Discovery of Proline-Based p300/CBP Inhibitors Using DNA-Encoded Library Technology in Combination with High-Throughput Screening.

E1A binding protein (p300) and CREB binding protein (CBP) are two highly homologous and multidomain histone acetyltransferases. These two proteins are involved in many cellular processes by acting as coactivators of a large number of transcription factors. Dysregulation of p300/CBP has been found in a variety of cancers and other diseases, and inhibition has been shown to decrease Myc expression. Herein, we report the identification of a series of highly potent, proline-based small-molecule p300/CBP histone acetyltransferase (HAT) inhibitors using DNA-encoded library technology in combination with high-throughput screening. The strategy of reducing ChromlogD and fluorination of metabolic soft spots was explored to improve the pharmacokinetic properties of potent p300 inhibitors. Fluorination of both cyclobutyl and proline rings of 22 led to not only reduced clearance but also improved cMyc cellular potency.

Improving the developability profile of pyrrolidine progesterone receptor partial agonists.

The previously reported pyrrolidine class of progesterone receptor partial agonists demonstrated excellent potency but suffered from serious liabilities including hERG blockade and high volume of distribution in the rat. The basic pyrrolidine amine was intentionally converted to a sulfonamide, carbamate, or amide to address these liabilities. The evaluation of the degree of partial agonism for these non-basic pyrrolidine derivatives and demonstration of their efficacy in an in vivo model of endometriosis is disclosed herein.

Evaluation of surgically placed gastrojejunostomy feeding tubes in critically ill dogs.

OBJECTIVE: To evaluate complications and outcomes associated with surgical placement of gastrojejunostomy feeding tubes in dogs with naturally occurring disease. DESIGN: Prospective study. ANIMALS: 26 dogs. Multiple preoperative, intraoperative, and postoperative variables were evaluated. Daily postoperative abdominal radiographic examinations were performed to determine the presence of the following mechanical tube complications: kinking, coiling, knotting, and migration. Tube stoma abnormalities (erythema, cellulitis, and discharge) were observed daily and recorded by use of a standardized visual analog grading scale. Additionally, presence of complications was compared with median survival times. RESULTS: The most common indication for gastrojejunostomy tube placement was gastrointestinal disease (n = 11), with confirmed septic peritonitis in 8 of 11 dogs. Other indications for gastrojejunostomy tube placement included extrahepatic biliary surgery (n = 6) and pancreatic disease (9). Mean +/- SD surgical time required for tube placement was 26 +/- 14 minutes. Overall, mechanical tube complication rate was 46% (12/26), including coiling (7), migration (4), and kinking (2). Overall minor tube stoma complication rate was 77% (20/26) and included erythema (16), cellulitis (13), and discharge (17). Dislodgement or self-induced tube trauma resulted in accidental tube removal in 2 of 26 dogs, and inadvertent tube damage necessitated premature removal by the clinician in 1 of 26 dogs. Kaplan-Meier median survival time was 39 days with 13 of 26 dogs still alive. CONCLUSIONS AND CLINICAL RELEVANCE: Gastrojejunostomy tube placement affords flexibility in the postoperative nutritional regimen by allowing for postgastric feeding with simultaneous access to the stomach.

Localization of pancreatic inflammation and necrosis in dogs.

Few studies of the prevalence of histologic lesions of the exocrine pancreas in dogs have been reported, and none of them systematically evaluate the localization of these lesions. The purpose of this study was to describe the anatomic localization of pancreatic inflammation, necrosis, and fibrosis in dogs presented for postmortem examination. Seventy-three pancreata from dogs presented for postmortem examination were evaluated and investigated for the presence of suppurative inflammation (SI), pancreatic necrosis (PN), and lymphocytic inflammation (LI). Sections that showed evidence of SI, PN, or LI also were evaluated for pancreatic fibrosis (F). The tendency for a preferred localization for SI, PN, and LI was evaluated by chi-square analysis. A total of 47 pancreata with histologic evidence of pancreatitis (SI, PN, or LI; F alone was not considered evidence of pancreatitis) were identified. Twenty-four (51.1%) had SI, 23 (48.9%) had PN, and 34 (72.3%) had LI. Of the 47 dogs with SI, PN, or LI, 28 (59.6%) had F. The distribution of SI, PN, and LI between the right and the left limb of the pancreas and among the 5 anatomic regions was random, based on a goodness-of-fit test. We conclude that pancreatic inflammation tends to occur in discrete areas within the pancreas rather than diffusely throughout the whole organ. These findings suggest that a single biopsy is insufficient to exclude subclinical pancreatitis and that there is no preferred site for pancreatic biopsy collection unless gross lesions are apparent.

The scl +18/19 stem cell enhancer is not required for hematopoiesis: identification of a 5' bifunctional hematopoietic-endothelial enhancer bound by Fli-1 and Elf-1.

Analysis of cis-regulatory elements is central to understanding the genomic program for development. The scl/tal-1 transcription factor is essential for lineage commitment to blood cell formation and previous studies identified an scl enhancer (the +18/19 element) which was sufficient to target the vast majority of hematopoietic stem cells, together with hematopoietic progenitors and endothelium. Moreover, expression of scl under control of the +18/19 enhancer rescued blood progenitor formation in scl(-/-) embryos. However, here we demonstrate by using a knockout approach that, within the endogenous scl locus, the +18/19 enhancer is not necessary for the initiation of scl transcription or for the formation of hematopoietic cells. These results led to the identification of a bifunctional 5' enhancer (-3.8 element), which targets expression to hematopoietic progenitors and endothelium, contains conserved critical Ets sites, and is bound by Ets family transcription factors, including Fli-1 and Elf-1. These data demonstrate that two geographically distinct but functionally related enhancers regulate scl transcription in hematopoietic progenitors and endothelial cells and suggest that enhancers with dual hematopoietic-endothelial activity may represent a general strategy for regulating blood and endothelial development.

Comparison of peritoneal fluid and peripheral blood pH, bicarbonate, glucose, and lactate concentration as a diagnostic tool for septic peritonitis in dogs and cats.

OBJECTIVE: To establish a reliable diagnostic tool for septic peritonitis in dogs and cats using pH, bicarbonate, lactate, and glucose concentrations in peritoneal fluid and venous blood. STUDY DESIGN: Prospective clinical study. ANIMALS: Eighteen dogs and 12 cats with peritoneal effusion. METHODS: pH, bicarbonate, electrolyte, lactate, and glucose concentrations were measured on 1- to 2-mL samples of venous blood and peritoneal fluid collected at admission. The concentration difference between blood and peritoneal fluid for pH, bicarbonate, glucose, and lactate concentrations were calculated by subtracting the peritoneal fluid concentration from the blood concentration. Peritoneal fluid was submitted for cytologic examination and bacterial culture. Peritonitis was classified as septic or nonseptic based on cytology and bacterial culture results. RESULTS: In dogs, with septic effusion, peritoneal fluid glucose concentration was always lower than the blood glucose concentration. A blood-to-fluid glucose (BFG) difference > 20 mg/dL was 100% sensitive and 100% specific for the diagnosis of septic peritoneal effusion in dogs. In 7 dogs in which it was evaluated, a blood-to-fluid lactate (BFL) difference < -2.0 mmol/L was also 100% sensitive and specific for a diagnosis of septic peritoneal effusion. In cats, the BFG difference was 86% sensitive and 100% specific for a diagnosis of septic peritonitis. In dogs and cats, the BFG difference was more accurate for a diagnosis of septic peritonitis than peritoneal fluid glucose concentration alone. CONCLUSIONS: A concentration difference > 20 mg/dL between blood and peritoneal fluid glucose concentration provides a rapid and reliable means to differentiate a septic peritoneal effusion from a nonseptic peritoneal effusion in dogs and cats. CLINICAL RELEVANCE: The difference between blood and peritoneal fluid glucose concentrations should be used as a more reliable diagnostic indicator of septic peritoneal effusion than peritoneal fluid glucose concentration alone.

Transcriptional regulation of the stem cell leukemia gene (SCL)--comparative analysis of five vertebrate SCL loci.

The stem cell leukemia (SCL) gene encodes a bHLH transcription factor with a pivotal role in hematopoiesis and vasculogenesis and a pattern of expression that is highly conserved between mammals and zebrafish. Here we report the isolation and characterization of the zebrafish SCL locus together with the identification of three neighboring genes, IER5, MAP17, and MUPP1. This region spans 68 kb and comprises the longest zebrafish genomic sequence currently available for comparison with mammalian, chicken, and pufferfish sequences. Our data show conserved synteny between zebrafish and mammalian SCL and MAP17 loci, thus suggesting the likely genomic domain necessary for the conserved pattern of SCL expression. Long-range comparative sequence analysis/phylogenetic footprinting was used to identify noncoding conserved sequences representing candidate transcriptional regulatory elements. The SCL promoter/enhancer, exon 1, and the poly(A) region were highly conserved, but no homology to other known mouse SCL enhancers was detected in the zebrafish sequence. A combined homology/structure analysis of the poly(A) region predicted consistent structural features, suggesting a conserved functional role in mRNA regulation. Analysis of the SCL promoter/enhancer revealed five motifs, which were conserved from zebrafish to mammals, and each of which is essential for the appropriate pattern or level of SCL transcription.