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Showing papers by "Torrey Pines Institute for Molecular Studies published in 2017"


Journal ArticleDOI
TL;DR: Mechanistic studies have begun to elucidate how divergent mechanisms that regulate tolerance are controlled, and single-cell antibody cloning has revealed defects of B cell central tolerance in human autoimmune diseases and in several human immunodeficiency diseases caused by single gene mutations.
Abstract: Receptor editing and apoptosis have crucial roles in promoting the central tolerance of B cells to self-antigens. Defects of these processes can result in autoimmunity or immunodeficiency disease in humans and mice.

292 citations


Journal ArticleDOI
TL;DR: The most heritable oral bacteria were not associated with caries state, did not tend to co-occur with other taxa, and decreased in abundance with age and sugar consumption frequency, suggesting cariogenic taxa are likely not controlled by genetic factors.

149 citations


Journal ArticleDOI
TL;DR: The strategies that led to the development of biased μ-OR agonists, and potential areas for improvement, are discussed, with an emphasis on structural aspects of the ligand-receptor recognition process.

81 citations


Journal ArticleDOI
TL;DR: AVB-620 imaging visualizes primary tumors and demonstrated high in vivo diagnostic sensitivity and specificity for identifying breast cancer metastases to lymph nodes in two immunocompetent syngeneic mouse models and a safety profile that has enabled it to advance into clinical evaluation in breast cancer patients.
Abstract: With the goal of improving intraoperative cancer visualization, we have developed AVB-620, a novel intravenously administered, in vivo fluorescent peptide dye conjugate that highlights malignant tissue and is optimized for human use. Matrix metalloproteinases (MMPs) hydrolyze AVB-620 triggering tissue retention and a ratiometric fluorescence color change which is visualized using camera systems capable of imaging fluorescence and white light simultaneously. AVB-620 imaging visualizes primary tumors and demonstrated high in vivo diagnostic sensitivity and specificity (both >95%) for identifying breast cancer metastases to lymph nodes in two immunocompetent syngeneic mouse models. It is well tolerated and single-dose toxicology studies in rats determined a no-observed-adverse-effect-level (NOAEL) at >110-fold above the imaging and estimated human dose. Protease specificity and hydrolysis kinetics were characterized and compared using recombinant MMPs. To understand the human translation potential, an in vitro diagnostic study was conducted to evaluate the ability of AVB-620 to differentiate human breast cancer tumor from healthy adjacent tissue. Patient tumor tissue and healthy adjacent breast tissue were homogenized, incubated with AVB-620, and fluorogenic responses were compared. Tumor tissue had 2-3 fold faster hydrolysis than matched healthy breast tissue; generating an assay sensitivity of 96% and specificity of 88%. AVB-620 has excellent sensitivity and specificity for identifying breast cancer in mouse and human tissue. Significant changes were made in the design of AVB-620 relative to previous ratiometric protease-activated agents. AVB-620 has pharmaceutical properties, fluorescence ratio dynamic range, usable diagnostic time window, a scalable synthesis, and a safety profile that have enabled it to advance into clinical evaluation in breast cancer patients.

65 citations


Journal ArticleDOI
TL;DR: Evidence from phase 3 clinical trials show re-ductions in glycated hemoglobin with a low risk of hypoglycemia except when used with insulin or insulin secretagogues, and moderate reductions in body weight and systolic blood pressure, supporting the use of empagliflozin as mono-therapy or in addition to other glucose-lowering agents.
Abstract: Introduction: Sodium glucose cotransporter 2 (SGLT2) inhibitors have a unique mecha-nism of action leading to excretion of glucose in the urine and subsequent lowering of plasma glu-cose. This mechanism is independent of β-cell function; thus, these agents are effective treatment for type 2 diabetes mellitus (T2DM) at theoretically any disease stage. This class should not confer an additional risk of hypoglycemia (unless combined with insulin or an insulin secretagogue) and has the potential to be combined with other classes of glucose-lowering agents. Empagliflozin is one of three currently approved SGLT2 inhibitors in the United States, and has shown a favorable benefit-risk ratio in phase 3 clinical trials as monotherapy and as add-on to other glucose-lowering therapy in broad patient populations. In addition to its glucose-lowering effects, empagliflozin has been shown to reduce body weight and blood pressure without a compensatory increase in heart rate. Moreover, on top of standard of care, empagliflozin is the first glucose-lowering agent to demonstrate cardiovas-cular risk reduction in patients at high risk of cardiovascular disease in a prospective outcomes trial: a 14% reduction in risk of the 3-point composite endpoint of death from cardiovascular causes, nonfa-tal myocardial infarction, or nonfatal stroke. Like other SGLT2 inhibitors, empagliflozin is associated with a higher rate of genital mycotic infections than placebo and has the potential for volume deple-tion–associated events.

63 citations


Journal ArticleDOI
TL;DR: It is hypothesized that brain‐penetrant κ receptor ligands possessing biased agonism towards G protein signalling over β‐arrestin2 recruitment would produce robust antinociception with fewer associated liabilities.
Abstract: Background and purpose The κ opioid (KOP) receptor has a central role in modulating neurotransmission in central and peripheral neuronal circuits that subserve pain and other behavioural responses. Although KOP agonists do not produce euphoria or lead to respiratory suppression, they induce dysphoria and sedation. We hypothesized that brain-penetrant KOP receptor ligands possessing biased agonism towards G protein signalling over β-arrestin2 recruitment would produce robust antinociception with fewer liabilities of use. Experimental approach Two new diphenethylamines with high KOP receptor selectivity, HS665 and HS666, were assessed following i.c.v. administration in mouse assays of antinociception with the 55°C warm-water tail-withdrawal test, locomotor activity in the rotorod and conditioned place preference. The [35S]GTPγS binding and β-arrestin2 recruitment in vitro assays were used to characterize biased agonism. Key results HS665 (KOP agonist) and HS666 (KOP partial agonist) demonstrated dose-dependent antinociception after i.c.v. administration mediated by the KOP receptor. We found that the two highly selective KOP ligands display in vitro varying bias signalling toward G protein coupling consistent with a reduced liability profile reflected by the reduced sedation and absence of conditioned place aversion for HS666. Conclusions and implications HS665 and HS666 activate central KOP receptors to produce potent antinociception, with HS666 displaying pharmacological characteristics of a KOP analgesic with reduced liability for aversive and dysphoric effects correlating to its low efficacy in the β-arrestin2 signalling pathways. Our data provide further understanding of the contribution of activation of central KOP receptors in pain suppression, and the prospect of dissociating the antinociceptive effects of HS665 and HS666 from KOP-mediated adverse effects.

54 citations


Journal ArticleDOI
TL;DR: This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.
Abstract: The ability to rewrite large stretches of genomic DNA enables the creation of new organisms with customized functions. However, few methods currently exist for accumulating such widespread genomic changes in a single organism. In this study, we demonstrate a rapid approach for rewriting bacterial genomes with modified synthetic DNA. We recode 200 kb of the Salmonella typhimurium LT2 genome through a process we term SIRCAS (stepwise integration of rolling circle amplified segments), towards constructing an attenuated and genetically isolated bacterial chassis. The SIRCAS process involves direct iterative recombineering of 10-25 kb synthetic DNA constructs which are assembled in yeast and amplified by rolling circle amplification. Using SIRCAS, we create a Salmonella with 1557 synonymous leucine codon replacements across 176 genes, the largest number of cumulative recoding changes in a single bacterial strain to date. We demonstrate reproducibility over sixteen two-day cycles of integration and parallelization for hierarchical construction of a synthetic genome by conjugation. The resulting recoded strain grows at a similar rate to the wild-type strain and does not exhibit any major growth defects. This work is the first instance of synthetic bacterial recoding beyond the Escherichia coli genome, and reveals that Salmonella is remarkably amenable to genome-scale modification.

54 citations


Journal ArticleDOI
TL;DR: It is proposed that the induction of neurotoxicity by Aβ oligomers is a reversible process, which has important implications for the development of AD therapies.
Abstract: Alzheimer's disease (AD) is characterized by the accumulation of extracellular amyloid β-protein (Aβ) and intracellular hyperphosphorylated tau proteins. Recent evidence suggests that soluble Aβ oligomers elicit neurotoxicity and synaptotoxicity, including tau abnormalities, and play an initiating role in the development of AD pathology. In this study, we focused on the unclarified issue of whether the neurotoxicity of Aβ oligomers is a reversible process. Using a primary neuron culture model, we examined whether the neurotoxic effects induced by 2-day treatment with Aβ42 oligomers (Aβ-O) are reversible during a subsequent 2-day withdrawal period. Aβ-O treatment resulted in activation of caspase-3 and eIF2α, effects that were considerably attenuated following Aβ-O removal. Immunocytochemical analyses revealed that Aβ-O induced aberrant phosphorylation and caspase-mediated cleavage of tau, both of which were mostly reversed by Aβ-O removal. Furthermore, Aβ-O caused intraneuronal dislocation of β-catenin protein and a reduction in its levels, and these alterations were partially reversed upon Aβ-O withdrawal. The dislocation of β-catenin appeared to reflect synaptic disorganization. These findings indicate that removal of extracellular Aβ-O can fully or partially reverse Aβ-O-induced neurotoxic alterations in our neuron model. Accordingly, we propose that the induction of neurotoxicity by Aβ oligomers is a reversible process, which has important implications for the development of AD therapies.

45 citations


Journal ArticleDOI
TL;DR: It is shown that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice, which establishes BET inhibition as a promising antifungal therapeutic strategy and identifies BDF1 as an antIfungal drug target that can be selectively inhibited without antagonizing human BET function.
Abstract: Invasive fungal infections cause significant morbidity and mortality among immunocompromised individuals, posing an urgent need for new antifungal therapeutic strategies. Here we investigate a chromatin-interacting module, the bromodomain (BD) from the BET family of proteins, as a potential antifungal target in Candida albicans, a major human fungal pathogen. We show that the BET protein Bdf1 is essential in C. albicans and that mutations inactivating its two BDs result in a loss of viability in vitro and decreased virulence in mice. We report small-molecule compounds that inhibit C. albicans Bdf1 with high selectivity over human BDs. Crystal structures of the Bdf1 BDs reveal binding modes for these inhibitors that are sterically incompatible with the human BET-binding pockets. Furthermore, we report a dibenzothiazepinone compound that phenocopies the effects of a Bdf1 BD-inactivating mutation on C. albicans viability. These findings establish BET inhibition as a promising antifungal therapeutic strategy and identify Bdf1 as an antifungal drug target that can be selectively inhibited without antagonizing human BET function.

37 citations



Journal ArticleDOI
TL;DR: A novel role of Reg3g as an immunosuppressive promoter that weakens tumor-specific antigenicity and suppresses antitumor effects of CD8+ T cells in a murine model of pancreatic cancer is revealed.
Abstract: Reg3g is a potential risk for pancreatic ductal adenocarcinoma (PDAC). We previously demonstrated that Reg3g promoted pancreatic carcinogenesis via a STAT3 signaling pathway in a murine model of chronic pancreatitis. Whether the immune response is involved in tumorigenesis induced by Reg3g remains unknown. In this study, Reg3g-regulated tumor immunity was evaluated in tumor-implanted murine models, immune cells, and tumor microenvironment. In mice that had been orthotopically or ectopically implanted with Panc02 cells, Reg3g overexpression increased EGFR and Ki67, diminished MHC-I and caspase-3 expression, and accelerated growth of tumors. By interacting with PD-1/PD-L1, Reg3g also promoted differentiation of Tregs and recruitment of MDSC, retarded maturation of DCs and inactivation of CD8+ T cells, and suppressed cross-priming of CD8+ T-cell responses by DCs in tumor-bearing mice. Knockdown of Reg3g delayed tumor development in normal mice, but not in CD8+ T-cell-deficient mice. In vitro, Reg3g upregulated EGFR in DCs, activated heme oxygenase-1 (Hmox1) involved JAK2/STAT3 signaling, raised levels of Th2 cytokines in and suppressed maturation of DCs, and enhanced tumor cell proliferation. These results reveal a novel role of Reg3g as an immunosuppressive promoter that weakens tumor-specific antigenicity and suppresses antitumor effects of CD8+ T cells in a murine model of pancreatic cancer. Reg3g produces these effects by activating the JAK2/STAT3 signaling pathway in DCs, triggering the generation of an immunosuppressive tumor microenvironment.

Journal ArticleDOI
TL;DR: It is demonstrated that NOD but not diabetes‐resistant mice developed anti‐Env autoantibodies that increase in titer as disease progresses, and abnormal ERV activation and secretion of MVs may induce anti‐retroviral responses to trigger autoimmunity.
Abstract: Secreted microvesicles (MVs) are potent inflammatory triggers that stimulate autoreactive B and T cells, causing Type 1 Diabetes in non-obese diabetic (NOD) mice. Proteomic analysis of purified MVs released from islet cells detected the presence of endogenous retrovirus (ERV) antigens, including Env and Gag sequences similar to the well-characterized murine leukemia retroviruses. This raises the possibility that ERV antigens may be expressed in the pancreatic islets via MV secretion. Using virus-like particles produced by co-expressing ERV Env and Gag antigens, and a recombinant gp70 Env protein, we demonstrated that NOD but not diabetes-resistant mice developed anti-Env autoantibodies that increase in titer as disease progresses. A lentiviral-based RNA interference knockdown of Gag revealed that Gag contributes to the MV-induced T-cell response, whose diabetogenic function can be demonstrated via cell-transfer into immune-deficient mice. Finally, we observed that Gag and Env are expressed in NOD islet-derived primary mesenchymal stem cells (MSCs). However, MSCs derived from the islets of diabetes-resistant mice do not express the antigens. Taken together, abnormal ERV activation and secretion of MVs may induce anti-retroviral responses to trigger autoimmunity.

Journal ArticleDOI
TL;DR: Improvements are sought in the first generation of triple-helical peptide inhibitors by enhancing thermal stability and selectivity by redesigned to incorporate non-native amino acids, resulting in an increase of 18 °C in thermal stability.
Abstract: The design of selective matrix metalloproteinase (MMP) inhibitors that also possess favorable solubility properties has proved to be especially challenging. A prior approach using collagen-model templates combined with transition state analogs produced a first generation of triple-helical peptide inhibitors (THPIs) that were effective in vitro against discrete members of the MMP family. These THPI constructs were also highly water-soluble. The present study sought improvements in the first generation THPIs by enhancing thermal stability and selectivity. A THPI selective for MMP-2 and MMP-9 was redesigned to incorporate non-native amino acids (Flp and mep), resulting in an increase of 18 °C in thermal stability. This THPI was effective in vivo in a mouse model of multiple sclerosis, reducing clinical severity and weight loss. Two other THPIs were developed to be more selective within the collagenolytic members of the MMP family. One of these THPIs was serendipitously more effective against MMP-8 than MT1-M...

Journal ArticleDOI
TL;DR: In this paper, the authors examined the effects on QOL-relevant psychosocial measures of a widely available commercial weight loss program enhanced for individuals with Type 2 diabetes mellitus.
Abstract: Aims Type 2 diabetes mellitus (T2DM) can substantially decrease quality of life (QOL). This study examined the effects on QOL-relevant psychosocial measures of a widely available commercial weight loss program enhanced for individuals with T2DM. Methods A year-long multi-site randomized clinical trial compared the Weight Watchers (WW) approach, supplemented with phone and email counseling with a certified diabetes educator (CDE), to brief standard diabetes nutrition counseling and education (Standard Care; SC). Participants were 400 women and 163 men (N = 279 WW; 284 SC) with T2DM [mean (± SD) HbA1c 8.32 ± 1%; BMI = 37.1 ± 5.7 kg/m2; age = 55.1 ± 9.1 years]. Psychosocial outcomes were assessed at baseline, month 6, and month 12 using a diabetes specific psychosocial measure (Diabetes Distress Scale [DDS]), Impact of Weight on Quality of Life-Lite scale (IWQOL), a generic QOL measure (SF-36), and a depression screen (PHQ-9). Results WW participants showed significantly greater improvements than did SC participants on all DDS subscales and total score and on IWQOL total score and physical function, sex life and work domains (all ps Conclusions WW enhanced for individuals with T2DM was superior to SC in improving psychosocial outcomes most specific to T2DM and obesity. Available commercial WL programs, combined with scalable complementary program-specific diabetes counseling, may have benefits that extend to diabetes-related distress and weight-relevant QOL.

Journal ArticleDOI
TL;DR: An approach is introduced to systematically characterize the structure-bioassay ac- tivity relationships in PubChem using the concept of bioassay activity landscape, which can be applied to any data set screened across multiple bioassays.
Abstract: Public compound databases annotated with biological activ- ity are increasingly being used in drug discovery programs. A promi- nent example is of such databases is PubChem. Herein, we introduce an approach to systematically characterize the structure-bioassay ac- tivity relationships in PubChem using the concept of bioassay activity landscape. This strategy is general and can be applied to any data set screened across multiple bioassays. We also present a visual represen- tation of the chemical space of an in-house data set using a recently developed web-based public tool.

Journal ArticleDOI
TL;DR: A hit-to-lead effort is described resulting in the discovery of a potent and selective class of benzimidazole-based direct AMPK activators, exemplified by 5-((5-([1,1'-biphenyl]-4-yl)-6-chloro-1H-benzo[d]imidrazol-2-yl)oxy)-2-methylbenzoic acid, 42 (MK-3903).
Abstract: AMP-activated protein kinase (AMPK) plays an essential role as a cellular energy sensor and master regulator of metabolism in eukaryotes. Dysregulated lipid and carbohydrate metabolism resulting from insulin resistance leads to hyperglycemia, the hallmark of type 2 diabetes mellitus (T2DM). While pharmacological activation of AMPK is anticipated to improve these parameters, the discovery of selective, direct activators has proven challenging. We now describe a hit-to-lead effort resulting in the discovery of a potent and selective class of benzimidazole-based direct AMPK activators, exemplified by 5-((5-([1,1′-biphenyl]-4-yl)-6-chloro-1H-benzo[d]imidazol-2-yl)oxy)-2-methylbenzoic acid, 42 (MK-3903). Compound 42 exhibited robust target engagement in mouse liver following oral dosing, leading to improved lipid metabolism and insulin sensitization in mice.

Journal ArticleDOI
TL;DR: It was found that visit activity was highly variable across individual physicians, and the observed activity metrics ranged widely as correlates to subjective work-load as measured by the task load survey.

Journal ArticleDOI
10 Mar 2017-Science
TL;DR: Using a variety of phenotypic assays and structural and functional genomics techniques, the researchers observed that the synthetic chromosomes drive biological processes just like the natural, native chromosomes.
Abstract: A core theme in synthetic biology, “understanding by creating,” inspired the effort to generate the first synthetic cell, JCVI-Syn1.0 ( 1 ). The project Sc2.0 is elevating this concept by attempting to create a synthetic version of a more evolved organism, Saccharomyces cerevisiae , a eukaryotic single-celled yeast. In a set of papers in this issue ( 2 – 8 ), scientists of the Sc2.0 project who previously constructed a single yeast chromosome ( 9 ) now report constructing five additional yeast chromosomes (more than one-third of the entire genome) (see the photo). Using a variety of phenotypic assays and structural and functional genomics techniques, the researchers observed that the synthetic chromosomes drive biological processes just like the natural, native chromosomes.

Journal ArticleDOI
TL;DR: It is argued that abnormal release of exosomes might be the trigger of the early inflammatory and autoimmune responses in the islets, particularly those containing endogenous retroviral antigens that can stimulate innate and adaptive immune responses.
Abstract: The initial autoimmune trigger of type 1 diabetes (T1D) remains unclear. In non-obese diabetic (NOD) mice, islet inflammation starts early in life, suggesting the presence of an endogenous trigger for the spontaneous autoimmune response in this T1D mouse model. In this review, we argue that abnormal release of exosomes might be the trigger of the early inflammatory and autoimmune responses in the islets. Exosomes are nano-sized membrane complexes that are secreted by cells following fusion of late endosomes and/or multivesicular bodies with the plasma membrane. They are known extracellular messengers, communicating among neighboring cells via transporting large molecules from parent cells to recipient cells. Recent evidence demonstrates that these extracellular vesicles can modulate immune responses. It has been shown that insulinoma and islet mesenchymal stem cell-released exosomes are potent immune stimuli that can induce autoreactive B and T cells. Searching for candidate antigens in the exosomes identified endogenous retrovirus (ERV) Env and Gag antigens, which are homologous to an endogenous murine leukemia retrovirus. Autoantibodies and autoreactive T cells spontaneously developed in NOD mice can react to these retroviral antigens. More importantly, expression of the retroviral antigens in the islet mesenchymal stem cells is associated with disease susceptibility, and the expression is restricted to T1D-susceptible but not resistant mouse strains. Exosomes are novel autoimmune targets, carrying autoantigens that can stimulate innate and adaptive immune responses. An abnormal or excess release of exosomes, particularly those ones containing endogenous retroviral antigens might be responsible for triggering tissue-specific inflammatory and autoimmune responses.

Journal ArticleDOI
TL;DR: Results indicate that mixed opioid receptor partial agonists can produce potent antinociceptive activity with a lack of aversion in mice and without being self-administered in rats.
Abstract: Opiates are still the most effective and widely used treatments for acute and chronic pain. However, the problems associated with morphine and other standard opioid analgesics severely limit their effectiveness in the clinic. PPL-101 and PPL-103 derived from morphine and morphinan ring systems contain a chiral N-substituent, which confers it with a unique combination of high-binding affinities and partial agonist activities at mu, delta, and kappa opioid receptors, leading to unique in vivo pharmacology compared to other conventional opioids. Acute antinociceptive and reward acquisition of PPL-101 and PPL-103 were assessed in mice using the tail flick assay and conditioned place preference (CPP) paradigm, respectively. The reinforcing effects of these compounds were assessed in rats using the self-administration paradigm. In mice, PPL-101 and PPL-103 produced antinociception reaching maximal effects that were equivalent to morphine at approximately 1/3 and 1/10 of morphine's dose, respectively. PPL-101-induced antinociception was attenuated following pretreatment with the kappa antagonist JDTic, but not the mu opioid antagonist beta-FNA. In mice, PPL-101 and PPL-103 produced dose-dependent decreases in activity, similar to other kappa agonists; however, they did not produce conditioned place aversion, and in fact elicited a trend toward CPP. In rats, neither PPL-101 nor PPL-103 were self-administered when substituted for morphine and PPL-101 attenuated morphine self-administration, when administered systemically prior to the self-administration session. Collectively, these results indicate that mixed opioid receptor partial agonists can produce potent antinociceptive activity with a lack of aversion in mice and without being self-administered in rats. Compounds with this profile could be superior analgesics with greatly reduced addiction liability and fewer side-effects compared to traditional opiates.

Journal ArticleDOI
TL;DR: The ANTISTAPHYBASE database contains 596 sequences of antimicrobial peptides produced by diverse organisms and 287 essential oil records, which permits a quick and easy search of peptides based on their activity as well as their general, physicochemical properties and literature data.
Abstract: Staphylococcus aureus and methicillin-resistant S. aureus are major pathogens. The antimicrobial peptides and essential oils (EOs) display narrow- or broad-spectrum activity against bacteria including these strains. A centralized resource, such as a database, designed specifically for anti-S. aureus/anti-methicillin-resistant S. aureus antimicrobial peptides and EOs is therefore needed to facilitate the comprehensive investigation of their structure/activity associations and combinations. The database ANTISTAPHYBASE is created to facilitate access to important information on antimicrobial peptides and essential peptides against methicillin-resistant S. aureus and S. aureus. At the moment, the database contains 596 sequences of antimicrobial peptides produced by diverse organisms and 287 essential oil records. It permits a quick and easy search of peptides based on their activity as well as their general, physicochemical properties and literature data. These data are very useful to perform further bioinformatic or chemometric analysis and would certainly be useful for the development of new drugs for medical use. The ANTISTAPHYBASE database is freely available at: https://www.antistaphybase.com/ .

Journal ArticleDOI
07 Mar 2017-Vaccine
TL;DR: The prevalence of V3C-type and V3L-type Ab responses in HIV-infected individuals and in HIV envelope-immunized humans and animals is analyzed and the need for improving immunogen designs and vaccination strategies to broaden the diversity of Abs in order to target the different conserved epitopes in the V3 loop and, by extension, in the entire HIV envelope is implied.

Journal ArticleDOI
TL;DR: A novel tetrapeptide scaffold that contains a sequentially reversed "Arg-(pI)DPhe" motif with respect to the classical "Phe-Arg" melanocortin signaling motif results in pharmacology that is first-in-class for the central melanoc Cortin receptors.
Abstract: The centrally expressed melanocortin-3 and -4 receptors (MC3R/MC4R) have been studied as possible targets for weight management therapies, with a preponderance of studies focusing on the MC4R. Herein, a novel tetrapeptide scaffold [Ac-Xaa1-Arg-(pI)DPhe-Xaa4-NH2] is reported. The scaffold was derived from results obtained from a MC3R mixture-based positional scanning campaign. From these results, a set of 48 tetrapeptides were designed and pharmacologically characterized at the mouse melanocortin-1, -3, -4, and -5 receptors. This resulted in the serendipitous discovery of nine compounds that were MC3R agonists (EC50 < 1000 nM) and MC4R antagonists (5.7 < pA2 < 7.8). The three most potent MC3R agonists, 18 [Ac-Arg-Arg-(pI)DPhe-Tic-NH2], 1 [Ac-His-Arg-(pI)DPhe-Tic-NH2], and 41 [Ac-Arg-Arg-(pI)DPhe-DNal(2′)-NH2] were more potent (EC50 < 73 nM) than the melanocortin tetrapeptide Ac-His-DPhe-Arg-Trp-NH2. This template contains a sequentially reversed “Arg-(pI)DPhe” motif with respect to the classical “Phe-Arg” ...

Journal ArticleDOI
26 May 2017-PLOS ONE
TL;DR: The results describe a culture method for isolating T. cruzi specific T cell clones from patients with Chagas disease, which enable the acquisition of information on functionality and specificity of individual T cells.
Abstract: Fil: Acevedo, Gonzalo Raul Consejo Nacional de Investigaciones Cientificas y Tecnicas Instituto de Investigaciones en Ingenieria Genetica y Biologia Molecular "Dr Hector N Torres"; Argentina

Journal ArticleDOI
TL;DR: Collectively, these studies suggest that an RARα specific agonist may afford a new strategy for lipid-lowering and CVD risk reduction.
Abstract: Elevated triglyceride (TG) levels are well-correlated with the risk for cardiovascular disease (CVD). Apolipoprotein CIII (ApoC-III) is a key regulator of plasma TG levels through regulation of lipolysis and lipid synthesis. To identify novel regulators of TG levels, we carried out a high throughput screen (HTS) using an ApoC-III homogenous time resolved fluorescence (HTRF) assay. We identified several retinoic acid receptor (RAR) agonists that reduced secreted ApoC-III levels in human hepatic cell lines. The RARα specific agonist AM580 inhibited secreted ApoC-III by >80% in Hep3B cells with an EC50 ~2.9 nM. In high-fat diet induced fatty-liver mice, AM580 reduced ApoC-III levels in liver as well as in plasma (~60%). In addition, AM580 treatment effectively reduced body weight, hepatic and plasma TG, and total cholesterol (TC) levels. Mechanistically, AM580 suppresses ApoC-III synthesis by downregulation of HNF4α and upregulation of SHP1 expression. Collectively, these studies suggest that an RARα specific agonist may afford a new strategy for lipid-lowering and CVD risk reduction.

Journal ArticleDOI
TL;DR: In vivo studies show that 5 can significantly reduce operant nicotine self-administration and nicotine relapse-like behavior in rats at doses of 0.3 and 1 mg/kg, and pharmacokinetic data indicate that 5, via sc administration, is rapidly absorbed into the blood, reaching maximal concentration within 10 min with a half-life of less than 1 h.
Abstract: The α4β2 nAChR is the most predominant subtype in the brain and is a well-known culprit for nicotine addiction. Previously we presented a series of α4β2 nAChR selective compounds that were discovered from a mixture-based positional-scanning combinatorial library. Here we report further optimization identified highly potent and selective α4β2 nAChR antagonists 5 (AP-202) and 13 (AP-211). Both compounds are devoid of in vitro agonist activity and are potent inhibitors of epibatidine-induced changes in membrane potential in cells containing α4β2 nAChR, with IC50 values of approximately 10 nM, but are weak agonists in cells containing α3β4 nAChR. In vivo studies show that 5 can significantly reduce operant nicotine self-administration and nicotine relapse-like behavior in rats at doses of 0.3 and 1 mg/kg. The pharmacokinetic data also indicate that 5, via sc administration, is rapidly absorbed into the blood, reaching maximal concentration within 10 min with a half-life of less than 1 h.

Journal ArticleDOI
16 Nov 2017-PLOS ONE
TL;DR: Rhesus θ-defensin 1 (RTD-1) is highly effective in arresting and reversing joint disease in a rodent model of rheumatoid arthritis (RA) and has potential as retroevolutionary biologics for the treatment of RA.
Abstract: θ-defensins constitute a family of macrocyclic peptides expressed exclusively in Old World monkeys. The peptides are pleiotropic effectors of innate immunity, possessing broad spectrum antimicrobial activities and immunoregulatory properties. Here we report that rhesus θ-defensin 1 (RTD-1) is highly effective in arresting and reversing joint disease in a rodent model of rheumatoid arthritis (RA). Parenteral RTD-1 treatment of DA/OlaHsd rats with established pristane-induced arthritis (PIA) rapidly suppressed joint disease progression, restored limb mobility, and preserved normal joint architecture. RTD-1 significantly reduced joint IL-1β levels compared with controls. RTD-1 dose-dependently inhibited fibroblast-like synoviocyte (FLS) invasiveness and FLS IL-6 production. Consistent with the inhibition of FLS invasiveness, RTD-1 was a potent inhibitor of arthritogenic proteases including ADAMs 17 and 10 which activate TNFα, and inhibited matrix metalloproteases, and cathepsin K. RTD-1 was non-toxic, non-immunogenic, and effective when administered as infrequently as once every five days. Thus θ-defensins, which are absent in humans, have potential as retroevolutionary biologics for the treatment of RA.

Journal ArticleDOI
24 Jan 2017
TL;DR: In this paper, two fluorescent calcium indicator dyes, FLIPR Calcium 6 and Cal-520 AM, were compared for drug-screening hit-selection; difference in average (unstandardized) and standardized difference.
Abstract: Calcium (Ca2+) plays a central role in regulating many biological processes in the cell from muscle contraction to neurotransmitter release. The need for reliable fluorescent calcium indicator dyes is of vast importance for studying many aspects of cell biology as well as screening compounds using phenotypic high throughput assays. We have assessed two of the latest generation of calcium indicator dyes, FLIPR Calcium 6 and Cal-520 AM for studying calcium transients (CaTs) in induced pluripotent stem cell (iPSC) -derived human cardiomyocytes. FLIPR Calcium 6 and Cal-520 dyes both displayed robust CaTs with a high signal-to-noise ratio (SNR) and were non-toxic to the cells. The analysis showed that CaT amplitudes were stable between measurements, but CaT duration was more variable and tended to increase between reads. Two methods were compared for drug-screening hit-selection; difference in average (unstandardized) and standardized difference. The unstandardized difference was better for assessing CaT amplitude, whereas standardized difference was equal to or better for assessing CaT duration. In summary, FLIPR Calcium 6 and Cal-520 are suitable dyes for drug-screening using iPSC-derived human cardiomyocytes.

Journal ArticleDOI
TL;DR: It is demonstrated that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multifunctional antibodies for therapeutic applications.
Abstract: We report a method to generate bifunctional antibodies by grafting full-length proteins into constant region loops of a full-length antibody or an antigen-binding fragment (Fab). The fusion proteins retain the antigen binding activity of the parent antibody but have an additional activity associated with the protein insert. The engineered antibodies have excellent in vitro activity, physiochemical properties, and stability. Among these, a Her2 × CD3 bispecific antibody (BsAb) was constructed by inserting an anti-Her2 single-chain variable fragment (ScFv) into an anti-CD3 Fab. This bispecific antibody efficiently induces targeted cell lysis in the presence of effector cells at as low as sub-picomolar concentrations in vitro. Moreover, the Her2 × CD3 BsAb shows potent in vivo antitumor activity in mouse Her22+ and Her21+ xenograft models. These results demonstrate that insertion of a full-length protein into non-CDR loops of antibodies provides a feasible approach to generate multifunctional antibodies for therapeutic applications.

Journal ArticleDOI
TL;DR: The reported data demonstrate the feasibility of designing lectinomimics based on cyclic peptides and show that the disulfide bond found in 1 can be replaced with a lactam bridge, however, the orientation of the lactam Bridge influenced cycling peptide‘s conformation and thus these peptides’ ability to bind carbohydrates.
Abstract: The transformation from normal to malignant phenotype in human cancers is associated with aberrant cell-surface glycosylation. Thus, targeting glycosylation changes in cancer is likely to provide not only better insight into the roles of carbohydrates in biological systems, but also facilitate the development of new molecular probes for bioanalytical and biomedical applications. In the reported study, we have synthesized lectinomimics based on odorranalectin 1; the smallest lectin-like cyclic peptide isolated from the frog Odorrana grahami skin, and assessed the ability of these peptides to bind specific carbohydrates on molecular and cellular levels. In addition, we have shown that the disulfide bond found in 1 can be replaced with a lactam bridge. However, the orientation of the lactam bridge, peptides 2 and 3, influenced cyclic peptide's conformation and thus these peptides' ability to bind carbohydrates. Naturally occurring 1 and its analog 3 that adopt similar conformation in water bind preferentially L-fucose, and to a lesser degree D-galactose and N-acetyl-D-galactosamine, typically found within the mucin O-glycan core structures. In cell-based assays, peptides 1 and 3 showed a similar binding profile to Aleuria aurantia lectin and these two peptides inhibited the migration of metastatic breast cancer cell lines in a Transwell assay. Altogether, the reported data demonstrate the feasibility of designing lectinomimics based on cyclic peptides.