Kelvin Lam

A Small-Molecule Inhibitor of Tgf-β Signaling Replaces Sox2 in Reprogramming by Inducing Nanog

The combined activity of three transcription factors can reprogram adult cells into induced pluripotent stem cells (iPSCs). However, the transgenic methods used for delivering reprogramming factors have raised concerns regarding the future utility of the resulting stem cells. These uncertainties could be overcome if each transgenic factor were replaced with a small molecule that either directly activated its expression from the somatic genome or in some way compensated for its activity. To this end, we have used high-content chemical screening to identify small molecules that can replace Sox2 in reprogramming. We show that one of these molecules functions in reprogramming by inhibiting Tgf-beta signaling in a stable and trapped intermediate cell type that forms during the process. We find that this inhibition promotes the completion of reprogramming through induction of the transcription factor Nanog.

Zebrafish Behavioral Profiling Links Drugs to Biological Targets and Rest/Wake Regulation

Behavioral Profiling The complexity of the brain makes it difficult to predict how a drug will affect behavior without direct testing in live animals. Rihel et al. (p. 348) developed a high-throughput assay to assess the effects of thousands of drugs on sleep/wake behaviors of zebrafish larvae. The data set reveals a broad conservation of zebrafish and mammalian sleep/wake pharmacology and identifies pathways that regulate sleep. Moreover, the biological targets of poorly characterized small molecules can be predicted by matching their behavioral profiles to those of well-known drugs. Thus, behavioral profiling in zebrafish offers a cost-effective way to characterize neuroactive drugs and to predict biological targets of novel compounds. The effects of most neuroactive drugs are conserved and can be detected by behavioral screening. A major obstacle for the discovery of psychoactive drugs is the inability to predict how small molecules will alter complex behaviors. We report the development and application of a high-throughput, quantitative screen for drugs that alter the behavior of larval zebrafish. We found that the multidimensional nature of observed phenotypes enabled the hierarchical clustering of molecules according to shared behaviors. Behavioral profiling revealed conserved functions of psychotropic molecules and predicted the mechanisms of action of poorly characterized compounds. In addition, behavioral profiling implicated new factors such as ether-a-go-go–related gene (ERG) potassium channels and immunomodulators in the control of rest and locomotor activity. These results demonstrate the power of high-throughput behavioral profiling in zebrafish to discover and characterize psychotropic drugs and to dissect the pharmacology of complex behaviors.

A small molecule that directs differentiation of human ESCs into the pancreatic lineage

Stepwise differentiation from embryonic stem cells (ESCs) to functional insulin-secreting beta cells will identify key steps in beta-cell development and may yet prove useful for transplantation therapy for diabetics. An essential step in this schema is the generation of pancreatic progenitors--cells that express Pdx1 and produce all the cell types of the pancreas. High-content chemical screening identified a small molecule, (-)-indolactam V, that induces differentiation of a substantial number of Pdx1-expressing cells from human ESCs. The Pdx1-expressing cells express other pancreatic markers and contribute to endocrine, exocrine and duct cells, in vitro and in vivo. Further analyses showed that (-)-indolactam V works specifically at one stage of pancreatic development, inducing pancreatic progenitors from definitive endoderm. This study describes a chemical screening platform to investigate human ESC differentiation and demonstrates the generation of a cell population that is a key milepost on the path to making beta cells.

Adenosine kinase inhibition selectively promotes rodent and porcine islet β-cell replication

Diabetes is a pathological condition characterized by relative insulin deficiency, persistent hyperglycemia, and, consequently, diffuse micro- and macrovascular disease. One therapeutic strategy is to amplify insulin-secretion capacity by increasing the number of the insulin-producing β cells without triggering a generalized proliferative response. Here, we present the development of a small-molecule screening platform for the identification of molecules that increase β-cell replication. Using this platform, we identify a class of compounds [adenosine kinase inhibitors (ADK-Is)] that promote replication of primary β cells in three species (mouse, rat, and pig). Furthermore, the replication effect of ADK-Is is cell type-selective: treatment of islet cell cultures with ADK-Is increases replication of β cells but not that of α cells, PP cells, or fibroblasts. Short-term in vivo treatment with an ADK-I also increases β-cell replication but not exocrine cell or hepatocyte replication. Therefore, we propose ADK inhibition as a strategy for the treatment of diabetes.

A screen for regulators of survival of motor neuron protein levels

The motor neuron disease spinal muscular atrophy (SMA) results from mutations that lead to low levels of the ubiquitously expressed protein survival of motor neuron (SMN). An ever-increasing collection of data suggests that therapeutics that elevate SMN may be effective in treating SMA. We executed an image-based screen of annotated chemical libraries and discovered several classes of compounds that were able to increase cellular SMN. Among the most important was the RTK-PI3K-AKT-GSK-3 signaling cascade. Chemical inhibitors of glycogen synthase kinase 3 (GSK-3) and short hairpin RNAs (shRNAs) directed against this target elevated SMN levels primarily by stabilizing the protein. It was particularly notable that GSK-3 chemical inhibitors were also effective in motor neurons, not only in elevating SMN levels, but also in blocking the death that was produced when SMN was acutely reduced by an SMN-specific shRNA. Thus, we have established a screen capable of detecting drug-like compounds that correct the main phenotypic change underlying SMA.

Glucocorticoid Compounds Modify Smoothened Localization and Hedgehog Pathway Activity

The Hedgehog signaling pathway is linked to a variety of diseases, notably a range of cancers. The first generation of drug screens identified Smoothened (Smo), a membrane protein essential for signaling, as an attractive drug target. Smo localizes to the primary cilium upon pathway activation, and this transition is critical for the response to Hedgehog ligands. In a high content screen directly monitoring Smo distribution in Hedgehog-responsive cells, we identified different glucocorticoids as specific modulators of Smo ciliary accumulation. One class promoted Smo accumulation, conferring cellular hypersensitivity to Hedgehog stimulation. In contrast, a second class inhibited Smo ciliary localization and signaling activity by both wild-type Smo, and mutant forms of Smo, SmoM2, and SmoD473H, that are refractory to previously identified Smo antagonists. These findings point to the potential for developing glucocorticoid-based pharmacological modulation of Smo signaling to treat mutated drug-resistant forms of Smo, an emerging problem in long-term cancer therapy. They also raise a concern about potential crosstalk of glucocorticoid drugs in the Hedgehog pathway, if therapeutic administration exceeds levels associated with on-target transcriptional mechanisms of glucocorticoid action.

Structure and expression of the Xenopus retinoblastoma gene

We have cloned a Xenopus homology (XRb1) of the human retinoblastoma susceptibility gene. DNA sequence analysis shows that the XRb1 gene product is highly conserved in many regions. The leucine repeat motif and many of the potential cdc2 phosphorylation sites, as well as potential sites for other kinases, are retained. The region of the protein homologous to the SV40 T antigen binding site and the basic region directly C-terminal to the E1A binding site are all conserved. XRb1 gene expression at the RNA level was studied by Northern blot analysis. Transcripts of 4.2 and 10-kb are present as maternal RNA stores in the oocyte. While the 4.2-kb product is stable until at least the mid-blastula stage, the 10-kb transcript is selectively degraded. Between stages 11 and 13 the 10-kb transcript reappears and also a minor product of approximately 11 kb becomes apparent. Both the 4.2- and the 10-kb transcripts remain present until later stages of development and are also present in all adult tissues examined, although at differing levels. Antibodies raised against human p105Rb which recognize the protein product of the XRb1 gene, pXRb1, detect the Xenopus 99-kDa protein prior to the mid-blastula stage, but at lower levels than at later stages in development.

hsp70 binds specifically to a peptide derived from the highly conserved domain (I) region of p53.

Products of a number of mutant p53 genes bind with high affinity to members of the hsp70 family of chaperonin proteins, whereas wild type p53 lacks this type of association. Examination of the sequences of p53 genes from five different species enabled us to predict domains on p53 which may be involved in the association with hsp70 family members. A synthetic polypeptide (Pro-17-Gly) corresponding to the candidate hsp70 binding domain bound to in vitro translated hsp70 as determined by affinity chromatography and nondenaturing gel mobility shift assays. In addition, the Pro-17-Gly peptide competitively inhibited association between hsp70 and p53, an activity which was determined by immunoprecipitation with anti-p53 monoclonal antibody PAb240. The data indicate that p53 contains a hsp70 binding domain, which is located in a highly conserved region at the amino terminus of the protein, and may participate in the cellular function of wild-type p53 or in the transforming capacity of p53 mutants.

Selective Identification of Hedgehog Pathway Antagonists by Direct Analysis of Smoothened Ciliary Translocation

Hedgehog (Hh) signaling promotes tumorigenesis. The accumulation of the membrane protein Smoothened (Smo) within the primary cilium (PC) is a key event in Hh signal transduction, and many pharmacological inhibitors identified to date target Smos actions. Smo ciliary translocation is inhibited by some pathway antagonists, while others promote ciliary accumulation, an outcome that can lead to a hypersensitive state on renewal of Hh signaling. To identify novel inhibitory compounds acting on the critical mechanistic transition of Smo accumulation, we established a high content screen to directly analyze Smo ciliary translocation. Screening thousands of compounds from annotated libraries of approved drugs and other agents, we identified several new classes of compounds that block Sonic hedgehog-driven Smo localization within the PC. Selective analysis was conducted on two classes of Smo antagonists. One of these, DY131, appears to inhibit Smo signaling through a common binding site shared by previously reported Smo agonists and antagonists. Antagonism by this class of compound is competed by high doses of Smo-binding agonists such as SAG and impaired by a mutation that generates a ligand-independent, oncogenic form of Smo (SmoM2). In contrast, a second antagonist of Smo accumulation within the PC, SMANT, was less sensitive to SAG-mediated competition and inhibited SmoM2 at concentrations similar to those that inhibit wild-type Smo. Our observations identify important differences among Hh antagonists and the potential for development of novel therapeutic approaches against mutant forms of Smo that are resistant to current therapeutic strategies.