qPCR validation of RNA-Seq data.

<p>(A) A subset of genes that were differentially regulated by OX₁ signaling in the RNA-Seq experiment were chosen for qPCR verification. GT1-7-OX₁ cells were treated with 100nM OxA for 3 hours. The fold-change from the RNA-Seq data is included as a reference to demonstrate similarity of effects. (B) An OX₁-specific antagonist inhibits OxA-dependent changes in transcription. GT1-7-OX₁ cells were treated with 20μM SB-334867 for 10 minutes prior to addition of 100nM OxA for 3 hours. RNA was purified from each sample and analyzed via qPCR. Mean fold change is presented (n = 1, reads done in triplicate) with error bars representing SEM.</p

Inhibition of Sgk1 depresses the OxA-dependent induction of a small set of transcripts.

<p>GT1-7-OX₁ cells were treated with an Sgk1 inhibitor, GSK-650394, prior to addition of OxA. Total RNA was purified from lysates and used for qPCR analysis. The genes displayed had their level of orexin-induced transcription inhibited by GSK-650394. Bars represent averages (n = 1, reads done in triplicate) while error bars represent SEM.</p

Solid-Phase Synthesis of β‑Hydroxy Ketones Via DNA-Compatible Organocatalytic Aldol Reactions

One-bead one-compound (OBOC) libraries constructed by solid-phase split-and-pool synthesis are a valuable source of protein ligands. Most OBOC libraries are composed of oligoamides, particularly peptides, peptoids, and peptoid-inspired molecules. Further diversification of the chemical space covered by OBOC libraries is desirable. Toward this end, we report here that the proline-catalyzed asymmetric aldol reaction, developed by List and Barbas for solution-phase synthesis, also works well for coupling immobilized aldehydes and soluble ketones. These reaction conditions do not compromise the amplification of DNA by the polymerase chain reaction. Thus, this chemistry should be useful for the construction of novel DNA-encoded OBOC libraries by solid-phase synthesis

Generation of a GT1-7-based cell line stably expressing OX₁.

<p>A lentiviral transduction system was used to generate GT1-7 cells that stably express OX₁. (A) Presence of OX₁ mRNA in the transduced cells was verified via qPCR. Data were analyzed by the 2^-ΔΔC_T method, using mouse GAPDH as the reference, and are expressed as relative quantity (RQ), normalized to the parental cell line. (B) Parental, mock-transduced, and OX₁-transduced GT1-7 cells were tested for the presence of functional OX₁ via the IP-One HTRF Assay. (C) An orexin receptor antagonist, SB-334867, blocked orexin signaling in GT1-7-OX₁ cells in a concentration-dependent manner. Data points are mean (n = 3), error bars represent SEM.</p

Partial list of transcription factors regulated by OxA in GT1-7-OX₁ cells.

<p>Partial list of transcription factors regulated by OxA in GT1-7-OX₁ cells.</p

Genes regulated by both sleep deprivation and OX₁ signaling.

<p>Genes regulated by both sleep deprivation and OX₁ signaling.</p

Characterization of orexin receptor expression via qPCR.

<p>Characterization of orexin receptor expression via qPCR.</p

Characterization of cell lines previously reported to express orexin receptors.

<p>Each cell line was assayed for the presence of functional orexin receptors via the IP-One HTRF Assay. Cells were incubated with orexin A at various concentrations for 45 min. A CHO-based cell line stably expressing OX₁ (CHO-OX₁) was used as a positive control. The data are presented as a percentage of the baseline HTRF ratio (A₆₆₅/A₆₂₀ x 10000). Data points are mean (n = 3) and error bars represent standard error of the mean (SEM).</p

Heat maps indicating the genes most highly regulated by OX₁ activation.

<p>(A) Vehicle-treated vs. 3-hour treatment with OxA. (B) Vehicle-treated vs. 8-hour treatment with OxA. The values (colors) shown are the regularized log transformations of the original count data.</p

Direct Comparison of Linear and Macrocyclic Compound Libraries as a Source of Protein Ligands

There has been much discussion of the potential desirability of macrocyclic molecules for the development of tool compounds and drug leads. But there is little experimental data comparing otherwise equivalent macrocyclic and linear compound libraries as a source of protein ligands. In this Letter, we probe this point in the context of peptoid libraries. Bead-displayed libraries of macrocyclic and linear peptoids containing four variable positions and 0–2 fixed residues, to vary the ring size, were screened against streptavidin and the affinity of every hit for the target was measured. The data show that macrocyclization is advantageous, but only when the ring contains 17 atoms, not 20 or 23 atoms. This technology will be useful for conducting direct comparisons between many different types of chemical libraries to determine their relative utility as a source of protein ligands