The Ferrous Awakens.

Recent discoveries provide a new hope that relapses of several types of cancer can be prevented by inducing ferroptosis.

σ1 receptor ligands control a switch between passive and active threat responses.

Humans and many animals show 'freezing' behavior in response to threatening stimuli. In humans, inappropriate threat responses are fundamental characteristics of several mental illnesses. To identify small molecules that modulate threat responses, we developed a high-throughput behavioral assay in zebrafish (Danio rerio) and evaluated 10,000 compounds for their effects on freezing behavior. We found three classes of compounds that switch the threat response from freezing to escape-like behavior. We then screened these for binding activity across 45 candidate targets. Using target profile clustering, we identified the sigma-1 (σ1) receptor as having a role in the mechanism of behavioral switching and confirmed that known σ1 ligands also disrupt freezing behavior. Furthermore, mutation of the gene encoding σ1 prevented the behavioral effect of escape-inducing compounds. One compound, which we call finazine, potently bound mammalian σ1 and altered threat-response behavior in mice. Thus, pharmacological and genetic interrogation of the freezing response revealed σ1 as a mediator of threat responses in vertebrates.

Zebrafish behavioral profiling identifies multitarget antipsychotic-like compounds.

Many psychiatric drugs act on multiple targets and therefore require screening assays that encompass a wide target space. With sufficiently rich phenotyping and a large sampling of compounds, it should be possible to identify compounds with desired mechanisms of action on the basis of behavioral profiles alone. Although zebrafish (Danio rerio) behavior has been used to rapidly identify neuroactive compounds, it is not clear what types of behavioral assays would be necessary to identify multitarget compounds such as antipsychotics. Here we developed a battery of behavioral assays in larval zebrafish to determine whether behavioral profiles can provide sufficient phenotypic resolution to identify and classify psychiatric drugs. Using the antipsychotic drug haloperidol as a test case, we found that behavioral profiles of haloperidol-treated zebrafish could be used to identify previously uncharacterized compounds with desired antipsychotic-like activities and multitarget mechanisms of action.

From phenotype to mechanism after zebrafish small molecule screens.

Small molecules screens conducted with living zebrafish have become a commonly practiced technique for small molecule discovery. Embryonic and larval zebrafish exhibit an almost limitless range of phenotypes, from the cellular to the organismal. Consequently, small molecule screens can be designed to discover compounds modifying any of these phenotypes. The compounds discovered by zebrafish screens pose unique challenges for target identification, but the zebrafish also provides several powerful approaches for identifying targets and determining mechanisms of action. Four major approaches have been used successfully, including methods based on comparison of chemical structures, genetic phenocopy, pharmacologic phenocopy, and compound affinity. These approaches will continue to facilitate target identification for compounds from zebrafish small molecule screens, and more importantly, to reveal their mechanisms of action.

Initiation of Epstein-Barr virus lytic replication requires transcription and the formation of a stable RNA-DNA hybrid molecule at OriLyt.

The genetic elements of herpesvirus origins of lytic replication have been characterized in detail; however, much remains to be elucidated concerning their functional role in replication initiation. In the case of the Epstein-Barr virus (EBV), we have found that in addition to the two well-defined critical elements required for lytic replication (the upstream and downstream essential elements, UEE and DEE), the origin of lytic replication (OriLyt) also requires the presence of a GC-rich RNA in cis. The BHLF1 transcript is similar to the essential K5 transcript identified at the Kaposi's sarcoma-associated herpesvirus OriLyt. We have found that truncation of the BHLF1 transcript or deletion of the TATA box, but not the putative ATG initiation codon, reduce OriLyt function to background levels. By using an antibody specific for RNA-DNA hybrid molecules, we found the BHLF1 RNA stably annealed to its DNA template during the early steps of lytic reactivation. Furthermore, expression of human RNase H1, which degrades RNA in RNA-DNA hybrids, drastically reduces OriLyt-dependent DNA replication as well as recruitment of the viral single-stranded DNA binding protein BALF2 to OriLyt. These studies suggest that a GC-rich OriLyt transcript is an important component of gammaherpesvirus lytic origins and is required for initial strand separation and loading of core replication proteins.

Evidence for DNA hairpin recognition by Zta at the Epstein-Barr virus origin of lytic replication.

The Epstein-Barr virus immediate-early protein (Zta) plays an essential role in viral lytic activation and pathogenesis. Zta is a basic zipper (b-Zip) domain-containing protein that binds multiple sites in the viral origin of lytic replication (OriLyt) and is required for lytic-cycle DNA replication. We present evidence that Zta binds to a sequence-specific, imperfect DNA hairpin formed by an inverted repeat within the upstream essential element (UEE) of OriLyt. Mutations in the OriLyt sequence that are predicted to disrupt hairpin formation also disrupt Zta binding in vitro. Restoration of the hairpin rescues the defect. We also show that OriLyt DNA isolated from replicating cells contains a nuclease-sensitive region that overlaps with the inverted-repeat region of the UEE. Furthermore, point mutations in Zta that disrupt specific recognition of the UEE hairpin are defective for activation of lytic replication. These data suggest that Zta acts by inducing and/or stabilizing a DNA hairpin structure during productive infection. The DNA hairpin at OriLyt with which Zta interacts resembles DNA structures formed at other herpesvirus origins and may therefore represent a common secondary structure used by all herpesvirus family members during the initiation of DNA replication.

Topoisomerase I and RecQL1 function in Epstein-Barr virus lytic reactivation.

Cellular topoisomerases and helicases are thought to play an essential role in herpesvirus replication and gene expression and are considered to be potential targets for antiviral therapies. Topoisomerase I (Topo I) and Topo II inhibitors can selectively inhibit Epstein-Barr virus (EBV) lytic cycle DNA replication. We found that the Topo I inhibitor camptothecin and, to a lesser extent, the Topo II inhibitor etoposide are potent inhibitors of the transcription and replication function of the EBV-encoded immediate-early protein Zta (also referred to as ZEBRA, EB1, and BZLF1). Camptothecin inhibited the Zta transcription activation of endogenous and reporter-linked viral promoters. Small interfering RNA depletion of Topo I also inhibited the Zta-dependent activation of lytic cycle DNA replication. Topo I could be coimmunoprecipitated with Zta, but this interaction was restricted to EBV-positive cells, suggesting that other viral proteins stabilize the interaction between Zta and Topo I. We also found that the RecQL1 helicase, which is known to associate with Kaposi's sarcoma-associated herpesvirus (KSHV) OriLyt, interacts with EBV OriLyt. Treatment with camptothecin reduced both Zta and RecQL1 binding to OriLyt in vivo, suggesting that Topo I promotes replication protein assembly at OriLyt.

Epstein-Barr virus immediate-early protein Zta co-opts mitochondrial single-stranded DNA binding protein to promote viral and inhibit mitochondrial DNA replication.

Disruption of cellular metabolic processes and usurpation of host proteins are hallmarks of herpesvirus lytic infection. Epstein-Barr virus (EBV) lytic replication is initiated by the immediate-early protein Zta. Zta is a multifunctional DNA binding protein that stimulates viral gene transcription, nucleates a replication complex at the viral origin of lytic replication, and inhibits cell cycle proliferation. To better understand these functions and identify cellular collaborators of Zta, we purified an epitope-tagged version of Zta in cells capable of supporting lytic replication. FLAG-tagged Zta was purified from a nuclear fraction using FLAG antibody immunopurification and peptide elution. Zta-associated proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by mass spectrometry. The Zta-associated proteins included members of the HSP70 family and various single-stranded DNA and RNA binding proteins. The nuclear replication protein A subunits (RPA70 and RPA32) and the human mitochondrial single-stranded DNA binding protein (mtSSB) were confirmed by Western blotting to be specifically enriched in the FLAG-Zta immunopurified complex. mtSSB coimmunoprecipitated with endogenous Zta during reactivation of EBV-positive Burkitt lymphoma and lymphoblastoid cell lines. Small interfering RNA depletion of mtSSB reduced Zta-induced lytic replication of EBV but had only a modest effect on transcription activation function. A point mutation in the Zta DNA binding domain (C189S), which is known to reduce lytic cycle replication, eliminated mtSSB association with Zta. The predominantly mitochondrial localization of mtSSB was shifted to partly nuclear localization in cells expressing Zta. Mitochondrial DNA synthesis and genome copy number were reduced by Zta-induced EBV lytic replication. We conclude that Zta interaction with mtSSB serves the dual function of facilitating viral and blocking mitochondrial DNA replication.

Modulation of Threat Response in Larval Zebrafish.

High-throughput, whole-organism phenotypic drug screening is made possible using live zebrafish larvae. Many human drugs have now been shown to affect zebrafish larvae in similar ways, through homologous molecular mechanisms. At this stage in life, zebrafish are small enough to fit in multi-well, microliter plates, yet developed enough to exhibit complex phenotypes, such as hunting behaviors and avoidance of predators. Importantly, zebrafish larvae can be easily dosed via automated pipetting of chemical compounds directly into their liquid medium, without injection. Only microgram amounts of small molecules are required, making animal husbandry and dosing regimens cost effective. This chapter describes how the stereotyped zebrafish larval responses to darkness and strobe light-which cause hyperactivity and freezing behavior, respectively-can be used to efficiently screen small molecules for brain and behavior-modulating activity.

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

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.

Chemical Screening in Zebrafish.

Phenotypic small molecule screens in zebrafish have gained popularity as an unbiased approach to probe biological processes. In this chapter we outline basic methods for performing chemical screens with larval zebrafish including breeding large numbers of embryos, plating larval fish into multi-well dishes, and adding small molecules to these wells. We also highlight important considerations when designing and interpreting the results of a phenotypic screen and possible follow-up approaches, including popular methods used to identify the mechanism of action of a chemical compound.

15 years of zebrafish chemical screening.

In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies.

Single Amino Acid Variation Underlies Species-Specific Sensitivity to Amphibian Skin-Derived Opioid-like Peptides.

It has been suggested that the evolution of vertebrate opioid receptors (ORs) follow a vector of increased functionality. Here, we test this idea by comparing human and frog ORs. Interestingly, some of the most potent opioid peptides known have been isolated from amphibian skin secretions. Here we show that such peptides (dermorphin and deltorphin) are highly potent in the human receptors and inactive in frog ORs. The molecular basis for the insensitivity of the frog ORs to these peptides was studied using chimeras and molecular modeling. The insensitivity of the delta OR (DOR) to deltorphin was due to variation of a single amino acid, Trp7.35, which is a leucine in mammalian DORs. Notably, Trp7.35 is completely conserved in all known DOR sequences from lamprey, fish, and amphibians. The deltorphin-insensitive phenotype was verified in fish. Our results provide a molecular explanation for the species selectivity of skin-derived opioid peptides.

Behavioral barcoding in the cloud: embracing data-intensive digital phenotyping in neuropharmacology.

For decades, studying the behavioral effects of individual drugs and genetic mutations has been at the heart of efforts to understand and treat nervous system disorders. High-throughput technologies adapted from other disciplines (e.g., high-throughput chemical screening, genomics) are changing the scale of data acquisition in behavioral neuroscience. Massive behavioral datasets are beginning to emerge, particularly from zebrafish labs, where behavioral assays can be performed rapidly and reproducibly in 96-well, high-throughput format. Mining these datasets and making comparisons across different assays are major challenges for the field. Here, we review behavioral barcoding, a process by which complex behavioral assays are reduced to a string of numeric features, facilitating analysis and comparison within and across datasets.

Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion.

Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.

Initiation of lytic DNA replication in Epstein-Barr virus: search for a common family mechanism.

Herpesviruses are a complex family of dsDNA viruses that are a major cause of human disease. All family members share highly related viral replication proteins, such as DNA polymerase, ssDNA-binding proteins and processivity factors. Consequently, it is generally thought that lytic replication occurs through a common and conserved mechanism. However, considerable evidence indicates that proteins controlling initiation of DNA replication vary greatly among the herepesvirus subfamilies. In this article, we focus on some of the known mechanisms that regulate Epstein-Barr virus lytic-cycle replication, and compare this to other herpesvirus family members. Our reading of the literature leads us to conclude that diverse viral mechanisms generate a common nucleoprotein prereplication structure that can be recognized by a highly conserved family of viral replication enzymes.

Chromatin profiling of Epstein-Barr virus latency control region.

Epstein-Barr virus (EBV) escapes host immunity by the reversible and epigenetic silencing of immunogenic viral genes. We previously presented evidence that a dynamic chromatin domain, which we have referred to as the latency control region (LCR), contributes to the reversible repression of EBNA2 and LMP1 gene transcription. We now explore the protein-DNA interaction profiles for a few known regulatory factors and histone modifications that regulate LCR structure and activity. A chromatin immunoprecipitation assay combined with real-time PCR analysis was used to analyze protein-DNA interactions at approximately 500-bp intervals across the first 60,000 bp of the EBV genome. We compared the binding patterns of EBNA1 with those of the origin recognition complex protein ORC2, the chromatin boundary factor CTCF, the linker histone H1, and several histone modifications. We analyzed three EBV-positive cell lines (MutuI, Raji, and LCL3459) with distinct transcription patterns reflecting different latency types. Our findings suggest that histone modification patterns within the LCR are complex but reflect differences in each latency type. The most striking finding was the identification of CTCF sites immediately upstream of the Qp, Cp, and EBER transcription initiation regions in all three cell types. In transient assays, CTCF facilitated EBNA1-dependent transcription activation of Cp, suggesting that CTCF coordinates interactions between different chromatin domains. We also found that histone H3 methyl K4 clustered with CTCF and EBNA1 at sites of active transcription or DNA replication initiation. Our findings support a model where CTCF delineates multiple domains within the LCR and regulates interactions between these domains that correlate with changes in gene expression.