An RNA-centric dissection of host complexes controlling flavivirus infection.
RACK1 on and off the ribosome
2019; 25 (7): 881?95
eIF5B gates the transition from translation initiation to elongation.
Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.
View details for DOI 10.1038/s41564-019-0518-2
View details for PubMedID 31384002
Chemical structure-guided design of dynapyrazoles, potent cell-permeable dynein inhibitors with a unique mode of action.
Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear1,2. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli3-5. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.
View details for DOI 10.1038/s41586-019-1561-0
View details for PubMedID 31534220
Dynamics of IRES-mediated translation
PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES
2017; 372 (1716)
Cytoplasmic dyneins are motor proteins in the AAA+ superfamily that power transport of cellular cargos towards microtubule minus-ends. Recently, ciliobrevins were reported as selective cell-permeable inhibitors of cytoplasmic dyneins. As is often true for first-in-class inhibitors, the use of ciliobrevins has been limited by low potency. Moreover, suboptimal chemical properties, such as the potential to isomerize, have hindered efforts to improve ciliobrevins. Here, we characterized the structure of ciliobrevins and designed conformationally-constrained isosteres. We identified dynapyrazoles, inhibitors more potent than ciliobrevins in vitro, and find that while ciliobrevins inhibit both dynein's microtubule-stimulated and basal ATPase activity, dynapyrazoles block only microtubule-stimulated activity. Single-digit micromolar concentrations of dynapyrazoles block intraflagellar transport in the cilium and lysosome motility in the cytoplasm, processes that depend on cytoplasmic dyneins. Together, our studies suggest that chemical structure-based analyses can lead to inhibitors with distinct modes of inhibition and improved properties.
View details for DOI 10.7554/eLife.25174
View details for PubMedID 28524820
Fluorescently-tagged human eIF3 for single-molecule spectroscopy.
Nucleic acids research
Viral internal ribosome entry sites (IRESs) are unique RNA elements, which use stable and dynamic RNA structures to recruit ribosomes and drive protein synthesis. IRESs overcome the high complexity of the canonical eukaryotic translation initiation pathway, often functioning with a limited set of eukaryotic initiation factors. The simplest types of IRESs are typified by the cricket paralysis virus intergenic region (CrPV IGR) and hepatitis C virus (HCV) IRESs, both of which independently form high-affinity complexes with the small (40S) ribosomal subunit and bypass the molecular processes of cap-binding and scanning. Owing to their simplicity and ribosomal affinity, the CrPV and HCV IRES have been important models for structural and functional studies of the eukaryotic ribosome during initiation, serving as excellent targets for recent technological breakthroughs in cryogenic electron microscopy (cryo-EM) and single-molecule analysis. High-resolution structural models of ribosome : IRES complexes, coupled with dynamics studies, have clarified decades of biochemical research and provided an outline of the conformational and compositional trajectory of the ribosome during initiation. Here we review recent progress in the study of HCV- and CrPV-type IRESs, highlighting important structural and dynamics insights and the synergy between cryo-EM and single-molecule studies.This article is part of the themed issue 'Perspectives on the ribosome'.
View details for DOI 10.1098/rstb.2016.0177
View details for PubMedID 28138065
Discovery of novel, orally bioavailable, antileishmanial compounds using phenotypic screening.
PLoS neglected tropical diseases
2017; 11 (12): e0006157
Human translation initiation relies on the combined activities of numerous ribosome-associated eukaryotic initiation factors (eIFs). The largest factor, eIF3, is an ?800 kDa multiprotein complex that orchestrates a network of interactions with the small 40S ribosomal subunit, other eIFs, and mRNA, while participating in nearly every step of initiation. How these interactions take place during the time course of translation initiation remains unclear. Here, we describe a method for the expression and affinity purification of a fluorescently-tagged eIF3 from human cells. The tagged eIF3 dodecamer is structurally intact, functions in cell-based assays, and interacts with the HCV IRES mRNA and the 40S-IRES complex in vitro. By tracking the binding of single eIF3 molecules to the HCV IRES RNA with a zero-mode waveguides-based instrument, we show that eIF3 samples both wild-type IRES and an IRES that lacks the eIF3-binding region, and that the high-affinity eIF3-IRES interaction is largely determined by slow dissociation kinetics. The application of single-molecule methods to more complex systems involving eIF3 may unveil dynamics underlying mRNA selection and ribosome loading during human translation initiation.
View details for PubMedID 29136179
Identification of Selective Inhibitors of the Plasmodium falciparum Hexose Transporter PfHT by Screening Focused Libraries of Anti-Malarial Compounds.
2015; 10 (4): e0123598
Leishmaniasis is a parasitic infection that afflicts approximately 12 million people worldwide. There are several limitations to the approved drug therapies for leishmaniasis, including moderate to severe toxicity, growing drug resistance, and the need for extended dosing. Moreover, miltefosine is currently the only orally available drug therapy for this infection. We addressed the pressing need for new therapies by pursuing a two-step phenotypic screen to discover novel, potent, and orally bioavailable antileishmanials. First, we conducted a high-throughput screen (HTS) of roughly 600,000 small molecules for growth inhibition against the promastigote form of the parasite life cycle using the nucleic acid binding dye SYBR Green I. This screen identified approximately 2,700 compounds that inhibited growth by over 65% at a single point concentration of 10 ?M. We next used this 2700 compound focused library to identify compounds that were highly potent against the disease-causing intra-macrophage amastigote form and exhibited limited toxicity toward the host macrophages. This two-step screening strategy uncovered nine unique chemical scaffolds within our collection, including two previously described antileishmanials. We further profiled two of the novel compounds for in vitro absorption, distribution, metabolism, excretion, and in vivo pharmacokinetics. Both compounds proved orally bioavailable, affording plasma exposures above the half-maximal effective concentration (EC50) concentration for at least 12 hours. Both compounds were efficacious when administered orally in a murine model of cutaneous leishmaniasis. One of the two compounds exerted potent activity against trypanosomes, which are kinetoplastid parasites related to Leishmania species. Therefore, this compound could help control multiple parasitic diseases. The promising pharmacokinetic profile and significant in vivo efficacy observed from our HTS hits highlight the utility of our two-step phenotypic screening strategy and strongly suggest that medicinal chemistry optimization of these newly identified scaffolds will lead to promising candidates for an orally available anti-parasitic drug.
View details for PubMedID 29287089
Dyotropic Rearrangements of Fused Tricyclic beta-Lactones: Application to the Synthesis of (-)-Curcumanolide A and (-)-Curcumalactone
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2012; 134 (32): 13348-13356
Development of resistance against current antimalarial drugs necessitates the search for novel drugs that interact with different targets and have distinct mechanisms of action. Malaria parasites depend upon high levels of glucose uptake followed by inefficient metabolic utilization via the glycolytic pathway, and the Plasmodium falciparum hexose transporter PfHT, which mediates uptake of glucose, has thus been recognized as a promising drug target. This transporter is highly divergent from mammalian hexose transporters, and it appears to be a permease that is essential for parasite viability in intra-erythrocytic, mosquito, and liver stages of the parasite life cycle. An assay was developed that is appropriate for high throughput screening against PfHT based upon heterologous expression of PfHT in Leishmania mexicana parasites that are null mutants for their endogenous hexose transporters. Screening of two focused libraries of antimalarial compounds identified two such compounds that are high potency selective inhibitors of PfHT compared to human GLUT1. Additionally, 7 other compounds were identified that are lower potency and lower specificity PfHT inhibitors but might nonetheless serve as starting points for identification of analogs with more selective properties. These results further support the potential of PfHT as a novel drug target.
View details for PubMedID 25894322
Dyotropic rearrangements of fused, tricyclic ?-lactones are described that proceed via unprecedented stereospecific, 1,2-acyl migrations delivering bridged, spiro-?-butyrolactones. A unique example of this dyotropic process involves a fused bis-lactone possessing both ?- and ?-lactone moieties which enabled rapid access to the core structures of curcumanolide A and curcumalactone. Our current mechanistic understanding of the latter dyotropic process, based on computational studies, is also described. Other key transformations in the described divergent syntheses of (-)-curcumanolide A and (-)-curcumalactone from a common intermediate (11 and 12 steps from 2-methyl-1,3-cyclopentanedione, respectively), include a catalytic, asymmetric nucleophile (Lewis base)-catalyzed aldol-lactonization (NCAL) leading to a tricyclic ?-lactone, a Baeyer-Villiger oxidation in the presence of a ?-lactone, and highly facial-selective and stereocomplementary reductions of an intermediate spirocyclic enoate. The described dyotropic rearrangements significantly alter the topology of the starting tricyclic ?-lactone, providing access to complex spirocyclic cyclopentyl-?-lactones and bis-?-lactones in a single synthetic operation.
View details for DOI 10.1021/ja303414a
View details for Web of Science ID 000307487200039
View details for PubMedID 22853802