Phenothiazine Neuroleptics Signal to the Human Insulin Promoter as Revealed by a Novel High-Throughput Screen

Abstract

A number of diabetogenic stimuli interact to influence insulin promoter activity, making it an attractive target for both mechanistic studies and therapeutic interventions. High-throughput screening (HTS) for insulin promoter modulators has the potential to reveal novel inputs into the control of that central element of the pancreatic β-cell. A cell line from human islets in which the expression of insulin and other β-cell-restricted genes are modulated by an inducible form of the bHLH transcription factor E47 was developed. This cell line, T6PNE, was adapted for HTS by transduction with a vector expressing green fluorescent protein under the control of the human insulin promoter. The resulting cell line was screened against a library of known drugs for those that increase insulin promoter activity. Members of the phenothiazine class of neuroleptics increased insulin gene expression upon short-term exposure. Chronic treatment, however, resulted in suppression of insulin promoter activity, consistent with the effect of phenothiazines observed clinically to induce diabetes in chronically treated patients. In addition to providing insights into previously unrecognized targets and mechanisms of action of phenothiazines, the novel cell line described here provides a broadly applicable platform for mining new molecular drug targets and central regulators of β-cell differentiated function.

Id3 upregulates BrdU incorporation associated with a DNA damage response, not replication, in human pancreatic β-cells

“…40 In this study Id3 efficiently suppressed p57 Kip2 . Together the data establish that the E47/Id axis controls p57…”

“…39 Due to the fact that duct cells act as β-cell progenitors during development and possibly during regeneration, 5 we hypothesized that β-cells might respond similarly to Id3. Additional support for a role for Id3 in β-cell replication came from the fact that E47 upregulates p57 Kip2 expression and induces growth arrest in a cell line derived from human fetal islets, 40 and that E47 and Ids control p57 Kip2 expression in other tissues. 41 Thus, we hypothesized that Id3 inhibition of E47 activity would downregulate p57…”

“…Recently we reported that E47 activated transcription of the cyclin dependent kinase inhibitor (cdki) p57 Kip2 , inducing cell cycle arrest in a human islet cell line. 40 Here, we investigated whether repressing E47 activity would inhibit p57…”

A Comparison of Ypet and Firefly Luciferase as Reporter Proteins for High-Throughput Screening

“…1) Reporter assay is the readout of choice for HTS in developing modulators of a particular transcription factor and transcription regulators of a gene of interest. 2,3) Firefly luciferase is the preferred reporter protein by most researchers due to its higher sensitivity, but cell lysis and chemiluminescent substrate addition are necessary for luciferase assay. Since light produced by luciferase decays with time, the assay should be performed wellby-well to minimize the decaying effect.…”

Modern Psychopharmacology and Psychiatric Treatment

“…Moreover, many steps in the chemical synthesis of variants of a desired compound (to seek more ideal candidate molecules or potential follow-on agents for later development), as well as molecular screening procedures that seem to be highly ef fi cient, are often automated and robotically controlled. These modern procedures involve high-capacity or combinatorial chemistry and high-throughput screening methods that are technically impressive but expensive and liable to produce large numbers of uninteresting candidate molecules [ 26,27,48,49,84 ] . In addition, pharmacokinetic modeling can identify oral bioavailability, elimination half-life and clearance rates as well as likely enzymatic routes of metabolic conversion, based usually on animal models [ 59 ] .…”