Novel Long-Acting Oxytocin Analog with Increased Efficacy in Reducing Food Intake and Body Weight.

Oxytocin (OXT) analogues have been designed to overcome the limitation of the short half-life of the native OXT peptide. Here, we tested ASK2131 on obesity related outcomes in diet-induced obese (DIO) Sprague Dawley rats. In vitro function assays were conducted. The effects of daily subcutaneous injections of ASK2131 vs. OXT and pair-feeding were assessed on food intake and body weight in vivo. ASK2131 is a longer-lasting OXT analog with improved pharmacokinetics compared to OXT (T1/2: 2.3 vs. 0.12 h). In chronic 22-day administration, ASK2131 was administered at 50 nmol/kg, while OXT doses were titrated up to 600 nmol/kg because OXT appeared to be less effective at reducing energy intake relative to ASK2131 at equimolar doses. After 22 days, vehicle-treated animals gained 4.5% body weight, OXT rats maintained their body weight, while those treated with ASK2131 declined in weight continuously over the 22-day period, leading to a 6.6 ± 1.3% reduction (mean ± standard error) compared to baseline. Compared to their pair-fed counterparts, ASK2131-treated rats showed a more pronounced reduction in body weight through most of the study. In summary, ASK2131 is a promising OXT-based therapeutic, with extended in vivo stability and improved potency leading to a profound reduction in body weight partly explained by reduced food intake.

3-Amino-thieno[2,3-b]pyridines as microtubule-destabilising agents: Molecular modelling and biological evaluation in the sea urchin embryo and human cancer cells.

A series of 3-amino-thieno[2,3-b]pyridines was prepared and tested in a phenotypic sea urchin embryo assay to identify potent and specific molecules that affect tubulin dynamics. The most active compounds featured a tricyclic core ring system with a fused cycloheptyl or cyclohexyl substituent and unsubstituted or alkyl-substituted phenyl moiety tethered via a carboxamide. Low nano-molar potency was observed in the sea urchin embryos for the most active compounds (1-5) suggestive of a microtubule-destabilising effect. The molecular modelling studies indicated that the tubulin colchicine site is inhibited, which often leads to microtubule-destabilisation in line with the sea urchin embryo results. Finally, the identified hits displayed a robust growth inhibition (GI50 of 50-250nM) of multidrug-resistant melanoma MDA-MB-435 and breast MDA-MB-468 human cancer cell lines. This work demonstrates that for the thieno[2,3-b]pyridines the most effective mechanism of action is microtubule-destabilisation initiated by binding to the colchicine pocket.

Key signaling pathways in the muscle-invasive bladder carcinoma: Clinical markers for disease modeling and optimized treatment.

In this review, we evaluate key molecular pathways and markers of muscle-invasive bladder cancer (MIBC). Overexpression and activation of EGFR, p63, and EMT genes are suggestive of basal MIBC subtype generally responsive to chemotherapy. Alterations in PPARγ, ERBB2/3, and FGFR3 gene products and their signaling along with deregulated p53, cytokeratins KRT5/6/14 in combination with the cellular proliferation (Ki-67), and cell cycle markers (p16) indicate the need for more radical treatment protocols. Similarly, the "bell-shape" dynamics of Shh expression levels may suggest aggressive MIBC. A panel of diverse biological markers may be suitable for simulation studies of MIBC and development of an optimized treatment protocol. We conducted a critical evaluation of PubMed/Medline and SciFinder databases related to MIBC covering the period 2009-2015. The free-text search was extended by adding the following keywords and phrases: bladder cancer, metastatic, muscle-invasive, basal, luminal, epithelial-to-mesenchymal transition, cancer stem cell, mutations, immune response, signaling, biological markers, molecular markers, mathematical models, simulation, epigenetics, transmembrane, transcription factor, kinase, predictor, prognosis. The resulting selection of ca 500 abstracts was further analyzed in order to select the latest publications relevant to MIBC molecular markers of immediate clinical significance.

Efficient Synthesis of Glaziovianin A Isoflavone Series from Dill and Parsley Extracts and Their in Vitro/in Vivo Antimitotic Activity.

A concise six-step protocol for the synthesis of isoflavone glaziovianin A (GVA) and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. The reaction sequence involved an efficient conversion of the key intermediate epoxides 7 into the respective ß-ketoaldehydes 8 followed by their Cu(I)-mediated cyclization into the target series 9. The biological activity of GVA and its derivatives was evaluated using a panel of seven human cancer cell lines and an in vivo sea urchin embryo assay. Both screening platforms confirmed the antimitotic effect of the parent GVA (9cg) and its alkoxy derivatives. Structure-activity relationship studies suggested that compounds 9cd and 9cf substituted with trimethoxy- and dillapiol-derived B-rings, respectively, were less active than the parent 9cg. Of the evaluated human cancer cell lines, the A375 melanoma cell line was the most sensitive to the tested molecules. Notably, the target compounds were not cytotoxic against human peripheral blood mononuclear cells up to 10 µM concentration. Phenotypic readouts from the sea urchin assay unequivocally suggest a direct microtubule-destabilizing effect of isoflavones 9cg, 9cd, and 9cf.

Comparative in vivo evaluation of polyalkoxy substituted 4H-chromenes and oxa-podophyllotoxins as microtubule destabilizing agents in the phenotypic sea urchin embryo assay.

A series of polyalkoxy substituted 7-hydroxy- and 7-methoxy-4-aryl-4H-chromenes were evaluated using the sea urchin embryo model to yield several compounds exhibiting potent antimitotic microtubule destabilizing activity. Data obtained by the assay were further confirmed in the NCI60 human cancer cell screen. The replacement of methylenedioxy ring A and lactone ring D in podophyllotoxin analogues by 7-methoxy, 2-NH2, and 3-CN groups in 4-aryl-4H-chromenes resulted in potent antimitotic microtubule destabilizing agents. Feasible synthesis and high yields render 7-methoxy-4H-chromenes to be a promising series for further anticancer drug development.

Synthesis and antiproliferative activity of conformationally restricted 1,2,3-triazole analogues of combretastatins in the sea urchin embryo model and against human cancer cell lines.

A series of 1,5-diaryl- and 4,5-diaryl-1,2,3-triazole derivatives of combretastatin A4 were synthesized and evaluated as antimitotic microtubule destabilizing agents using the sea urchin embryo model. Structure-activity relationship studies identified compounds substituted with 3,4,5-trimethoxyphenyl and 3,4-methylenedioxy-5-methoxyphenyl ring A and 4-methoxyphenyl ring B as potent antiproliferative agents with high cytotoxicity against a panel of human cancer cell lines including multi-drug resistant cells. 4,5-Diaryl-1,2,3-triazoles (C-C geometry) were found to be considerably more active than the respective 1,5-diaryl-1,2,3-triazoles (N-C geometry). Compound 10ad' induced G2/M cell cycle arrest and apoptosis in human T-leukemia Jurkat cells via caspase 2/3/9 activation and downregulation of the antiapoptotic protein XIAP. A mitotic catastrophe has been evaluated as another possible cell death mode.

cis-Restricted 3-aminopyrazole analogues of combretastatins: synthesis from plant polyalkoxybenzenes and biological evaluation in the cytotoxicity and phenotypic sea urchin embryo assays.

We have synthesized a series of novel cis-restricted 4,5-polyalkoxydiaryl-3-aminopyrazole analogues of combretastatins via short synthetic sequences using building blocks isolated from dill and parsley seed extracts. The resulting compounds were tested in vivo in the phenotypic sea urchin embryo assay to reveal their antimitotic and antitubulin effects. The most potent aminopyrazole, 14a, altered embryonic cell division at 10 nM concentration, exhibiting microtubule-destabilizing properties. Compounds 12a and 14a displayed pronounced cytotoxicity in the NCI60 anticancer drug screen, with the ability to inhibit growth of multi-drug-resistant cancer cells.

Synthesis and comparative evaluation of 4-oxa- and 4-aza-podophyllotoxins as antiproliferative microtubule destabilizing agents.

A series of novel 4-oxa-podophyllotoxin derivatives 7 featuring the intact lactone ring D and various substituents in rings B and E has been synthesized and evaluated in a phenotypic sea urchin embryo assay along with the representative 4-aza-analogs 5 for their antimitotic and microtubule destabilizing activity. The most active compounds exhibited myristicin-derived or a 3',5'-dimethoxy substitution pattern in the ring E and a 6-methoxy moiety replacing the methylenedioxy ring A. Compounds 5xb, 5xe, 5yb, 7xa, 7xb, and 7xc showed potent antiproliferative effects in the NCI60 cytotoxicity screen. Notably, growth of the multi-drug resistant NCI/ADR-RES cells was more affected by these agents than the parent OVCAR-8 cell line. Although generally 4-oxa-podophyllotoxins were less potent than the respective 4-aza-derivatives in these assays, stability of the former series towards oxidation may prove to be of interest for the development of anticancer agents with in vivo activity.

iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease modeling.

Skeletal muscle myoblasts (iMyoblasts) were generated from human induced pluripotent stem cells (iPSCs) using an efficient and reliable transgene-free induction and stem cell selection protocol. Immunofluorescence, flow cytometry, qPCR, digital RNA expression profiling, and scRNA-Seq studies identify iMyoblasts as a PAX3+/MYOD1+ skeletal myogenic lineage with a fetal-like transcriptome signature, distinct from adult muscle biopsy myoblasts (bMyoblasts) and iPSC-induced muscle progenitors. iMyoblasts can be stably propagated for >12 passages or 30 population doublings while retaining their dual commitment for myotube differentiation and regeneration of reserve cells. iMyoblasts also efficiently xenoengrafted into irradiated and injured mouse muscle where they undergo differentiation and fetal-adult MYH isoform switching, demonstrating their regulatory plasticity for adult muscle maturation in response to signals in the host muscle. Xenograft muscle retains PAX3+ muscle progenitors and can regenerate human muscle in response to secondary injury. As models of disease, iMyoblasts from individuals with Facioscapulohumeral Muscular Dystrophy revealed a previously unknown epigenetic regulatory mechanism controlling developmental expression of the pathological DUX4 gene. iMyoblasts from Limb-Girdle Muscular Dystrophy R7 and R9 and Walker Warburg Syndrome patients modeled their molecular disease pathologies and were responsive to small molecule and gene editing therapeutics. These findings establish the utility of iMyoblasts for ex vivo and in vivo investigations of human myogenesis and disease pathogenesis and for the development of muscle stem cell therapeutics.

Muscular dystrophies are a group of inherited genetic diseases characterised by progressive muscle weakness. They lead to disability or even death, and no cure exists against these conditions. Advances in genome sequencing have identified many mutations that underly muscular dystrophies, opening the door to new therapies that could repair incorrect genes or rebuild damaged muscles. However, testing these ideas requires better ways to recreate human muscular dystrophy in the laboratory. One strategy for modelling muscular dystrophy involves coaxing skin or other cells from an individual into becoming 'induced pluripotent stem cells'; these can then mature to form almost any adult cell in the body, including muscles. However, this approach does not usually create myoblasts, the 'precursor' cells that specifically mature into muscle during development. This limits investigations into how disease-causing mutations impact muscle formation early on. As a response, Guo et al. developed a two-step protocol of muscle maturation followed by stem cell growth selection to isolate and grow 'induced myoblasts' from induced pluripotent stem cells taken from healthy volunteers and muscular dystrophy patients. These induced myoblasts can both make more of themselves and become muscle, allowing Guo et al. to model three different types of muscular dystrophy. These myoblasts also behave as stem cells when grafted inside adult mouse muscles: some formed human muscle tissue while others remained as precursor cells, which could then respond to muscle injury and start repair. The induced myoblasts developed by Guo et al. will enable scientists to investigate the impacts of different mutations on muscle tissue and to better test treatments. They could also be used as part of regenerative medicine therapies, to restore muscle cells in patients.

Structure of human caspase-6 in complex with Z-VAD-FMK: New peptide binding mode observed for the non-canonical caspase conformation.

Caspase-6 is a cysteine protease implicated in neuronal survival and apoptosis. Deregulation of caspase-6 activity was linked to several neurodegenerative disorders including Alzheimer's and Huntington's Diseases. Several recent studies on the structure of caspase-6 feature the caspase-6 zymogen, mature apo-caspase-6 as well as the Ac-VEID-CHO peptide complex. All structures share the same typical dimeric caspase conformation. However, mature apo-caspase-6 crystallized at low pH revealed a novel, non-canonical inactive caspase conformation speculated to represent a latent state of the enzyme suitable for the design of allosteric inhibitors. In this treatise we present the structure of caspase-6 in the non-canonical inactive enzyme conformation bound to the irreversible inhibitor Z-VAD-FMK. The complex features a unique peptide binding mode not observed previously.

Robust Reductions of Body Weight and Food Intake by an Oxytocin Analog in Rats.

Background: Oxytocin is a hypothalamic neuropeptide that participates in the network of appetite regulation. Recently the oxytocin signaling pathway has emerged as an attractive target for treating obesity. However, the short half-life limits its development as a clinical therapeutic. Here we provide results from testing a long-lasting, potent and selective oxytocin analog ASK1476 on its efficacy to reduce food intake and body weight in comparison to the native oxytocin peptide. Methods: ASK1476 features two specific amino acid substitutions in positions 7 and 8 combined with a short polyethylene glycol spacer. Short time dose escalation experiments testing increasing doses of 3 days each were performed in diet-induced overweight (DIO) male rats assessing effects on body weight as well as changes in food intake. Furthermore, DIO rats were tested for changes in body weight, food intake, temperature, and locomotor activity over 28 days of treatment (oxytocin, ASK1476, or vehicle). Results: In dose escalation experiments, significant reductions in food intake relative to baseline were detected beginning with doses of 15 nmol/kg ASK1476 (-15.2 ± 2.3 kcal/d, p = 0.0017) and 20 nmol/kg oxytocin (-11.2.9 ± 2.4 kcal/d, p = 0.0106) with corresponding significant changes in body weight (ASK1476: -5.2 ± 0.8 g, p = 0.0016; oxytocin: -2.6 ± 0.7 g, p = 0.0326). In long-term experiments, there was no difference on body weight change between 120 nmol/kg/d ASK1476 (-71.4 ± 34.2 g, p = 0.039) and 600 nmol/kg/d oxytocin (-91.8 ± 32.2 g, p = 0.035) relative to vehicle (706.9 ± 28.3 g), indicating a stronger dose response for ASK1476. Likewise, both ASK1476 and oxytocin at these doses resulted in similar reductions in 28-day cumulative food intake (ASK1476: -562.7 ± 115.0 kcal, p = 0.0001; oxytocin: -557.1 ± 101.3 kcal, p = 0.0001) relative to vehicle treatment (2716 ± 75.4 kcal), while no effects were detected on locomotor activity or body temperature. Conclusion: This study provides proof-of-concept data demonstrating an oxytocin analog with extended in vivo stability and improved potency to reduce food intake and body weight in DIO animals which could mark a new avenue in anti-obesity drug interventions.

Pharmacological characterization of mutant huntingtin aggregate-directed PET imaging tracer candidates.

Huntington's disease (HD) is caused by a CAG trinucleotide repeat expansion in the first exon of the huntingtin (HTT) gene coding for the huntingtin (HTT) protein. The misfolding and consequential aggregation of CAG-expanded mutant HTT (mHTT) underpin HD pathology. Our interest in the life cycle of HTT led us to consider the development of high-affinity small-molecule binders of HTT oligomerized/amyloid-containing species that could serve as either cellular and in vivo imaging tools or potential therapeutic agents. We recently reported the development of PET tracers CHDI-180 and CHDI-626 as suitable for imaging mHTT aggregates, and here we present an in-depth pharmacological investigation of their binding characteristics. We have implemented an array of in vitro and ex vivo radiometric binding assays using recombinant HTT, brain homogenate-derived HTT aggregates, and brain sections from mouse HD models and humans post-mortem to investigate binding affinities and selectivity against other pathological proteins from indications such as Alzheimer's disease and spinocerebellar ataxia 1. Radioligand binding assays and autoradiography studies using brain homogenates and tissue sections from HD mouse models showed that CHDI-180 and CHDI-626 specifically bind mHTT aggregates that accumulate with age and disease progression. Finally, we characterized CHDI-180 and CHDI-626 regarding their off-target selectivity and binding affinity to beta amyloid plaques in brain sections and homogenates from Alzheimer's disease patients.

Discovery of novel leukotriene A4 hydrolase inhibitors based on piperidine and piperazine scaffolds.

Novel piperidine and piperazine derivatives have been designed and tested as inhibitors of LTA(4) hydrolase (LTA(4)H). Most potent compounds showed good potency in both enzymatic and functional human whole blood assay. Crystallography studies further confirmed observed structure-activity relationship and LTA(4)H binding mode for analogs from the piperidine series.

1,7-Disubstituted oxyindoles are potent and selective EP(3) receptor antagonists.

A series of novel 1,7-disubstituted oxyindoles were shown to be potent and selective EP(3) receptor antagonists. Variation of substitution pattern at the C-3 position of indole enhanced in vitro metabolic stability of the resulting derivatives. Series 27a-c showed >1000-fold selectivity over a panel of prostanoid receptors including IP, FP, EP(1), EP(2) and EP(4). These agents also featured low CYP inhibition and good activity in the functional rat platelet aggregation assay.

Peri-substituted hexahydro-indolones as novel, potent and selective human EP3 receptor antagonists.

A series of peri-substituted [4.3.0] bicyclic non-aromatic heterocycles have been identified as potent and selective hEP(3) receptor antagonists. These molecules adopt a hair-pin conformation that overlaps with the endogenous ligand PGE(2) and fits into an internally generated EP(3) pharmacophore model. Optimized compounds show good metabolic stability and improved solubility over their corresponding bicyclic aromatic analogs.

3,4-Disubstituted indole acylsulfonamides: a novel series of potent and selective human EP3 receptor antagonists.

A series of potent and selective EP(3) receptor antagonists are described. Utilizing a pharmacophore model developed for the EP(3) receptor, a series of 3,4-disubstituted indoles were shown to be high affinity ligands for this target. These compounds showed high selectivity over IP, FP and other EP receptors and are potent antagonists in functional assays.

3-Acrylamide-4-aryloxyindoles: synthesis, biological evaluation and metabolic stability of potent and selective EP3 receptor antagonists.

A series of potent and selective EP(3) receptor antagonists are described. Utilizing a pharmacophore model developed for the EP(3) receptor, a series of 3,4-disubstituted indoles were found to be efficient ligands for this target. These compounds showed high selectivity over IP, FP and other EP receptors. An optimized molecule 7c featured a sound profile and potency in the functional rat and human platelet aggregation assays.

Synthesis and structural assignment of two major metabolites of the LTA4H inhibitor DG-051.

The same two major CYP mediated metabolites of DG-051 were produced in the presence of rat, dog, monkey and human liver microsomes. Their respective structures were hypothesized based on mass spectrometry data correlated with the parent structure and confirmed by comparison with authentic synthetic samples. The number of regioisomers synthesized as candidates for metabolite M1 was narrowed down using a metabolic study of a selectively deuterated DG-051 analogue.

Heterocyclic 1,7-disubstituted indole sulfonamides are potent and selective human EP3 receptor antagonists.

We have developed a pharmacophore model for the EP(3) receptor antagonists based on its endogenous ligand PGE(2). This ligand-based design yielded a series of novel peri-substituted [4.3.0] bicyclic aromatics featuring 1-alklyaryl 7-heterocyclic sulfonamide substituents. The synthesized molecules are potent antagonists of human EP(3) receptor in vitro and show inhibition of rat platelets aggregation. Optimized derivatives display high selectivity over IP, FP, and other EP receptor panels.

Benzimidazolyl-pyrazolo[3,4-b]pyridinones, Selective Inhibitors of MOLT-4 Leukemia Cell Growth and Sea Urchin Embryo Spiculogenesis: Target Quest.

1,3-Substituted pyrazolo[3,4-b]pyridinones 11-18 were synthesized by a three-component condensation of Meldrum's acid with aryl aldehydes and 1,3-substituted 5-aminopyrazoles. Their biological activity was evaluated using the in vivo phenotypic sea urchin embryo assay and the in vitro cytotoxicity screen against human cancer cell lines. In the sea urchin embryo model, 1-benzimidazolyl-pyrazolo[3,4-b]pyridinones 11 caused inhibition of hatching and spiculogenesis at sub-micromolar concentrations. These compounds also selectively and potently inhibited growth of the MOLT-4 leukemia cell line. Subsequent structure-activity relationship studies determined the benzimidazolyl fragment as an essential pharmacophore for both effects. We applied numerous techniques for target identification. A preliminary QSAR target identification search did not result in tangible leads. Attempts to prepare a relevant photoaffinity probe that retained potency in both assays were not successful. Compounds 11 were further characterized for their activity in a wild-type versus Notch-mutant leukemia cell lines, and in in vitro panels of kinases and matrix metalloproteinases. Using a series of diverse modulators of spiculogenesis as standards, we excluded multiple signaling networks including Notch, Wnt/ß-catenin, receptor tyrosine kinases (VEGF/VEGFR, FGF/FGFR), PI3K, and Raf-MEK-ERK as possible targets of 11. On the other hand, matrix metalloproteinase-9/hatching enzyme was identified as one potential target.