Publications by authors named "Randall T Peterson"

123 Publications

The 5α-reductase inhibitor finasteride reduces opioid self-administration in animal models of opioid use disorder.

J Clin Invest 2021 May;131(10)

Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, Utah, USA.

Opioid use disorder (OUD) has become a leading cause of death in the United States, yet current therapeutic strategies remain highly inadequate. To identify potential treatments for OUD, we screened a targeted selection of over 100 drugs using a recently developed opioid self-administration assay in zebrafish. This paradigm showed that finasteride, a steroidogenesis inhibitor approved for the treatment of benign prostatic hyperplasia and androgenetic alopecia, reduced self-administration of multiple opioids without affecting locomotion or feeding behavior. These findings were confirmed in rats; furthermore, finasteride reduced the physical signs associated with opioid withdrawal. In rat models of neuropathic pain, finasteride did not alter the antinociceptive effect of opioids and reduced withdrawal-induced hyperalgesia. Steroidomic analyses of the brains of fish treated with finasteride revealed a significant increase in dehydroepiandrosterone sulfate (DHEAS). Treatment with precursors of DHEAS reduced opioid self-administration in zebrafish in a fashion akin to the effects of finasteride. These results highlight the importance of steroidogenic pathways as a rich source of therapeutic targets for OUD and point to the potential of finasteride as a new treatment option for this disorder.
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http://dx.doi.org/10.1172/JCI143990DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8121512PMC
May 2021

Discovery of a Potent Conorfamide from Using a Novel Zebrafish Larvae Assay.

J Nat Prod 2021 04 25;84(4):1232-1243. Epub 2021 Mar 25.

School of Biological Sciences, University of Utah, 257 S 1400 E, Salt Lake City, Utah 84112, United States.

Natural products such as conotoxins have tremendous potential as tools for biomedical research and for the treatment of different human diseases. Conotoxins are peptides present in the venoms of predatory cone snails that have a rich diversity of pharmacological functions. One of the major bottlenecks in natural products research is the rapid identification and evaluation of bioactive molecules. To overcome this limitation, we designed a set of light-induced behavioral assays in zebrafish larvae to screen for bioactive conotoxins. We used this screening approach to test several unique conotoxins derived from different cone snail clades and discovered that a conorfamide from , CNF-Ep1, had the most dramatic alterations in the locomotor behavior of zebrafish larvae. Interestingly, CNF-Ep1 is also bioactive in several mouse assay systems when tested and . Our novel screening platform can thus accelerate the identification of bioactive marine natural products, and the first compound discovered using this assay has intriguing properties that may uncover novel neuronal circuitry.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01297DOI Listing
April 2021

Emerimicins V-X, 15-Residue Peptaibols Discovered from an sp. through Integrated Genomic and Chemical Approaches.

J Nat Prod 2021 04 22;84(4):1113-1126. Epub 2021 Feb 22.

Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.

Fermentation of W. Gams isolated from a soil sample collected from the University of Utah led to the isolation and characterization of six new linear pentadecapeptides, emerimicins V-X (-). Peptaibols containing 15-residues are quite rare, with only 22 reported. Genome mining and bioinformatic analysis were used to identify the emerimicin 60 kbp biosynthetic cluster harboring a single 16-module hybrid polyketide-nonribosomal peptide synthetase. A detailed bioinformatic investigation of the corresponding 15 adenylation domains, combined with 1D and 2D NMR experiments, LC-MS/MS data, and advanced Marfey's method, allowed for the elucidation and absolute configuration of all proteinogenic and nonproteinogenic amino acid residues in -. As some peptaibols possess cytotoxic activity, a zebrafish embryotoxicity assay was used to evaluate the toxicity of the six emerimicins and showed that emerimicin V () and VI () exhibit the most potent activity. Additionally, out of the six emerimicins, displayed modest activity against , methicillin-resistant , and vancomycin-resistant with MIC values of 64, 32, and 64 μg/mL, respectively.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01186DOI Listing
April 2021

Chemical Genetics: Manipulating the Germline with Small Molecules.

Methods Mol Biol 2021 ;2218:61-73

College of Pharmacy, University of Utah, Salt Lake City, UT, USA.

Primordial germ cells (PGCs) are the precursor cells that form during early embryogenesis and later differentiate into oocytes or spermatozoa. Abnormal development of PGCs is frequently a causative factor of infertility and germ cell tumors. However, our understanding of PGC development remains insufficient, and we have few pharmacological tools for manipulating PGC development for biological study or therapy. The zebrafish (Danio rerio) embryos provide an excellent in vivo animal model to study PGCs, because zebrafish embryos are transparent and develop outside the mother. Importantly, the model is also amenable to facile chemical manipulations, including scalable screening to discover novel compounds that alter PGC development. This chapter describes methodologies for manipulating the germline (i.e., PGCs) with small molecules and for monitoring PGC development. Utilizing the 3'UTR of PGC marker genes such as nanos3 and ddx4/vasa is a key component of these methodologies, which consist of expressing fluorescent or luminescent proteins in PGCs, treatment with small molecules, and quantitative observation of PGC development.
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http://dx.doi.org/10.1007/978-1-0716-0970-5_6DOI Listing
March 2021

TRPswitch-A Step-Function Chemo-optogenetic Ligand for the Vertebrate TRPA1 Channel.

J Am Chem Soc 2020 10 5;142(41):17457-17468. Epub 2020 Oct 5.

Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah 84112, United States.

Chemo-optogenetics has produced powerful tools for optical control of cell activity, but current tools suffer from a variety of limitations including low unitary conductance, the need to modify the target channel, or the inability to control both on and off switching. Using a zebrafish behavior-based screening strategy, we discovered "TRPswitch", a photoswitchable nonelectrophilic ligand scaffold for the transient receptor potential ankyrin 1 (TRPA1) channel. TRPA1 exhibits high unitary channel conductance, making it an ideal target for chemo-optogenetic tool development. Key molecular determinants for the activity of TRPswitch were elucidated and allowed for replacement of the TRPswitch azobenzene with a next-generation azoheteroarene. The TRPswitch compounds enable reversible, repeatable, and nearly quantitative light-induced activation and deactivation of the vertebrate TRPA1 channel with violet and green light, respectively. The utility of TRPswitch compounds was demonstrated in larval zebrafish hearts exogenously expressing zebrafish Trpa1b, where the heartbeat could be controlled using TRPswitch and light. Therefore, TRPA1/TRPswitch represents a novel step-function chemo-optogenetic system with a unique combination of high conductance, high efficiency, activity against an unmodified vertebrate channel, and capacity for bidirectional optical switching. This chemo-optogenetic system will be particularly applicable in systems where a large depolarization current is needed or sustained channel activation is desirable.
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http://dx.doi.org/10.1021/jacs.0c06811DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011302PMC
October 2020

Efficacy of Ciprofloxacin/Celecoxib combination in zebrafish models of amyotrophic lateral sclerosis.

Ann Clin Transl Neurol 2020 10 11;7(10):1883-1897. Epub 2020 Sep 11.

The Dead Sea Arava Science Center, Auspices of Ben Gurion University, Central Arava, 86815, Israel.

Objective: To evaluate the efficacy of a fixed-dose combination of two approved drugs, Ciprofloxacin and Celecoxib, as a potential therapeutic treatment for amyotrophic lateral sclerosis (ALS).

Methods: Toxicity and efficacy of Ciprofloxacin and Celecoxib were tested, each alone and in distinct ratio combinations in SOD1 G93R transgenic zebrafish model for ALS. Quantification of swimming measures following stimuli, measurements of axonal projections from the spinal cord, neuromuscular junction structure and morphometric analysis of microglia cells were performed in the combination- treated vs nontreated mutant larvae. Additionally, quantifications of touch-evoked locomotor escape response were conducted in treated vs nontreated zebrafish expressing the TARDBP G348C ALS variant.

Results: When administered individually, Ciprofloxacin had a mild effect and Celecoxib had no therapeutic effect. However, combined Ciprofloxacin and Celecoxib (Cipro/Celecox) treatment caused a significant increase of ~ 84% in the distance the SOD1 G93R transgenic larvae swam. Additionally, Cipro/Celecox elicited recovery of impaired motor neurons morphology and abnormal neuromuscular junction structure and preserved the ramified morphology of microglia cells in the SOD1 mutants. Furthermore, larvae expressing the TDP-43 mutation displayed evoked touch responses that were significantly longer in swim distance (110% increase) and significantly higher in maximal swim velocity (~44% increase) when treated with Cipro/Celecox combination.

Interpretation: Cipro/Celecox combination improved locomotor and cellular deficits of ALS zebrafish models. These results identify this novel combination as effective, and may prove promising for the treatment of ALS.
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http://dx.doi.org/10.1002/acn3.51174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545590PMC
October 2020

A small-molecule allosteric inhibitor of BAX protects against doxorubicin-induced cardiomyopathy.

Nat Cancer 2020 Mar 2;1(3):315-328. Epub 2020 Mar 2.

Department of Medicine, Albert Einstein College of Medicine, Bronx NY, USA.

Doxorubicin remains an essential component of many cancer regimens, but its use is limited by lethal cardiomyopathy, which has been difficult to target, owing to pleiotropic mechanisms leading to apoptotic and necrotic cardiac cell death. Here we show that BAX is rate-limiting in doxorubicin-induced cardiomyopathy and identify a small-molecule BAX inhibitor that blocks both apoptosis and necrosis to prevent this syndrome. By allosterically inhibiting BAX conformational activation, this compound blocks BAX translocation to mitochondria, thereby abrogating both forms of cell death. When co-administered with doxorubicin, this BAX inhibitor prevents cardiomyopathy in zebrafish and mice. Notably, cardioprotection does not compromise the efficacy of doxorubicin in reducing leukemia or breast cancer burden in vivo, primarily due to increased priming of mitochondrial death mechanisms and higher BAX levels in cancer cells. This study identifies BAX as an actionable target for doxorubicin-induced cardiomyopathy and provides a prototype small-molecule therapeutic.
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http://dx.doi.org/10.1038/s43018-020-0039-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413180PMC
March 2020

Fetal alcohol spectrum disorder predisposes to metabolic abnormalities in adulthood.

J Clin Invest 2020 05;130(5):2252-2269

Division of Genetics, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

Prenatal alcohol exposure (PAE) affects at least 10% of newborns globally and leads to the development of fetal alcohol spectrum disorders (FASDs). Despite its high incidence, there is no consensus on the implications of PAE on metabolic disease risk in adults. Here, we describe a cohort of adults with FASDs that had an increased incidence of metabolic abnormalities, including type 2 diabetes, low HDL, high triglycerides, and female-specific overweight and obesity. Using a zebrafish model for PAE, we performed population studies to elucidate the metabolic disease seen in the clinical cohort. Embryonic alcohol exposure (EAE) in male zebrafish increased the propensity for diet-induced obesity and fasting hyperglycemia in adulthood. We identified several consequences of EAE that may contribute to these phenotypes, including a reduction in adult locomotor activity, alterations in visceral adipose tissue and hepatic development, and persistent diet-responsive transcriptional changes. Taken together, our findings define metabolic vulnerabilities due to EAE and provide evidence that behavioral changes and primary organ dysfunction contribute to resultant metabolic abnormalities.
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http://dx.doi.org/10.1172/JCI132139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7190939PMC
May 2020

Boholamide A, an APD-Class, Hypoxia-Selective Cyclodepsipeptide.

J Nat Prod 2020 04 18;83(4):1249-1257. Epub 2020 Mar 18.

Department of Medicinal Chemistry, University of Utah, Salt Lake City, Utah 84112, United States.

Calcium homeostasis is implicated in some cancers, leading to the possibility that selective control of calcium might lead to new cancer drugs. On the basis of this idea, we designed an assay using a glioblastoma cell line and screened a collection of 1000 unique bacterial extracts. Isolation of the active compound from a hit extract led to the identification of boholamide A (), a 4-amido-2,4-pentadieneoate (APD)-class peptide. Boholamide A () applied in the nanomolar range induces an immediate influx of Ca in glioblastoma and neuronal cells. APD-class natural products are hypoxia-selective cytotoxins that primarily target mitochondria. Like other APD-containing compounds, is hypoxia selective. Since APD natural products have received significant interest as potential chemotherapeutic agents, provides a novel APD scaffold for the development of new anticancer compounds.
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http://dx.doi.org/10.1021/acs.jnatprod.0c00038DOI Listing
April 2020

Isonitrile-responsive and bioorthogonally removable tetrazine protecting groups.

Chem Sci 2020 Jan 5;11(1):169-179. Epub 2019 Nov 5.

Department of Medicinal Chemistry , College of Pharmacy , University of Utah , Salt Lake City , 84112 , USA . Email:

compatible reactions have a broad range of possible applications in chemical biology and the pharmaceutical sciences. Here we report tetrazines that can be removed by exposure to isonitriles under very mild conditions. Tetrazylmethyl derivatives are easily accessible protecting groups for amines and phenols. The isonitrile-induced removal is rapid and near-quantitative. Intriguingly, the deprotection is especially effective with (trimethylsilyl)methyl isocyanide, and serum albumin can catalyze the elimination under physiological conditions. NMR and computational studies revealed that an imine-tautomerization step is often rate limiting, and the unexpected cleavage of the Si-C bond accelerates this step in the case with (trimethylsilyl)methyl isocyanide. Tetrazylmethyl-removal is compatible with use on biomacromolecules, in cellular environments, and in living organisms as demonstrated by cytotoxicity experiments and fluorophore-release studies on proteins and in zebrafish embryos. By combining tetrazylmethyl derivatives with previously reported tetrazine-responsive 3-isocyanopropyl groups, it was possible to liberate two fluorophores in vertebrates from a single bioorthogonal reaction. This chemistry will open new opportunities towards applications involving multiplexed release schemes and is a valuable asset to the growing toolbox of bioorthogonal dissociative reactions.
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http://dx.doi.org/10.1039/c9sc04649fDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012038PMC
January 2020

Cyp1 Inhibition Prevents Doxorubicin-Induced Cardiomyopathy in a Zebrafish Heart-Failure Model.

Chembiochem 2020 07 6;21(13):1905-1910. Epub 2020 Mar 6.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, 30 S 2000 E, Salt Lake City, UT, 84112, USA.

Doxorubicin is a highly effective chemotherapy agent used to treat many common malignancies. However, its use is limited by cardiotoxicity, and cumulative doses exponentially increase the risk of heart failure. To identify novel heart failure treatment targets, a zebrafish model of doxorubicin-induced cardiomyopathy was previously established for small-molecule screening. Using this model, several small molecules that prevent doxorubicin-induced cardiotoxicity both in zebrafish and in mouse models have previously been identified. In this study, exploration of doxorubicin cardiotoxicity is expanded by screening 2271 small molecules from a proprietary, target-annotated tool compound collection. It is found that 120 small molecules can prevent doxorubicin-induced cardiotoxicity, including 7 highly effective compounds. Of these, all seven exhibited inhibitory activity towards cytochrome P450 family 1 (CYP1). These results are consistent with previous findings, in which visnagin, a CYP1 inhibitor, also prevents doxorubicin-induced cardiotoxicity. Importantly, genetic mutation of cyp1a protected zebrafish against doxorubicin-induced cardiotoxicity phenotypes. Together, these results provide strong evidence that CYP1 is an important contributor to doxorubicin-induced cardiotoxicity and highlight the CYP1 pathway as a candidate therapeutic target for clinical cardioprotection.
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http://dx.doi.org/10.1002/cbic.201900741DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500981PMC
July 2020

Genetic deletion of gpr27 alters acylcarnitine metabolism, insulin sensitivity, and glucose homeostasis in zebrafish.

FASEB J 2020 01 2;34(1):1546-1557. Epub 2019 Dec 2.

Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA.

G protein-coupled receptors (GPCRs) comprise the largest group of membrane receptors in eukaryotic genomes and collectively they regulate nearly all cellular processes. Despite the widely recognized importance of this class of proteins, many GPCRs remain understudied. G protein-coupled receptor 27 (Gpr27) is an orphan GPCR that displays high conservation during vertebrate evolution. Although, GPR27 is known to be expressed in tissues that regulate metabolism including the pancreas, skeletal muscle, and adipose tissue, its functions are poorly characterized. Therefore, to investigate the potential roles of Gpr27 in energy metabolism, we generated a whole body gpr27 knockout zebrafish line. Loss of gpr27 potentiated the elevation in glucose levels induced by pharmacological or nutritional perturbations. We next leveraged a mass spectrometry metabolite profiling platform to identify other potential metabolic functions of Gpr27. Notably, genetic deletion of gpr27 elevated medium-chain acylcarnitines, in particular C6-hexanoylcarnitine, C8-octanoylcarnitine, C9-nonanoylcarnitine, and C10-decanoylcarnitine, lipid species known to be associated with insulin resistance in humans. Concordantly, gpr27 deletion in zebrafish abrogated insulin-dependent Akt phosphorylation and glucose utilization. Finally, loss of gpr27 increased the expression of key enzymes in carnitine shuttle complex, in particular the homolog to the brain-specific isoform of CPT1C which functions as a hypothalamic energy senor. In summary, our findings shed light on the biochemical functions of Gpr27 by illuminating its role in lipid metabolism, insulin signaling, and glucose homeostasis.
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http://dx.doi.org/10.1096/fj.201901466RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6956728PMC
January 2020

Parallel Reaction Monitoring reveals structure-specific ceramide alterations in the zebrafish.

Sci Rep 2019 12 27;9(1):19939. Epub 2019 Dec 27.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, Utah, USA.

Extensive characterisations of the zebrafish genome and proteome have established a foundation for the use of the zebrafish as a model organism; however, characterisation of the zebrafish lipidome has not been as comprehensive. In an effort to expand current knowledge of the zebrafish sphingolipidome, a Parallel Reaction Monitoring (PRM)-based liquid chromatography-mass spectrometry (LC-MS) method was developed to comprehensively quantify zebrafish ceramides. Comparison between zebrafish and a human cell line demonstrated remarkable overlap in ceramide composition, but also revealed a surprising lack of most sphingadiene-containing ceramides in the zebrafish. PRM analysis of zebrafish embryogenesis identified developmental stage-specific ceramide changes based on long chain base (LCB) length. A CRISPR-Cas9-generated zebrafish model of Farber disease exhibited reduced size, early mortality, and severe ceramide accumulation where the amplitude of ceramide change depended on both acyl chain and LCB lengths. Our method adds an additional level of detail to current understanding of the zebrafish lipidome, and could aid in the elucidation of structure-function associations in the context of lipid-related diseases.
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http://dx.doi.org/10.1038/s41598-019-56466-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934720PMC
December 2019

Use of Zebrafish in Drug Discovery Toxicology.

Chem Res Toxicol 2020 01 16;33(1):95-118. Epub 2019 Nov 16.

Stem Cell Program and Division of Hematology/Oncology, Children's Hospital and Dana Farber Cancer Institute, Howard Hughes Medical Institute, Harvard Medical School, Harvard Stem Cell Institute, Stem Cell and Regenerative Biology Department , Harvard University , Boston , Massachusetts 02138 , United States.

Unpredicted human safety events in clinical trials for new drugs are costly in terms of human health and money. The drug discovery industry attempts to minimize those events with diligent preclinical safety testing. Current standard practices are good at preventing toxic compounds from being tested in the clinic; however, false negative preclinical toxicity results are still a reality. Continual improvement must be pursued in the preclinical realm. Higher-quality therapies can be brought forward with more information about potential toxicities and associated mechanisms. The zebrafish model is a bridge between in vitro assays and mammalian in vivo studies. This model is powerful in its breadth of application and tractability for research. In the past two decades, our understanding of disease biology and drug toxicity has grown significantly owing to thousands of studies on this tiny vertebrate. This Review summarizes challenges and strengths of the model, discusses the 3Rs value that it can deliver, highlights translatable and untranslatable biology, and brings together reports from recent studies with zebrafish focusing on new drug discovery toxicology.
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http://dx.doi.org/10.1021/acs.chemrestox.9b00335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162671PMC
January 2020

Zebrafish behavioural profiling identifies GABA and serotonin receptor ligands related to sedation and paradoxical excitation.

Nat Commun 2019 09 9;10(1):4078. Epub 2019 Sep 9.

Institute for Neurodegenerative Diseases, University of California, San Francisco, CA, 94143, USA.

Anesthetics are generally associated with sedation, but some anesthetics can also increase brain and motor activity-a phenomenon known as paradoxical excitation. Previous studies have identified GABA receptors as the primary targets of most anesthetic drugs, but how these compounds produce paradoxical excitation is poorly understood. To identify and understand such compounds, we applied a behavior-based drug profiling approach. Here, we show that a subset of central nervous system depressants cause paradoxical excitation in zebrafish. Using this behavior as a readout, we screened thousands of compounds and identified dozens of hits that caused paradoxical excitation. Many hit compounds modulated human GABA receptors, while others appeared to modulate different neuronal targets, including the human serotonin-6 receptor. Ligands at these receptors generally decreased neuronal activity, but paradoxically increased activity in the caudal hindbrain. Together, these studies identify ligands, targets, and neurons affecting sedation and paradoxical excitation in vivo in zebrafish.
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http://dx.doi.org/10.1038/s41467-019-11936-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6733874PMC
September 2019

The zebrafish subcortical social brain as a model for studying social behavior disorders.

Dis Model Mech 2019 08 6;12(8). Epub 2019 Aug 6.

Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, 30 S. 2000 East, Salt Lake City, UT 84112, USA

Social behaviors are essential for the survival and reproduction of social species. Many, if not most, neuropsychiatric disorders in humans are either associated with underlying social deficits or are accompanied by social dysfunctions. Traditionally, rodent models have been used to model these behavioral impairments. However, rodent assays are often difficult to scale up and adapt to high-throughput formats, which severely limits their use for systems-level science. In recent years, an increasing number of studies have used zebrafish () as a model system to study social behavior. These studies have demonstrated clear potential in overcoming some of the limitations of rodent models. In this Review, we explore the evolutionary conservation of a subcortical social brain between teleosts and mammals as the biological basis for using zebrafish to model human social behavior disorders, while summarizing relevant experimental tools and assays. We then discuss the recent advances gleaned from zebrafish social behavior assays, the applications of these assays to studying related disorders, and the opportunities and challenges that lie ahead.
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http://dx.doi.org/10.1242/dmm.039446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6737945PMC
August 2019

Intramuscular administration of hexachloroplatinate reverses cyanide-induced metabolic derangements and counteracts severe cyanide poisoning.

FASEB Bioadv 2019 Feb 8;1(2):81-92. Epub 2018 Oct 8.

Department of Cardiology, Beth Israel Deaconess Medical Center, Boston, MA 02115, USA.

Cyanide is a highly toxic industrial chemical that is widely used by manufactures. Smoke inhalation during household fires is the most common source of cyanide poisoning while additional risks to civilians include industrial accidents and terrorist attacks. Despite the risks to large numbers of individuals, an antidote capable of administration at scale adequate for a mass casualty, prehospital scenario does not yet exist. Previously, we demonstrated that intravenous cisplatin analogues accelerate recovery from cyanide poisoning in mice and rabbits. Of the dozens of platinum-based organometallic complexes tested, hexachloroplatinate (HCP) emerged as a promising lead compound, exhibiting strong affinity for cyanide and efficacy across model systems. Here, we show HCP is an antidote to lethal cyanide exposure and importantly is effective when delivered intramuscularly. The pharmacokinetic profile of HCP exhibited bioavailability in the systemic circulation 2.5 minutes post-treatment and subsequent renal clearance of HCP-cyanide. HCP restored parameters of cellular physiology including cytochrome oxidase redox state and TCA cycle metabolism. We next validated these findings in a large animal model (swine). Finally, preclinical safety studies in mice revealed minimal toxicity. Cumulatively, these findings demonstrate hexachloroplatinate is a promising lead compound for development of an intramuscular injectable cyanide antidote for mass casualty scenarios.
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http://dx.doi.org/10.1096/fba.1024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660183PMC
February 2019

Stable, Reactive, and Orthogonal Tetrazines: Dispersion Forces Promote the Cycloaddition with Isonitriles.

Angew Chem Int Ed Engl 2019 07 6;58(27):9043-9048. Epub 2019 Jun 6.

Department of Medicinal Chemistry, University of Utah, 30 S 2000 E, Salt Lake City, UT, 84112, USA.

The isocyano group is a structurally compact bioorthogonal functional group that reacts with tetrazines under physiological conditions. Now it is shown that bulky tetrazine substituents accelerate this cycloaddition. Computational studies suggest that dispersion forces between the isocyano group and the tetrazine substituents in the transition state contribute to the atypical structure-activity relationship. Stable asymmetric tetrazines that react with isonitriles at rate constants as high as 57 L mol  s were accessible by combining bulky and electron-withdrawing substituents. Sterically encumbered tetrazines react selectively with isonitriles in the presence of strained alkenes/alkynes, which allows for the orthogonal labeling of three proteins. The established principles will open new opportunities for developing tetrazine reactants with improved characteristics for diverse labeling and release applications with isonitriles.
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http://dx.doi.org/10.1002/anie.201903877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6615965PMC
July 2019

Developing zebrafish disease models for in vivo small molecule screens.

Curr Opin Chem Biol 2019 06 28;50:37-44. Epub 2019 Mar 28.

College of Pharmacy, University of Utah, Salt Lake City, UT 84112, United States. Electronic address:

The zebrafish is a model organism that allows in vivo studies to be performed at a scale usually restricted to in vitro studies. As such, the zebrafish is well suited to in vivo screens, in which thousands of small molecules are tested for their ability to modify disease phenotypes in zebrafish disease models. Numerous approaches have been developed for modeling human diseases in zebrafish, including mutagenesis, transgenesis, pharmacological approaches, wounding, and exposure to infectious or cancerous agents. We review the various strategies for modeling human diseases in zebrafish and discuss important considerations when developing zebrafish models for use in in vivo small molecule screens.
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http://dx.doi.org/10.1016/j.cbpa.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800242PMC
June 2019

5-Nitrofurans and Cancer: Teaching an Old Drug New Tricks.

Cell Chem Biol 2018 12;25(12):1439-1440

Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, UT 84112, USA. Electronic address:

Aldehyde dehydrogenases (ALDHs) are known as robust markers of tumor-initiating cells and as potential therapeutic targets for cancer. In this issue of Cell Chemical Biology, Sarvi et al. (2018) demonstrate that 5-nitrofuran antibiotics can target ALDH-expressing cancer cells in two distinct ways, effectively depleting melanomas of their tumor-initiating cells.
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http://dx.doi.org/10.1016/j.chembiol.2018.12.005DOI Listing
December 2018

High-throughput screening for selective appetite modulators: A multibehavioral and translational drug discovery strategy.

Sci Adv 2018 10 31;4(10):eaav1966. Epub 2018 Oct 31.

Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA.

How appetite is modulated by physiological, contextual, or pharmacological influence is still unclear. Specifically, the discovery of appetite modulators is compromised by the abundance of side effects that usually limit in vivo drug action. We set out to identify neuroactive drugs that trigger only their intended single behavioral change, which would provide great therapeutic advantages. To identify these ideal bioactive small molecules, we quantified the impact of more than 10,000 compounds on an extended series of different larval zebrafish behaviors using an in vivo imaging strategy. Known appetite-modulating drugs altered feeding and a pleiotropy of behaviors. Using this multibehavioral strategy as an active filter for behavioral side effects, we identified previously unidentified compounds that selectively increased or reduced food intake by more than 50%. The general applicability of this strategy is shown by validation in mice. Mechanistically, most candidate compounds were independent of the main neurotransmitter systems. In addition, we identified compounds with multibehavioral impact, and correlational comparison of these profiles with those of known drugs allowed for the prediction of their mechanism of action. Our results illustrate an unbiased and translational drug discovery strategy for ideal psychoactive compounds and identified selective appetite modulators in two vertebrate species.
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http://dx.doi.org/10.1126/sciadv.aav1966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209392PMC
October 2018

Noncanonical translation via deadenylated 3' UTRs maintains primordial germ cells.

Nat Chem Biol 2018 09 9;14(9):844-852. Epub 2018 Jul 9.

Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, MA, USA.

Primordial germ cells (PGCs) form during early embryogenesis with a supply of maternal mRNAs that contain shorter poly(A) tails. How translation of maternal mRNAs is regulated during PGC development remains elusive. Here we describe a small-molecule screen with zebrafish embryos that identified primordazine, a compound that selectively ablates PGCs. Primordazine's effect on PGCs arises from translation repression through primordazine-response elements in the 3' UTRs. Systematic dissection of primordazine's mechanism of action revealed that translation of mRNAs during early embryogenesis occurs by two distinct pathways, depending on the length of their poly(A) tails. In addition to poly(A)-tail-dependent translation (PAT), early embryos perform poly(A)-tail-independent noncanonical translation (PAINT) via deadenylated 3' UTRs. Primordazine inhibits PAINT without inhibiting PAT, an effect that was also observed in quiescent, but not proliferating, mammalian cells. These studies reveal that PAINT is an alternative form of translation in the early embryo and is indispensable for PGC maintenance.
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http://dx.doi.org/10.1038/s41589-018-0098-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6800240PMC
September 2018

Bioorthogonal Removal of 3-Isocyanopropyl Groups Enables the Controlled Release of Fluorophores and Drugs in Vivo.

J Am Chem Soc 2018 07 27;140(27):8410-8414. Epub 2018 Jun 27.

Department of Medicinal Chemistry, College of Pharmacy , University of Utah , 30 S 2000 E , Salt Lake City , Utah 84112 , United States.

Dissociative bioorthogonal reactions allow for chemically controlling the release of bioactive agents and reporter probes. Here we describe 3-isocyanopropyl substituents as masking groups that can be effectively removed in biological systems. 3-Isocyanopropyl derivatives react with tetrazines to afford 3-oxopropyl groups that eliminate diverse functionalities. The study shows that the reaction is rapid and can liberate phenols and amines near-quantitatively under physiological conditions. The reaction is compatible with living organisms as demonstrated by the release of a resorufin fluorophore and a mexiletine drug in zebrafish embryos implanted with tetrazine-modified beads. The combined benefits of synthetic ease, rapid kinetics, diversity of leaving groups, high release yields, and structural compactness, make 3-isocyanopropyl derivatives attractive chemical caging moieties for uses in chemical biology and drug delivery.
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http://dx.doi.org/10.1021/jacs.8b05093DOI Listing
July 2018

Small Molecule Inhibitors of NFkB Reverse Iron Overload and Hepcidin Deregulation in a Zebrafish Model for Hereditary Hemochromatosis Type 3.

ACS Chem Biol 2018 08 28;13(8):2143-2152. Epub 2018 Jun 28.

CSIR-Institute of Genomics and Integrative Biology , New Delhi , 110025 , India.

Hereditary hemochromatosis (HH) is one of the most common genetic disorders in Caucasian populations, with no viable therapeutic options except phlebotomy. We describe a zebrafish model of human HH (HH) created by targeted mutagenesis of the gene encoding transferrin receptor 2 ( tfr2). TFR2 mutations in humans lead to HH Type 3, a rare but severe form of the disease. The tfr2 mutant model in zebrafish recapitulates the defining features of HH3: iron overload and suppression of hepcidin, the iron regulatory hormone. Using in vivo chemical screens in zebrafish embryos, we identify a new small molecule inducer of hepcidin: SC-514, a specific chemical inhibitor of NFkB signaling. Using independent small molecule inhibitors of the NFkB pathway, we demonstrate that inhibition of NFkB signaling causes induction of hepcidin transcription and reduction of iron overload in the HH3 model. This first successful chemical intervention for hereditary hemochromatosis may also have relevance in treatment of other very prevalent iron regulatory iron overload disorders such as thalassemia.
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http://dx.doi.org/10.1021/acschembio.8b00317DOI Listing
August 2018

Identification of specific metabolic pathways as druggable targets regulating the sensitivity to cyanide poisoning.

PLoS One 2018 7;13(6):e0193889. Epub 2018 Jun 7.

Division of Cardiovascular Medicine, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, United States of America.

Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193889PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991913PMC
November 2018

Highly potent visnagin derivatives inhibit Cyp1 and prevent doxorubicin cardiotoxicity.

JCI Insight 2018 01 11;3(1). Epub 2018 Jan 11.

Cardiovascular Research Center, Cardiology Division, Massachusetts General Hospital.

Anthracyclines such as doxorubicin are highly effective chemotherapy agents used to treat many common malignancies. However, their use is limited by cardiotoxicity. We previously identified visnagin as protecting against doxorubicin toxicity in cardiac but not tumor cells. In this study, we sought to develop more potent visnagin analogs in order to use these analogs as tools to clarify the mechanisms of visnagin-mediated cardioprotection. Structure-activity relationship studies were performed in a zebrafish model of doxorubicin cardiomyopathy. Movement of the 5-carbonyl to the 7 position and addition of short ester side chains led to development of visnagin analogs with 1,000-fold increased potency in zebrafish and 250-fold increased potency in mice. Using proteomics, we discovered that doxorubicin caused robust induction of Cytochrome P450 family 1 (CYP1) that was mitigated by visnagin and its potent analog 23. Treatment with structurally divergent CYP1 inhibitors, as well as knockdown of CYP1A, prevented doxorubicin cardiomyopathy in zebrafish. The identification of potent cardioprotective agents may facilitate the development of new therapeutic strategies for patients receiving cardiotoxic chemotherapy. Moreover, these studies support the idea that CYP1 is an important contributor to doxorubicin cardiotoxicity and suggest that modulation of this pathway could be beneficial in the clinical setting.
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http://dx.doi.org/10.1172/jci.insight.96753DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821184PMC
January 2018

Dimethylguanidino valeric acid is a marker of liver fat and predicts diabetes.

J Clin Invest 2017 12 30;127(12):4394-4402. Epub 2017 Oct 30.

Division of Cardiovascular Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.

Unbiased, "nontargeted" metabolite profiling techniques hold considerable promise for biomarker and pathway discovery, in spite of the lack of successful applications to human disease. By integrating nontargeted metabolomics, genetics, and detailed human phenotyping, we identified dimethylguanidino valeric acid (DMGV) as an independent biomarker of CT-defined nonalcoholic fatty liver disease (NAFLD) in the offspring cohort of the Framingham Heart Study (FHS) participants. We verified the relationship between DMGV and early hepatic pathology. Specifically, plasma DMGV levels were correlated with biopsy-proven nonalcoholic steatohepatitis (NASH) in a hospital cohort of individuals undergoing gastric bypass surgery, and DMGV levels fell in parallel with improvements in post-procedure cardiometabolic parameters. Further, baseline DMGV levels independently predicted future diabetes up to 12 years before disease onset in 3 distinct human cohorts. Finally, we provide all metabolite peak data consisting of known and unidentified peaks, genetics, and key metabolic parameters as a publicly available resource for investigations in cardiometabolic diseases.
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http://dx.doi.org/10.1172/JCI95995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5707166PMC
December 2017

A high-conductance chemo-optogenetic system based on the vertebrate channel Trpa1b.

Sci Rep 2017 09 19;7(1):11839. Epub 2017 Sep 19.

Cardiovascular Research Center, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, 02129, USA.

Optogenetics is a powerful research approach that allows localized optical modulation of selected cells within an animal via the expression of genetically encoded photo-excitable ion channels. Commonly used optogenetic techniques rely on the expression of microbial opsin variants, which have many excellent features but suffer from various degrees of blue spectral overlap and limited channel conductance. Here, we expand the optogenetics toolbox in the form of a tunable, high-conductance vertebrate cation channel, zTrpa1b, coupled with photo-activated channel ligands, such as optovin and 4g6. Our results demonstrate that zTrpa1b/ligand pairing offers high light sensitivity, millisecond-scale response latency in vivo, as well as adjustable channel off latency. Exogenous in vivo expression of zTrpa1b in sensory neurons allowed subcellular photo-activation, enabling light-dependent motor control. zTrpa1b/ligand was also suitable for cardiomyocyte pacing, as shown in experiments performed on zebrafish hearts in vivo as well as in human stem cell-derived cardiomyocytes in vitro. Therefore, zTrpa1b/optovin represents a novel tool for flexible, high-conductance optogenetics.
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http://dx.doi.org/10.1038/s41598-017-11791-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5605526PMC
September 2017

Development of an opioid self-administration assay to study drug seeking in zebrafish.

Behav Brain Res 2017 09 12;335:158-166. Epub 2017 Aug 12.

Department of Pharmacology and Toxicology,University of Utah, Salt Lake City, UT 84112, USA. Electronic address:

The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction.
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http://dx.doi.org/10.1016/j.bbr.2017.08.001DOI Listing
September 2017

Bonnevillamides, Linear Heptapeptides Isolated from a Great Salt Lake-Derived Streptomyces sp.

Mar Drugs 2017 Jun 24;15(7). Epub 2017 Jun 24.

Department of Medicinal Chemistry, University of Utah, Salt Lake City, UT 84112, USA.

sp. GSL-6B was isolated from sediment collected from the Great Salt Lake and investigation of its organic extract led to the isolation of three new linear heptapeptides, bonnevillamides A (), B (), and C (). The bonnevillamides represent a new class of linear peptides featuring unprecedented non-proteinogenic amino acids. All three peptides contain the newly characterized bonnevillic acid moiety (3-(3,5-dichloro-4-methoxyphenyl)-2-hydroxyacrylic acid), as well as a heavily modified proline residue. Moreover, in bonnevillamide A, the terminal proline residue found in bonnevillamides B and C is replaced with 4-methyl-azetidine-2-carboxylic acid methyl ester. The structures of the three heptapeptides were elucidated by NMR, high-resolution electrospray ionization mass spectroscopy (HRESIMS), and LC-MS/MS, and the absolute configuration of all proteinogenic amino acid residues were determined by advanced Marfey's method. Bonnevillamides A, B and C were evaluated for their effects on zebrafish embryo development. All three heptapeptides were shown to modulate heart growth and cardiac function, with bonnevillamide B having the most pronounced effect.
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http://dx.doi.org/10.3390/md15070195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5532637PMC
June 2017