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A new series of pyrazolo[1,5-a]pyrimidines exemplified by compound 1, has been identified with moderate activity (IC50=165nM), following GSK256066 rescaffolding. Compound 1 optimization at positions 2, 3, 6 and 7 gave compound 10 with high in vitro activity (IC50=0.7nM). Modeling studies based on the PDB structure 3GWT with compound 5 showed the expected overlay with the carboxamide, the aryl moiety and the sulfone. Cyclisation of the primary amide to the 5 position of the pyrazolo[1,5-a]pyrimidines scaffold afforded compounds 15 and 16 with 200-fold enhancement in activity and cellular potency., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A novel class of heat shock protein 90 (Hsp90) inhibitors was developed after a low throughput screen (LTS) of a focused library containing approximately 21K compounds selected by virtual screening. The initial [1-{3-H-imidazo[4-5-c]pyridin-2-yl}-3,4-dihydro-2H-pyrido[2,1-a]isoindole-6-one] (1) compound showed moderate activity (IC(50) = 7.6 μM on Hsp82, the yeast homologue of Hsp90). A high-resolution X-ray structure shows that compound 1 binds into an "induced" hydrophobic pocket, 10-15 Å away from the ATP/resorcinol binding site. Iterative cycles of structure-based drug design (SBDD) and chemical synthesis led to the design and preparation of analogues with improved affinity. These optimized molecules make productive interactions within the ATP binding site as reported by other Hsp90 inhibitors. This resulted in compound 8, which is a highly potent inhibitor in biochemical and cellular assays (K(d) = 0.35 nM on Hsp90; IC(50) = 30 nM on SKBr3 mammary carcinoma cells) and in an in vivo leukemia model.

Pheochromocytoma-12 (PC12) cells recapitulate the program of neuronal differentiation by developing neurites after about 12 days of nerve growth factor (NGF) treatment. This model can be used to evaluate the neuroprotective/neurotrophic effect of compounds. Specific mRNAs such as cfos and c-jun are early biomarkers of the irreversible commitment into the differentiation program as they appear after only 30-40 min of NGF treatment. Monitoring the level of these mRNAs instead of the neurite outgrowth dramatically reduces the time needed to identify the drug potential of compounds. The electrophoretic tags, or eTag reporters (ACLARA Biosciences, Inc., Mountain View, CA), are a new class of fluorescent reporters that have unique migration properties in capillary electrophoresis, which allows for their separation and identification. (The eTag Multiplex Invader Assay and products incorporate Invader technology and Cleavase enzyme licensed for use from Third Wave Technologies, Inc. [Madison, WI] for multiplexed gene expression applications.) Each eTag molecule used begins as a phosphoramidite that is incorporated into a specific oligonucleotide using standard oligonucleotide synthesis procedures. A set of distinct probes labeled with different eTag molecules can then be mixed together to simultaneously quantify the levels of different mRNAs from the same sample. When compared to existing methods for measuring multiplexed gene expression from the same sample, the eTag assay allows a direct quantification of the mRNA from cells without any extraction/purification and still provides multiplexing capability, high sensitivity, miniaturization, and reproducibility compatible with medium-throughput screening methods. The eTag technology was used to simultaneously measure the level of expression of four mRNAs-c-fos, c-jun, c-myc, and gapdh-in NGF-treated PC12 cells in a standard 96-well format. The experimental data shown here demonstrate the use of eTag technology as a new screening tool, which uniquely combines robustness, sensitivity, multiplexing capability, and direct measurement of mRNA without any sample preparation steps, such as RNA extraction/purification or a reverse transcription step.

DMSO-primed P19 pluripotent cells, which recapitulate the first stages of mammalian cardiogenesis and endodermal formation, were used as an in vitro model to analyze the variations in activity and expression of the different adenylyl cyclase (AC) isoforms during the early events of embryonic cell differentiation. Here, we show that the total AC activity, which increases up to 10-fold after differentiation of P19 cells, is mainly associated with increases in AC2, AC5, and AC6 mRNA levels. Particularly, the marked increase in AC5 mRNA correlates with the appearance of beating cardiomyocytes and with the transcription of the atrial myosin light chain (MLC1A) gene which encodes a protein specifically involved in the cardiac muscle cell contractile phenotype. Together, the results strongly suggest that 1) a rise in cyclic AMP (cAMP) may be associated with cardiomyocyte and endodermal cell differentiation during mammalian embryogenesis; and 2) AC5 gene expression starts very early during normal mouse cardiogenesis and correlates with the differentiation of cardiomyocytes.

Cardiac growth and contractility is profoundly altered in response to various hormones and neurotransmitters as well as by changes in electrolyte balance, blood volume, and blood pressure. In turn, the endocrine heart contributes to body homeostasis by secreting a number of biologically active peptide hormones that act on several target tissues. Atrial natriuretic peptide (ANP) and B-type natiuretic peptide (BNP) are the major secretory products of the endocrine myocardium. These peptide hormones act on many target organs via guanylate cyclase-linked membrane receptors to produce natriuresis, diuresis, vasodilatation, and hypotension. ANP and BNP receptors are found on most organs involved in cardiovascular homeostasis (e.g., kidney, adrenal, vasculature, brain). They are also present in gonads and in pituitary, where they alter steroidogenesis and pituitary hormone secretion. Not surprisingly, changes in blood pressure, volume, or hormone status influence ANP and BNP expression, which is also altered in almost all diseases that affect cardiac function. Thus, studies of ANP and BNP gene expression are relevant for many clinical settings. Moreover, transcription of the ANP and BNP genes characterizes cardiac cells at very early stages of development and is tightly linked to cardiac growth--be it proliferation, as in the fetal heart, or trophic growth, as in postnatal ventricular hypertrophy. Thus, analysis of the molecular circuitry that controls ANP and BNP expression might shed important insight into the complex regulatory pathways that underlie normal and pathologic heart development. Indeed, over the past few years, we have analysed transcriptional control of the ANP and BNP genes in embryonic and postnatal cardiomyocytes and in cardiomyocytes treated with hormones and neurotransmitters that cause cardiomyocyte hypertrophy. These studies have lead to the identification of distinct cis-acting DNA elements that modulate basal and hormone-stimulated transcription. Most importantly, the work also resulted in the isolation and characterization of cardiac-specific transcription factors that play critical roles for cardiac cell differentiation and survival.