Rationale The lack of measurable single cell contractility of human induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery. Objective To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult cardiac myocytes (CMs). Methods and Results Single hiPSC-CMs were cultured for 5 – 7 days on a 0.4 – 0.8 mm thick mattress of undiluted Matrigel (“mattress hiPSC-CM”) and compared to hiPSC-CMs maintained on control substrate (<0.1 mm thick 1:60 diluted matrigel, “control hiPSC-CM”). Compared to control hiPSC-CM, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection was comparable to that of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CM were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CM, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity due to a 2-fold increase in sodium current (INa). Conclusions The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single cell level, which should be valuable to disease modeling, drug discovery and preclinical cardiotoxicity testing.

We studied the mechanism of action of 3,5-dibromo-4-(3,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid ethyl ester and found that it is a potent microtubule depolymerizer. JG-03-14 caused a dose-dependent loss of cellular microtubules, formation of aberrant mitotic spindles, accumulation of cells in the G 2 /M phase of the cell cycle, and Bcl-2 phosphorylation. These events culminated in the initiation of apoptosis, as evidenced by the caspase 3-dependent cleavage of poly-(ADP-ribose) polymerase (PARP). JG-03-14 has antiproliferative activity against a wide range of cancer cell lines, with an average IC 50 value of 62 nM, and it is a poor substrate for transport by P-glycoprotein. JG-03-14 inhibited the polymerization of purified tubulin in vitro, consistent with a direct interaction between the compound and tubulin. JG-03-14 potently inhibited the binding of [ 3 H]colchicine to tubulin, suggesting that it bound to tubulin at a site overlapping the colchicine site. JG-03-14 had antitumor effects in the PC3 xenograft model, in which it caused greater than 50% reduction in tumor burden after 14 days of treatment. Molecular modeling studies indicated that the dimethoxyphenyl group of JG-03-14 occupies a space similar to that of the trimethoxyphenyl group of colchicine. However, the 2,3,5-trisubstituted pyrrole group, which is connected to the dimethoxyphenyl moiety, interacted with both ␣ and ␤ tubulin in space not shared with colchicine, suggesting significant differences compared with colchicine in the mechanism of binding to tubulin. Our results suggest that this tetrasubstituted pyrrole represents a new, biologically active chemotype for the colchicine site on tubulin.Microtubules are cellular structures that play a central role in metabolism, intracellular transport, and cell division. A wide range of chemicals have been identified that interrupt microtubule function. These compounds can be divided into microtubule stabilizers and microtubule depolymerizers. Microtubule stabilizers include paclitaxel, discodermolide, the epothilones, and the laulimalides. Microtubule stabilizers cause an increase in the density of cellular microtubules, and they stimulate the assembly of purified tubulin. In contrast, microtubule depolymerizers cause a loss of cellular microtubules, and they inhibit the assembly of purified tubulin. Microtubule depolymerizing compounds can be further subdivided into those that bind to tubulin within the colchicine site and those that bind within the vinca domain. Agents acting upon the colchicine site include 2ME2, combretastatin A-4, and podophyllotoxin. The phenotypic effects of microtubule stabilizing and depolymerizing agents are quite disparate when they are used at high concentrations in cells, but at their lowest antiproliferative concentrations, both classes of agents inhibit microtubule dynamics (Jordan and Wilson, 2004). In due course, inhibition of microtubule dynamics is believed to hinder the normal function of the mitotic spindle,

SUMMARY Hedgehog (Hh) signaling plays an integral role in vertebrate development, and its dysregulation has been widely accepted as a driver of numerous malignancies. While a variety of small molecules target Smoothened (Smo) as a strategy for Hh inhibition, Smo gain of function mutations have limited their clinical implementation. Modulation of targets downstream of Smo could define a paradigm for treatment of Hh-dependent cancers. Here, we describe eggmanone, a small molecule identified from a chemical genetic zebrafish screen which induced a Hh-null phenotype. Eggmanone exerts its Hh-inhibitory effects through selective antagonism of phosphodiesterase (PDE) 4, leading to protein kinase A activation and subsequent Hh blockade. Our study implicates PDE4 as a target for Hh inhibition, suggests an improved strategy for Hh-dependent cancer therapy and identifies a unique probe of downstream-of-Smo Hh modulation.

Studies directed at the synthesis of lamellarin G trimethyl ether and ningalin B via vinylogous iminium salt derivatives are described. The successful strategy relies on the formation of a 2,4-disubstituted pyrrole or a 1,2,3,4-tetrasubstituted pyrrole from a vinylogous iminium salt or vinylogous iminium salt derivative. Subsequent transformations of these highly substituted pyrroles lead to efficient and regiocontrolled formal syntheses of the respective pyrrole containing natural products.

A stereochemical feature of the hibarimicins is a central biaryl (HMP-Y6) or aryl-quinone (hibarimicinone) incorporated as a single atropodiastereomer. Herein, a chiral resolution and deracemization process to access optically enriched biaryls aR-3 and aS-3 is described. From these atropoenantiomers the BCD-EFG ring system of HMP-Y6 is constructed [(+)-aR-7]. Comparison of CD spectra of aR-7 to HMP-Y6 leads to the assignment of HMP-Y6 and hibarimicin B atropoisomers as aR and aS, respectively.