Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

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 CMs. METHODS AND RESULTS: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, 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 were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, 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 because of 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.

Development of thieno- and benzopyrimidinone inhibitors of the Hedgehog signaling pathway reveals PDE4-dependent and PDE4-independent mechanisms of action.

From a high content in vivo screen for modulators of developmental patterning in embryonic zebrafish, we previously identified eggmanone (EGM1, 3) as a Hedgehog (Hh) signaling inhibitor functioning downstream of Smoothened. Phenotypic optimization studies for in vitro probe development utilizing a Gli transcription-linked stable luciferase reporter cell line identified EGM1 analogs with improved potency and aqueous solubility. Mechanistic profiling of optimized analogs indicated two distinct scaffold clusters: PDE4 inhibitors able to inhibit downstream of Sufu, and PDE4-independent Hh inhibitors functioning between Smo and Sufu. Each class represents valuable in vitro probes for elucidating the complex mechanisms of Hh regulation.

Synthetic studies directed toward the AB decalin common to HMP-Y1 and hibarimicinone.

Efforts toward the synthesis of the decalin ring system common to the hibarimicin shunt metabolite HMP-Y1 and parent aglycone hibarimicinone are reported herein. An intramolecular Diels-Alder cyclization rapidly generated the decalin framework. Two approaches toward completion of the AB decalin were vetted. Incorporation of a phenylsulfonyl leaving group ß- to both a ketone and a γ-lactone followed by base-induced elimination of sulfinate led to the undesired α,ß-unsaturated lactone. Methanolysis of the γ-lactone followed by elimination produced the unexpected bridged cyclic ether by way of an intramolecular oxy-Michael addition of the endo oriented C13 alcohol.

The application of vinylogous iminium salt derivatives to efficient formal syntheses of the marine akaloids lamellarin G trimethyl ether and ningalin B.

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.

The Application of Vinylogous Iminium Salt Derivatives and Microwave Accelerated Vilsmeier-Haack Reactions to Efficient Relay Syntheses of the Polycitone and Storniamide Natural Products.

Studies directed at the synthesis of polycitone and storniamide natural products via vinylogous iminium salts and microwave accelerated Vilsmeier-Haack formylations are described. The successful strategy relies on the formation of a 2,4-disubstituted pyrrole or a 2,3,4-trisubstituted pyrrole from a vinamidinium salt or vinamidinium salt derivative followed by formylation at the 5-position of the pyrrole. Subsequent transformations of the selectively formylated pyrroles lead to efficient and regiocontrolled relay syntheses of the respective pyrrole containing natural products.

Practical strategies for small-molecule probe development in chemical biology.

The effective identification, selection, and implementation of small molecules for the interrogation of biological systems require an intricate understanding of the chemical principles underlying their cellular activities. While much has been published regarding the use of screening techniques in forward chemical genetics platforms and on small-molecule target identification, less emphasis has been placed on detailed strategies for evaluating, selecting, and optimizing screening hits. This chapter provides practical tools for identifying and developing promising screening hit compounds into effective tools for biological discovery.

An in vivo chemical genetic screen identifies phosphodiesterase 4 as a pharmacological target for hedgehog signaling inhibition.

Hedgehog (Hh) signaling plays an integral role in vertebrate development, and its dysregulation has been accepted widely 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 an Hh-null phenotype. Eggmanone exerts its Hh-inhibitory effects through selective antagonism of phosphodiesterase 4 (PDE4), 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.

Assignment and stereocontrol of hibarimicin atropoisomers.

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.