Identification of a ß-arrestin-biased negative allosteric modulator for the ß2-adrenergic receptor.

Catecholamine-stimulated ß2-adrenergic receptor (ß2AR) signaling via the canonical Gs-adenylyl cyclase-cAMP-PKA pathway regulates numerous physiological functions, including the therapeutic effects of exogenous ß-agonists in the treatment of airway disease. ß2AR signaling is tightly regulated by GRKs and ß-arrestins, which together promote ß2AR desensitization and internalization as well as downstream signaling, often antithetical to the canonical pathway. Thus, the ability to bias ß2AR signaling toward the Gs pathway while avoiding ß-arrestin-mediated effects may provide a strategy to improve the functional consequences of ß2AR activation. Since attempts to develop Gs-biased agonists and allosteric modulators for the ß2AR have been largely unsuccessful, here we screened small molecule libraries for allosteric modulators that selectively inhibit ß-arrestin recruitment to the receptor. This screen identified several compounds that met this profile, and, of these, a difluorophenyl quinazoline (DFPQ) derivative was found to be a selective negative allosteric modulator of ß-arrestin recruitment to the ß2AR while having no effect on ß2AR coupling to Gs. DFPQ effectively inhibits agonist-promoted phosphorylation and internalization of the ß2AR and protects against the functional desensitization of ß-agonist mediated regulation in cell and tissue models. The effects of DFPQ were also specific to the ß2AR with minimal effects on the ß1AR. Modeling, mutagenesis, and medicinal chemistry studies support DFPQ derivatives binding to an intracellular membrane-facing region of the ß2AR, including residues within transmembrane domains 3 and 4 and intracellular loop 2. DFPQ thus represents a class of biased allosteric modulators that targets an allosteric site of the ß2AR.

Preclinical study of the long-range safety and anti-inflammatory effects of high-dose oral meglumine.

Meglumine is a methylamino derivative of sorbitol that is an approved drug excipient. Recent preclinical studies suggest that administration of high-dose oral meglumine can exert beneficial medicinal effects to treat diabetes, obesity, and fatty liver disease (NAFLD/nonalcoholic steatohepatitis [NASH]). Here we address gaps in knowledge about the pharmacology and toxicology of this substance administered at high concentrations to explore its medicinal potential. We observed that high-dose meglumine limited secretion of proinflammatory cytokines and cell adhesion molecules from activated human THP-1 or murine RAW264.7 monocytes. Preclinical pharmacokinetic analysis in Swiss mice confirmed that meglumine was orally available. Informed by this data, oral doses of 18 to 75 mM meglumine were administered ad libitum in the drinking water of Sprague-Dawley rats and two cohorts of C57BL/6 mice housed in different vivariums. In a 32-week study, urinary isoprostane levels trended lower in subjects consistent with the possibility of anti-inflammatory effects. In full lifespan studies, there was no detrimental effect on longevity. Heart function evaluated in C57BL/6 mice using an established noninvasive cardiac imaging system showed no detrimental effects on ejection fraction, fractional shortening, left ventricle function or volume, and cardiac output in mice up to 15-month old, with a potential positive trend in heart function noted in elderly mice consistent with earlier reported benefits on muscle stamina. Finally, in a transgenic model of inflammation-associated skin carcinogenesis, the incidence, number, and growth of skin tumors trended lower in subjects receiving meglumine. Overall, the evidence obtained illustrating the long-range safety of high-dose oral meglumine support the rationale for its evaluation as a low-cost modality to limit diabetes, hypertriglyceridemia, and NAFLD/NASH.

Development and Validation of a Universal High-Throughput UDP-Glycosyltransferase Assay with a Time-Resolved FRET Signal.

Glycosyltransferase enzymes play diverse metabolic and regulatory roles by catalyzing the transfer of sugar molecules to protein, lipid, and carbohydrate acceptors, and they are increasingly of interest as therapeutic targets in a number of diseases, including metabolic disorders, cancer, and infectious diseases. The glycosyltransferases are a challenging target class from an assay development perspective because of the diversity of both donor and acceptor substrates and the lack of suitable glycan detection methods. However, many glycosyltransferases use uridine 5'-diphosphate (UDP) sugars as donor substrates, and detection of the free UDP reaction product provides a generic approach for measuring the activity of those enzymes. To exploit this approach for a broadly applicable high-throughput screening (HTS) assay for discovery of glycosyltransferase inhibitors, we developed a Transcreener(®) assay for immunodetection of UDP with a time-resolved Förster resonance energy transfer (TR-FRET) signal. We optimized the assay for detection of glycosyltransferase activity with nucleotide diphosphate (NDP) sugars at concentrations from 10 µM to 1 mM, achieving Z' values of 0.6 or higher. The assay was validated by orthogonal pooled screening with 8,000 compounds using polypeptide N-acetylgalactosaminyltransferase T3 as the target, and the hits were confirmed using an orthogonal readout. The reagents and signal were both stable for more than 8 h at room temperature, insuring robust performance in automated HTS environments. The TR-FRET-based UDP detection assay provides a broadly applicable approach for screening glycosyltransferases that use a UDP-sugar donor.

Open innovation in early drug discovery: roadmaps and roadblocks.

Open innovation in pharmaceutical R&D evolved from a triple helix of convergent paradigm shifts in academic, industrial and government research sectors. The birth of the biotechnology sector catalyzed shifts in location dynamics that led to the first wave of open innovation in pharmaceutical R&D between big pharma and startup companies. The National Institutes of Health (NIH) Roadmap was a crucial inflection point that set the stage for a new wave of open innovation models between pharmaceutical companies and universities that have the potential to transform the pharmaceutical R&D landscape. We highlight the attributes of leading protected open innovation models that foster the sharing of proprietary small molecule collections by lowering the risk of premature escape of intellectual property, particularly structure-activity data.

A High-Throughput Assay for Rho Guanine Nucleotide Exchange Factors Based on the Transcreener GDP Assay.

Ras homologous (Rho) family GTPases act as molecular switches controlling cell growth, movement, and gene expression by cycling between inactive guanosine diphosphate (GDP)- and active guanosine triphosphate (GTP)-bound conformations. Guanine nucleotide exchange factors (GEFs) positively regulate Rho GTPases by accelerating GDP dissociation to allow formation of the active, GTP-bound complex. Rho proteins are directly involved in cancer pathways, especially cell migration and invasion, and inhibiting GEFs holds potential as a therapeutic strategy to diminish Rho-dependent oncogenesis. Methods for measuring GEF activity suitable for high-throughput screening (HTS) are limited. We developed a simple, generic biochemical assay method for measuring GEF activity based on the fact that GDP dissociation is generally the rate-limiting step in the Rho GTPase catalytic cycle, and thus addition of a GEF causes an increase in steady-state GTPase activity. We used the Transcreener GDP Assay, which relies on selective immunodetection of GDP, to measure the GEF-dependent stimulation of steady-state GTP hydrolysis by small GTPases using Dbs (Dbl's big sister) as a GEF for Cdc42, RhoA, and RhoB. The assay is well suited for HTS, with a homogenous format and far red fluorescence polarization (FP) readout, and it should be broadly applicable to diverse Rho GEF/GTPase pairs.

Identification and characterization of small molecule human papillomavirus E6 inhibitors.

Cervical cancer is the sixth most common cancer in women worldwide and the leading cause of women's death in developing countries. Nearly all cervical cancers are associated with infection of the human papillomavirus (HPV). This sexually transmitted pathogen disrupts the cell cycle via two oncoproteins: E6 and E7. Cells respond to E7-mediated degradation of pRB by upregulating the p53 tumor suppressor pathway. However, E6 thwarts this response by binding to the cellular E6-Associating Protein (E6AP) and targeting p53 for degradation. These two virus-facilitated processes pave the way for cellular transformation. Prophylactic HPV vaccines are available, but individuals already infected with HPV lack drug-based therapeutic options. To fill this void, we sought to identify small molecule inhibitors of the E6-E6AP interaction. We designed an ELISA-based high throughput assay to rapidly screen compound libraries, and hits were confirmed in several orthogonal biochemical and cell-based assays. Over 88,000 compounds were screened; 30 had in vitro potencies in the mid-nanomolar to mid-micromolar range and were classified as validated hits. Seven of these hits inhibited p53 degradation in cell lines with HPV-integrated genomes. Two compounds of similar scaffold successfully blocked p53 degradation and inhibited cell proliferation in cells stably transfected with E6. Together, these studies suggest that small molecules can successfully block E6-dependent p53 degradation and restore p53 activity. The compounds identified here constitute attractive starting points for further medicinal chemistry efforts and development into beneficial therapeutics.

Meglumine exerts protective effects against features of metabolic syndrome and type II diabetes.

Metabolic syndrome, diabetes and diabetes complications pose a growing medical challenge worldwide, accentuating the need of safe and effective strategies for their clinical management. Here we present preclinical evidence that the sorbitol derivative meglumine (N-methyl-D-glucamine) can safely protect against several features of metabolic syndrome and diabetes, as well as elicit enhancement in muscle stamina. Meglumine is a compound routinely used as an approved excipient to improve drug absorption that has not been ascribed any direct biological effects in vivo. Normal mice (SV129) administered 18 mM meglumine orally for six weeks did not display any gastrointestinal or other observable adverse effects, but had a marked effect on enhancing muscle stamina and at longer times in limiting weight gain. In the established KK.Cg-Ay/J model of non-insulin dependent diabetes, oral administration of meglumine significantly improved glycemic control and significantly lowered levels of plasma and liver triglycerides. Compared to untreated control animals, meglumine reduced apparent diabetic nephropathy. Sorbitol can improve blood glucose uptake by liver and muscle in a manner associated with upregulation of the AMPK-related enzyme SNARK, but with undesirable gastrointestinal side effects not seen with meglumine. In murine myoblasts, we found that meglumine increased steady-state SNARK levels in a dose-dependent manner more potently than sorbitol. Taken together, these findings provide support for the clinical evaluation of meglumine as a low-cost, safe supplement offering the potential to improve muscle function, limit metabolic syndrome and reduce diabetic complications.

New informatics and automated infrastructure to accelerate new leads discovery by high throughput screening (HTS).

The Lankenau Institute for Medical Research Chemical Genomics Center, Inc. has developed a new (patents issued and pending) Nanotube Automated Repository System (NARS) for dynamic storage of millions of 'single-shot' samples stored in a new monolithic microtiter-storage tube plate of our own design we call 'nanotubes.' We have integrated the NARS with customized software to efficiently access up to 10,000,000 samples stored continuously frozen (-20°C) in a dehumidified enclosure and sealed in a new microtiter NARS plate that is SBS compliant. Additional software was developed to analyze HTS data from orthogonally pooled compound libraries. Following 'de-convolution' of pooled HTS data, the software designates confirmatory retest samples to be 'cherry-picked' using the NARS. The application of a new, fully-integrated infrastructure for new leads discovery is described in detail. Other applications for our technologies and new infrastructure are discussed.

Identification and characterization of small molecule antagonists of pRb inactivation by viral oncoproteins.

The retinoblastoma protein pRb is essential for regulating many cellular activities through its binding and inhibition of E2F transcription activators, and pRb inactivation leads to many cancers. pRb activity can be perturbed by viral oncoproteins including human papillomavirus (HPV) that share an LxCxE motif. Because there are no treatments for existing HPV infection leading to nearly all cervical cancers and other cancers to a lesser extent, we screened for compounds that inhibit the ability of HPV-E7 to disrupt pRb/E2F complexes. This lead to the identification of thiadiazolidinedione compounds that bind to pRb with mid-high nanomolar dissociation constants, are competitive with the binding of viral oncoproteins containing an LxCxE motif, and are selectively cytotoxic in HPV-positive cells alone and in mice. These inhibitors provide a promising scaffold for the development of therapies to treat HPV-mediated pathologies.

Development of a high-throughput screen for inhibitors of Epstein-Barr virus EBNA1.

Latent infection with Epstein-Barr virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small-molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work, the authors describe the development of a biochemical high-throughput screening (HTS) method using a homogeneous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. The authors demonstrate that EBNA1 binding to a fluorescent-labeled DNA probe provides a robust assay with a Z factor consistently greater than 0.6. A pilot screen of a small-molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1 but not Zta. All 3 compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof of concept that small-molecule inhibitors of EBNA1 can be identified by biochemical HTS of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.