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SummaryDiabetic cardiomyopathy is a complication of type 2 diabetes, with known contributions of lifestyle and genetics. We develop environmentally and genetically driven in vitro models of the condition using human-induced-pluripotent-stem-cell-derived cardiomyocytes. First, we mimic diabetic clinical chemistry to induce a phenotypic surrogate of diabetic cardiomyopathy, observing structural and functional disarray. Next, we consider genetic effects by deriving cardiomyocytes from two diabetic patients with variable disease progression. The cardiomyopathic phenotype is recapitulated in the patient-specific cells basally, with a severity dependent on their original clinical status. These models are incorporated into successive levels of a screening platform, identifying drugs that preserve cardiomyocyte phenotype in vitro during diabetic stress. In this work, we present a patient-specific induced pluripotent stem cell (iPSC) model of a complex metabolic condition, showing the power of this technique for discovery and testing of therapeutic strategies for a disease with ever-increasing clinical significance.

SummaryThe use of human pluripotent stem cells (hPSCs) in cell therapy is hindered by the tumorigenic risk from residual undifferentiated cells. Here we performed a high-throughput screen of over 52,000 small molecules and identified 15 pluripotent cell-specific inhibitors (PluriSIns), nine of which share a common structural moiety. The PluriSIns selectively eliminated hPSCs while sparing a large array of progenitor and differentiated cells. Cellular and molecular analyses demonstrated that the most selective compound, PluriSIn #1, induces ER stress, protein synthesis attenuation, and apoptosis in hPSCs. Close examination identified this molecule as an inhibitor of stearoyl-coA desaturase (SCD1), the key enzyme in oleic acid biosynthesis, revealing a unique role for lipid metabolism in hPSCs. PluriSIn #1 was also cytotoxic to mouse blastocysts, indicating that the dependence on oleate is inherent to the pluripotent state. Finally, application of PluriSIn #1 prevented teratoma formation from tumorigenic undifferentiated cells. These findings should increase the safety of hPSC-based treatments.

Design, synthesis, and SAR are described for a class of DPP-IV inhibitors based on aminobenzo[a]quinolizines with non-aromatic substituents in the S1 specificity pocket. One representative thereof, carmegliptin (8p), was chosen for clinical development. Its X-ray structure in complex with the enzyme and early efficacy data in animal models of type 2 diabetes are also presented.

Design, synthesis, and SAR of novel alpha-alkoxy-beta-arylpropionic acids as potent and balanced PPARalphagamma coagonists are described. One representative thereof, Aleglitazar ((S)-2Aa), was chosen for clinical development. Its X-ray structure in complex with both receptors as well as its high efficacy in animal models of T2D and dyslipidemia are also presented.

Dear Editor, According to the cancer stem cell (CSC) hypothesis, cancer cells are organized in a hierarchical manner (Reya et al., 2001). Indeed, the differentiation state of cancer cells has been linked to their proliferative potential, drug response, and ability to metastasize (Clevers, 2011). Targeting self-renewing, undifferentiated cancer cells could therefore be of importance for the success of cancer therapies. A classical case of a tumor composed of undifferentiated and differentiated cells is the tumor induced by pluripotent stem cells (PSCs) (Ben-David and Benvenisty, 2011). We have recently identified several small molecules termed pluripotent-specific inhibitors (PluriSIns) that selectively target undifferentiated tumorigenic PSCs (Ben-David et al., 2013). Several of these compounds, including PluriSIn#1, inhibit the activity of stearoyl-coA desaturase (SCD1) (Ben-David et al., 2013). SCD1 is an endoplasmic reticulum (ER)-membrane protein that plays a key role in the biosynthesis of the mono-unsaturated fatty acid oleate. This novel metabolic vulnerability is in line with the unique metabolome described in PSCs (Panopoulos et al., 2012). Inhibition of SCD1 was previously suggested as a potential cancer therapy (Hess et al., 2010; Minville-Walz et al., 2010; Roongta et al., 2011); however, why some cancer cells are highly sensitive to SCD1 inhibition, whereas others are completely resistant to it, remains unknown. As undifferentiated human PSCs were highly sensitive to SCD1 inhibition and rapidly lost this sensitivity upon differentiation (Ben-David et al., 2013), and the processes of pluripotency induction and oncogenic transformation share many characteristics (Ben-David and Benvenisty, 2011), we speculated that the sensitivity of some cancer cells to SCD1 inhibition might be related to their differentiation state as well. To test this hypothesis, we took advantage of a recently developed system of in vitro transformed CSC-like cells (Scaffidi and Misteli, 2011). When forced to stably express hTERT, H-RasV12 and SV40 LT and ST antigens, human primary skin fibroblasts undergo an oncogenic reprogramming process and acquire long-term self-renewal ability and differentiation capacity, in contrast to their hTERT-immortalized control counterparts (Scaffidi and Misteli, 2011). We therefore tested the response of these isogenic cell lines to PluriSIn#1. Interestingly, while the immortalized fibroblasts exhibited a very weak response, the transformed undifferentiated cells were extremely sensitive to PluriSIn#1, and massive cell death was observed in culture at 72 h post exposure (Figure 1A). To examine whether the direct cause of death following PluriSIn#1 exposure was indeed SCD1 inhibition, we exposed the cells to another specific inhibitor of SCD1, A939572, and obtained a highly similar differential response (Supplementary Figure S1A). Moreover, exogenous supplementation of oleate, the enzymatic product of SCD1 activity, completely rescued the cell death induced by PluriSIn#1 (Figure 1B). We repeated the experiment with a second isogenic pair of immortalized and transformed cell lines (Supplementary Figure S1B), ruling out the possibility that a cell line-specific difference unrelated to the cellular transformation underlies the differential response. Figure 1 Cellular transformation renders undifferentiated cancer cells sensitive to SCD1 inhibition, and this sensitivity is quickly lost upon differentiation. (A) Quantitated cellular viability of immortalized fibroblasts (blue) and undifferentiated transformed ... We next set out to determine whether the sensitivity of the transformed cells to SCD1 inhibition is related to their undifferentiated state. The transformed cells were differentiated into adipocytes (Scaffidi and Misteli, 2011), and were exposed to PluriSIn#1. We found that 6 days of adipogenic differentiation were sufficient to significantly reduce the sensitivity of the transformed cells to SCD1 inhibition (Figure 1C and D). To further examine the relationship between differentiation state and oleate dependency in cancer cells, we tested a cell line of undifferentiated stem-like glioma cells (SLGCs) (Campos et al., 2010). These cells grow as neurospheres in suspension, and efficiently differentiate when cultured in adhesion in the presence of retinoic acid (RA) (Campos et al., 2010). Consistent with the findings from the transformed cell lines, undifferentiated SLGCs were highly sensitive to PluriSIn#1, but 1 week of RA-induced differentiation rendered them completely resistant to the molecule (Figure 1E and F). To examine whether it is indeed differentiation, rather than cell cycle exit, that protects cells from SCD1 inhibitor-mediated death, human PSCs were cultured in a bFGF-free medium and exposed to PluriSIn#1 prior to their actual differentiation; the sensitivity to SCD1 inhibition was not reduced in the growth factor-depleted medium (Supplementary Figure S2), suggesting that cell cycle exit is not sufficient to protect the cells. Lastly, we examined whether undifferentiated cancer cells could be depleted from a heterogeneous population by inhibition of oleate synthesis. Transformed fibroblasts were treated with either 20 μM PluriSIn#1 or 0.02% DMSO as control for 72 h, in the presence of immortalized fibroblasts. Cells were then harvested and injected subcutaneously into immune-deficient mice (n = 4). Each mouse was injected with same number of control-treated cells into one side of the body and PluriSIn#1-treated cells into the other side. Six weeks later, mice were sacrificed, and tumors were measured and analyzed. PluriSIn#1-treated cells gave rise to much smaller tumors compared with control-treated cells (Figure 1G and H). Treatment with A939572 similarly reduced the size of the generated tumors (n = 3, Supplementary Figure S1C). These data indicate that SCD1 inhibition significantly decreases the tumorigenicity of the heterogeneous cell population. We have previously shown that fibroblasts resistant to SCD1 inhibition acquire the metabolic sensitivity when reprogrammed into induced PSCs, but this sensitivity is quickly lost upon differentiation (Ben-David et al., 2013). Here we demonstrate that these fibroblasts acquire the same metabolic vulnerability when they are transformed into undifferentiated stem cell-like cancer cells, but the resistance to SCD1 inhibition is conferred again once cancer cells are induced to differentiate (Figure 1I). These findings link, for the first time, the differentiation state of cancer cells with their response to inhibition of oleate synthesis, suggesting that SCD1 inhibition might be an especially effective cancer therapy for undifferentiated tumor types. This work also demonstrates that research on cellular reprogramming can yield insights into cancer biology, and validates that in vitro generated CSCs can be successfully used for screening anticancer drugs. [Supplementary material is available at Journal of Molecular Cell Biology online. We thank members of the Stem Cell Unit at the Hebrew University, and especially Tamar Golan-Lev for her assistance with the graphic design of the Figures, Ofra Yanuka for her help with SLGC culturing, and Ido Sagi and Daniel Ronen for critically reading the manuscript. N.B. is the Herbert Cohn Chair in Cancer Research. U.B.-D. and A.B. are Clore Fellows. This work was supported by a Hoffmann La Roche-Yissum Collaboration grant, a DKFZ-Israel Collaboration grant (#CA-146) from the Israel Ministry of Science, and the Intramural Research Program of the National Institutes of Health (NIH), NCI, Center for Cancer Research.]

Single-atom substrate modifications have revealed an intricate network of transition state interactions in the Tetrahymena ribozyme reaction. So far, these studies have targeted virtually every oxygen atom near the reaction center, except one, the 5'-bridging oxygen atom of the scissile phosphate. To address whether interactions with this atom play any role in catalysis, we used a new type of DNA substrate in which the 5'-oxygen is replaced with a methylene (-CH2-) unit. Under (kcat/Km)S conditions, the methylene phosphonate monoester substrate dCCCUCUT(mp)TA4 (where mp indicates the position of the phosphonate linkage) unexpectedly reacts approximately 10(3)-fold faster than the analogous control substrates lacking the -CH2- modification. Experiments with DNA-RNA chimeric substrates reveal that the -CH2- modification enhances docking of the substrates into the catalytic core of the ribozyme by approximately 10-fold and stimulates the chemical cleavage by approximately 10(2)-fold. The docking effect apparently arises from the ability of the -CH2- unit to suppress inherently deleterious effects caused by the thymidine residue that immediately follows the cleavage site. To analyze the -O- to -CH2- modification in the absence of this thymidine residue, we prepared oligonucleotide substrates containing methyl phosphate or ethyl phosphonate at the reaction center, thereby eliminating the 3'-terminal TA4 nucleotidyl group. In this context, the -O- to -CH2-modification has no effect on docking but retains the approximately 10(2)-fold effect on the chemical step. To investigate further the stimulatory influence on the chemical step, we measured the "intrinsic" effect of the -O- to -CH2- modification in nonenzymatic reactions with nucleophiles. We found that in solution, the -CH2- modification stimulates chemical reactivity of the phosphorus center by5-fold, substantially lower in magnitude than the stimulatory effect in the catalytic core of the ribozyme. The greater stimulatory effect of the -CH2- modification in the active site compared to in solution may arise from fortuitous changes in molecular geometry that allow the ribozyme to accommodate the phosphonate transition state better than the natural phosphodiester transition state. As the -CH2- unit lacks lone pair electrons, its effectiveness in the ribozyme reaction suggests that the 5'-oxygen of the scissile phosphate plays no role in catalysis via hydrogen bonding or metal ion coordination. Finally, we show by analysis of physical organic data that such interactions in general provide little catalytic advantage to RNA and protein phosphoryl transferases because the 5'-oxygen undergoes only a small buildup of negative charge during the reaction. In addition to its mechanistic significance for the Tetrahymena ribozyme reaction and phosphoryl transfer reactions in general, this work suggests that phosphonate monoesters may provide a novel molecular tool for determining whether the chemical step limits the rate of an enzymatic reaction. As methylene phosphonate monoesters react modestly faster than phosphate diesters in model reactions, a similarly modest stimulatory effect on an enzymatic reaction upon -CH2- substitution would suggest rate-limiting chemistry.

In a novel series of DPP-IV inhibitors, a large increase of inhibitory activity was achieved by optimisation of aromatic substituents and conformational restriction.

A recently identified DPP-IV inhibitor (1) was found to induce phospholipidosis and to inhibit CYP3A4. A small series of less lipophilic and less amphiphilic analogues was synthesized in an effort to overcome these issues. One compound from this series was equipotent to 1, did not induce phospholipidosis and showed a reduced CYP3A4 inhibition.

The influence of aromatic substitution on a newly discovered class of inhibitors of dipeptidyl peptidase IV was investigated. A 10(5)-fold increase in potency was achieved by the optimization of aromatic substituents in a parallel chemistry program. The observed SAR could be explained by an X-ray structure of the protein-ligand complex.