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.

Screening for Small-Molecule Inhibitors of Histone Methyltransferases.

Potent and highly selective small-molecule inhibitors are needed to unravel the biological complexities of histone methyltransferases and to reveal their therapeutic potential. A prerequisite to developing these inhibitors is the identification of validated chemical matter for initiating a medicinal chemistry campaign. For the most part, finding these initial starting points occurs through screening of large, unbiased compound libraries. The size and nature of these libraries, coupled with the complexities of the bisubstrate utilizing histone methyltransferases, necessitates that the primary screen and subsequent hit triage be carefully considered.In this chapter, using EZH2 as a representative example, we describe a screening and hit triage campaign that identified validated chemical matter allowing initiation of medicinal chemistry studies. Moreover, we discuss a cell-based assay to support lead identification and optimization. The approach described here entailing a mixture of biochemical, biophysical and cell-based assays should be applicable to identifying validated starting points for other histone methyltransferases.

Identification and characterization of second-generation EZH2 inhibitors with extended residence times and improved biological activity.

The histone methyltransferase EZH2 has been the target of numerous small-molecule inhibitor discovery efforts over the last 10+ years. Emerging clinical data have provided early evidence for single agent activity with acceptable safety profiles for first-generation inhibitors. We have developed kinetic methodologies for studying EZH2-inhibitor-binding kinetics that have allowed us to identify a unique structural modification that results in significant increases in the drug-target residence times of all EZH2 inhibitor scaffolds we have studied. The unexpected residence time enhancement bestowed by this modification has enabled us to create a series of second-generation EZH2 inhibitors with sub-pM binding affinities. We provide both biophysical evidence validating this sub-pM potency and biological evidence demonstrating the utility and relevance of such high-affinity interactions with EZH2.

Design, Synthesis, and Pharmacological Evaluation of Second Generation EZH2 Inhibitors with Long Residence Time.

Histone methyltransferase EZH2, which is the catalytic subunit of the PRC2 complex, catalyzes the methylation of histone H3K27-a transcriptionally repressive post-translational modification (PTM). EZH2 is commonly mutated in hematologic malignancies and frequently overexpressed in solid tumors, where its expression level often correlates with poor prognosis. First generation EZH2 inhibitors are beginning to show clinical benefit, and we believe that a second generation EZH2 inhibitor could further build upon this foundation to fully realize the therapeutic potential of EZH2 inhibition. During our medicinal chemistry campaign, we identified 4-thiomethyl pyridone as a key modification that led to significantly increased potency and prolonged residence time. Leveraging this finding, we optimized a series of EZH2 inhibitors, with enhanced antitumor activity and improved physiochemical properties, which have the potential to expand the clinical use of EZH2 inhibition.

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.

Identification of (R)-N-((4-Methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-2-methyl-1-(1-(1-(2,2,2-trifluoroethyl)piperidin-4-yl)ethyl)-1H-indole-3-carboxamide (CPI-1205), a Potent and Selective Inhibitor of Histone Methyltransferase EZH2, Suitable for Phase I Clinical Trials for B-Cell Lymphomas.

Polycomb repressive complex 2 (PRC2) has been shown to play a major role in transcriptional silencing in part by installing methylation marks on lysine 27 of histone 3. Dysregulation of PRC2 function correlates with certain malignancies and poor prognosis. EZH2 is the catalytic engine of the PRC2 complex and thus represents a key candidate oncology target for pharmacological intervention. Here we report the optimization of our indole-based EZH2 inhibitor series that led to the identification of CPI-1205, a highly potent (biochemical IC50 = 0.002 µM, cellular EC50 = 0.032 µM) and selective inhibitor of EZH2. This compound demonstrates robust antitumor effects in a Karpas-422 xenograft model when dosed at 160 mg/kg BID and is currently in Phase I clinical trials. Additionally, we disclose the co-crystal structure of our inhibitor series bound to the human PRC2 complex.

Fragment-Based Discovery of a Selective and Cell-Active Benzodiazepinone CBP/EP300 Bromodomain Inhibitor (CPI-637).

CBP and EP300 are highly homologous, bromodomain-containing transcription coactivators involved in numerous cellular pathways relevant to oncology. As part of our effort to explore the potential therapeutic implications of selectively targeting bromodomains, we set out to identify a CBP/EP300 bromodomain inhibitor that was potent both in vitro and in cellular target engagement assays and was selective over the other members of the bromodomain family. Reported here is a series of cell-potent and selective probes of the CBP/EP300 bromodomains, derived from the fragment screening hit 4-methyl-1,3,4,5-tetrahydro-2H-benzo[b][1,4]diazepin-2-one.

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.

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).

EZH2 inhibitor efficacy in non-Hodgkin's lymphoma does not require suppression of H3K27 monomethylation.

The histone lysine methyltransferase (MT) Enhancer of Zeste Homolog 2 (EZH2) is considered an oncogenic driver in a subset of germinal center B-cell-like diffuse large B cell lymphoma (GCB-DLBCL) and follicular lymphoma due to the presence of recurrent, monoallelic mutations in the EZH2 catalytic domain. These genomic data suggest that targeting the EZH2 MT activity is a valid therapeutic strategy for the treatment of lymphoma patients with EZH2 mutations. Here we report the identification of highly potent and selective EZH2 small molecule inhibitors, their validation by a cellular thermal shift assay, application across a large cell panel representing various non-Hodgkin's lymphoma (NHL) subtypes, and their efficacy in EZH2mutant-containing GCB-DLBCL xenograft models. Surprisingly, our EZH2 inhibitors selectively affect the turnover of trimethylated, but not monomethylated histone H3 lysine 27 at pharmacologically relevant doses. Importantly, we find that these inhibitors are broadly efficacious also in NHL models with wild-type EZH2.

A twenty-year experience with endoscopic therapy for symptomatic primary sclerosing cholangitis.

GOALS: The current study presents 1 tertiary endoscopy center's 20-year experience using endoscopic therapy to treat patients with symptomatic primary sclerosing cholangitis (PSC). BACKGROUND: Endoscopic therapy for patients with PSC and dominant strictures has been used for more than 20 years, but there is concern that instrumenting a sclerotic biliary tree induces risks that outweigh anticipated benefits. STUDY: In this retrospective chart review, 117 patients with PSC were identified using ICD-9 codes. Patients had a mean age of 47 years (range: 15 to 86 y). Mean duration of follow-up was 8 years (range: 2 to 20 y). Of the 117 identified patients, 106 underwent endoscopic retrograde cholangiopancreatography on one or more occasions (for a total of 317 endoscopic retrograde cholangiopancreatographies), and a subset of 84 patients received endoscopic therapy for treatment of dominant strictures and/or deteriorating clinical status. Actual survival for endoscopically treated patients was compared with predicted survival using the Mayo Clinic natural history model for PSC. RESULTS: Our chart review revealed 23 recognized complications among the 317 procedures performed (7.3%), and no procedure-related deaths. Observed patient survival at years 3 and 4 was significantly higher than that predicted by the Mayo Clinic natural history model for PSC (P=0.021). CONCLUSIONS: Patients with PSC who have a deteriorating clinical course benefited from endoscopic therapy to provide drainage of bile ducts, removal of stones, and/or temporary relief from obstructions, with acceptable procedure-related complications and higher than expected 3-year and 4-year survival.

Treatment of Barrett's esophagus with early neoplasia: a comparison of endoscopic therapy and esophagectomy.

BACKGROUND: Endoscopic therapies for early neoplasia in Barrett's esophagus may be a viable alternative to esophagectomy. OBJECTIVE: Our purpose was to compare endoscopic therapy and esophagectomy. DESIGN: Retrospective review from a single institution. SETTING: A medium-sized tertiary referral center. PATIENTS AND INTERVENTIONS: All patients with Barrett's esophagus and dysplasia or intramucosal carcinoma treated by photodynamic therapy (PDT), EMR, or argon plasma coagulation (APC) or esophagectomy with curative intent from May 1998 until November 2005. MAIN OUTCOME MEASUREMENTS: Survival, progression to cancer, eradication of dysplasia and Barrett's esophagus, major and minor complications, and costs were compared. RESULTS: Sixty-two patients who underwent endoscopic therapy (2 APC alone, 18 EMR + APC, 20 PDT + APC, and 22 EMR + PDT + APC) and 32 patients who underwent esophagectomy met the inclusion criteria. The 30-day mortality rate included 1 patient in the endotherapy group (2%) and none in the surgical group (P = .49). No deaths from esophageal cancer occurred in either group. Cancer developed in 6% of endotherapy patients and in none in the surgical cohort (P < .05). Major and minor complications occurred in 8% and 31% of endotherapy patients, respectively, and 13% and 63% of surgery patients (P = .50, P < .001). Median cost to date was $40,079 for endotherapy and $66,060 for esophagectomy (P < .001). LIMITATIONS: Retrospective study, relatively short follow-up, small numbers. CONCLUSIONS: Both endotherapy and esophagectomy can effectively treat high-grade dysplasia and intramucosal carcinoma associated with Barrett's esophagus. Endotherapy is associated with a higher risk of tumor progression, although this is uncommon. Esophagectomy incurs higher initial costs and results in more frequent minor complications but is usually curative.

Patient satisfaction scores for endoscopic procedures: impact of a survey-collection method.

BACKGROUND: Many endoscopy units administer patient satisfaction surveys. We hypothesized that the survey collection method would affect satisfaction scores. OBJECTIVE: To compare satisfaction scores obtained by using on-site (OS) surveys versus mail-back (MB) surveys. DESIGN: Quasi-randomization based on alternating weeks. SETTING: Teaching hospital. PATIENTS: Patients undergoing elective routine outpatient colonoscopy or upper endoscopy. INTERVENTIONS: Every patient was given an 11-question survey that asked about the patient's satisfaction with the nurses and the physician, wait times, the bowel-preparation process, patient education, procedural comfort, and sedation. Survey collection methods alternated weekly between an OS versus an MB method. MAIN OUTCOME MEASUREMENTS: Satisfaction scores on a Likert scale ranged from 1 (worst) to 7 (best). RESULTS: A total of 1698 subjects were included. The response rate was higher for the OS group (95%) than the MB group (62%). OS scores were significantly higher than MB scores for 5 of 11 questions, which concerned nurse satisfaction, physician satisfaction, bowel-preparation comfort, postprocedure education, and overall satisfaction (Bonferroni adjusted P < .05 for all). Younger patients gave lower scores than older patients for all questions, whereas women gave significantly lower scores than men for bowel-preparation satisfaction. LIMITATIONS: Lack of true randomization and formal validation of the satisfaction survey. CONCLUSIONS: Survey collection methods may bias not only response rates but also satisfaction scores. OS survey collection methods tend to result in higher satisfaction scores than MB methods. This bias should be noted when comparing scores among studies that used different survey collection methods.

Risk stratification for colon neoplasia: screening strategies using colonoscopy and computerized tomographic colonography.

BACKGROUND & AIMS: We developed a risk index to identify low-risk patients who may be screened for colorectal cancer with computerized tomographic colonography (CTC) instead of colonoscopy. METHODS: Asymptomatic persons aged 50 years or older who had undergone screening colonoscopy were randomized retrospectively to derivation (n = 1512) and validation (n = 1493) subgroups. We developed a risk index (based on age, sex, and family history) from the derivation group. The expected results of 3 screening strategies--universal colonoscopy, universal CTC, and a stratified strategy of colonoscopy for high-risk and CTC for low-risk patients--were then compared. Outcomes for the 3 strategies were extrapolated from the known colonic findings in each patient, using sensitivity/specificity values for CTC from the medical literature. Results were validated in the validation subgroup. RESULTS: In the derivation subgroup, universal colonoscopy detected 94% of advanced neoplasia and universal CTC detected only 70% and resulted in the largest total number of procedures and number of patients undergoing both procedures. The stratified strategy detected 92% of advanced neoplasia, requiring colonoscopy in 68% and CTC in 36% of patients, with only 4% having to undergo both procedures. In the validation subgroup, universal colonoscopy detected 94% and universal CTC detected 71% of advanced neoplasia, whereas the stratified strategy detected 89%, requiring colonoscopy in 64% and CTC in 40%. Unlike universal CTC, the stratified strategy was independent of assumptions for CTC sensitivity, specificity, and threshold for colonoscopy. CONCLUSIONS: The stratified strategy based on our risk index may optimize the yield of colonoscopic resources and reduce the number of patients undergoing colonoscopy.