanous-related formins (DRFs) regulate the nucleation and polymerization of unbranched actin filaments. The activity of DRFs is inhibited by an intramolecular interaction between their N-terminal regulatory region and a conserved C-terminal segment termed the Diaphanous autoinhibitory domain (DAD). Binding of GTP bound Rho to the mDia1 N terminus releases this autoinhibitory restraint. Here, we describe the crystal structure of the DAD segment of mDia1 in complex with the relevant N-terminal fragment, termed the DID domain. The structure reveals that the DAD segment forms an amphipathic helix that binds a conserved, concave surface on the DID domain. Comparison with the structure of the mDia1 N terminus bound to RhoC suggests that release of the autoinhibitory DAD interaction is accomplished largely by Rho-induced restructuring of the adjacent GTPase binding subdomain (GBD), but also by electrostatic repulsion and a small, direct steric occlusion of the DAD binding cleft by Rho itself.

Crystal structure of the FERM domain of focal adhesion kinase.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells. Through phosphorylation of proteins assembled at the cytoplasmic tails of integrins, FAK promotes signaling events that modulate cellular growth, survival, and migration. The amino-terminal region of FAK contains a region of sequence homology with band 4.1 and ezrin/radixin/moesin (ERM) proteins termed a FERM domain. FERM domains are found in a variety of signaling and cytoskeletal proteins and are thought to mediate intermolecular interactions with partner proteins and phospholipids at the plasma membrane and intramolecular regulatory interactions. Here we report two crystal structures of an NH2-terminal fragment of avian FAK containing the FERM domain and a portion of the regulatory linker that connects the FERM and kinase domains. The tertiary folds of the three subdomains (F1, F2, and F3) are similar to those of known FERM structures despite low sequence conservation. Differences in the sequence and relative orientation of the F3 subdomain alters the nature of the interdomain interface, and the phosphoinositide binding site found in ERM family FERM domains is not present in FAK. A putative protein interaction site on the F3 lobe is masked by the proximal region of the linker. Additionally, in one structure the adjacent Src SH3 and SH2 binding sites in the linker associate with the surfaces of the F3 and F1 lobes, respectively. These structural features suggest the possibility that protein interactions of the FAK FERM domain can be regulated by binding of Src kinases to the linker segment.