Library Synthesis: Building Block Selection, Handling, and Tracking.

Careful selection and manipulation of small molecule building blocks is crucial to the success of a DNA-encoded library. Building block selection impacts the quality of the hits arising out of a selection assay, while proper sample handling and tracking ensure follow-up synthetic work is done with the appropriate synthetic map in mind. In this chapter, possible strategies for building block selection are outlined, as well as best practices for handling and tracking samples to be used for validation and library synthesis.

Multifunctional Building Blocks Compatible with Photoredox-Mediated Alkylation for DNA-Encoded Library Synthesis.

DNA-encoded library (DEL) technology has emerged as a novel interrogation modality for ligand discovery in the pharmaceutical industry. Given the increasing demand for a higher proportion of C(sp3)-hybridized centers in DEL platforms, a photoredox-mediated cross-coupling and defluorinative alkylation process is introduced using commercially available alkyl bromides and structurally diverse α-silylamines. Notably, no protecting group strategies for amines are necessary for the incorporation of a variety of amino-acid-based organosilanes, providing crucial branching points for further derivatization.

Open-Air Alkylation Reactions in Photoredox-Catalyzed DNA-Encoded Library Synthesis.

DNA-encoded library (DEL) technology is a powerful tool commonly used by the pharmaceutical industry for the identification of compounds with affinity to biomolecular targets. Success in this endeavor lies in sampling diverse chemical libraries. However, current DELs tend to be deficient in C(sp3) carbon counts. We report unique solutions to the challenge of increasing both the chemical diversity of these libraries and their C(sp3) carbon counts by merging Ni/photoredox dual catalytic C(sp2)-C(sp3) cross-coupling as well as photoredox-catalyzed radical/polar crossover alkylation protocols with DELs. The successful integration of multiple classes of radical sources enables the rapid incorporation of a diverse set of alkyl fragments.

Insulin signaling displayed a differential tissue-specific response to low-dose dihydrotestosterone in female mice.

Hyperandrogenemia and hyperinsulinemia are believed to play prominent roles in polycystic ovarian syndrome (PCOS). We explored the effects of low-dose dihydrotestosterone (DHT), a model of PCOS, on insulin signaling in metabolic and reproductive tissues in a female mouse model. Insulin resistance in the energy storage tissues is associated with type 2 diabetes. Insulin signaling in the ovaries and pituitary either directly or indirectly stimulates androgen production. Energy storage and reproductive tissues were isolated and molecular assays were performed. Livers and white adipose tissue (WAT) from DHT mice displayed lower mRNA and protein expression of insulin signaling intermediates. However, ovaries and pituitaries of DHT mice exhibited higher expression levels of insulin signaling genes/proteins. Insulin-stimulated p-AKT levels were blunted in the livers and WAT of the DHT mice but increased or remained the same in the ovaries and pituitaries compared with controls. Glucose uptake decreased in liver and WAT but was unchanged in pituitary and ovary of DHT mice. Plasma membrane GLUTs were decreased in liver and WAT but increased in ovary and pituitary of DHT mice. Skeletal muscle insulin-signaling genes were not lowered in DHT mice compared with control. DHT mice did not display skeletal muscle insulin resistance. Insulin-stimulated glucose transport increased in skeletal muscles of DHT mice compared with controls. DHT mice were hyperinsulinemic. However, the differential mRNA and protein expression pattern was independent of hyperinsulinemia in cultured hepatocytes and pituitary cells. These findings demonstrate a differential effect of DHT on the insulin-signaling pathway in energy storage vs. reproductive tissues independent of hyperinsulinemia.

Small-molecule studies identify CDK8 as a regulator of IL-10 in myeloid cells.

Enhancing production of the anti-inflammatory cytokine interleukin-10 (IL-10) is a promising strategy to suppress pathogenic inflammation. To identify new mechanisms regulating IL-10 production, we conducted a phenotypic screen for small molecules that enhance IL-10 secretion from activated dendritic cells. Mechanism-of-action studies using a prioritized hit from the screen, BRD6989, identified the Mediator-associated kinase CDK8, and its paralog CDK19, as negative regulators of IL-10 production during innate immune activation. The ability of BRD6989 to upregulate IL-10 is recapitulated by multiple, structurally differentiated CDK8 and CDK19 inhibitors and requires an intact cyclin C-CDK8 complex. Using a highly parallel pathway reporter assay, we identified a role for enhanced AP-1 activity in IL-10 potentiation following CDK8 and CDK19 inhibition, an effect associated with reduced phosphorylation of a negative regulatory site on c-Jun. These findings identify a function for CDK8 and CDK19 in regulating innate immune activation and suggest that these kinases may warrant consideration as therapeutic targets for inflammatory disorders.

Low-Dose Dihydrotestosterone Drives Metabolic Dysfunction via Cytosolic and Nuclear Hepatic Androgen Receptor Mechanisms.

Androgen excess in women is associated with metabolic dysfunction (e.g., obesity, hyperinsulinemia, insulin resistance, and increased risk of type 2 diabetes) and reproductive dysfunction (e.g., polycystic ovaries, amenorrhea, dysregulated gonadotropin release, and infertility). We sought to identify the effects of androgen excess on glucose metabolic dysfunction and the specific mechanisms of action by which androgens are inducing pathology. We developed a mouse model that displayed pathophysiological serum androgen levels with normal body mass/composition to ensure that the phenotypes were directly from androgens and not an indirect consequence of obesity. We performed reproductive tests, metabolic tests, and hormonal assays. Livers were isolated and examined via molecular, biochemical, and histological analysis. Additionally, a low-dose dihydrotestosterone (DHT) cell model using H2.35 mouse hepatocytes was developed to study androgen effects on hepatic insulin signaling. DHT mice demonstrated impaired estrous cyclicity; few corpora lutea in the ovaries; glucose, insulin, and pyruvate intolerance; and lowered hepatic insulin action. Mechanistically, DHT increased hepatic androgen-receptor binding to phosphoinositide-3-kinase (PI3K)-p85, resulting in dissociation of PI3K-p85 from PI3K-p110, leading to reduced PI3K activity and decreased p-AKT and, thus, lowered insulin action. DHT increased gluconeogenesis via direct transcriptional regulation of gluconeogenic enzymes and coactivators. The hepatocyte model recapitulated the in vivo findings. The DHT-induced hepatocyte insulin resistance was reversed by the androgen-receptor antagonist, flutamide. These findings present a phenotype (i.e., impaired glucose tolerance and disrupted glucose metabolism) in a lean hyperandrogenemia model (low-dose DHT) and data to support 2 molecular mechanisms that help drive androgen-induced impaired glucose metabolism.

Synthesis and evaluation of antimigratory and antiproliferative activities of lipid-linked [13]-macro-dilactones.

The biological activities of a family of novel, lipid-linked 13-membered-ring macro-dilactones are reported. These [13]-macro-dilactones were synthesized by diacylation of functionalized diols, followed by ring-closing metathesis under conditions we had previously reported. Antimigratory, cytostatic and cytotoxic activities of the compounds against cancer cells were evaluated. Compound 13 was the most potent in the series, while compound 10 had the broadest concentration range of subtoxic antiproliferative activity. These compounds share common structural components, namely the [13]-macro-dilactone templated by an octyl alpha-glucoside 4,6-diol.