Priscilla Li-ning Yang

All Publications

• Targeted protein degradation as an antiviral approach. Antiviral research Chakravarty, A., Yang, P. L. 2022: 105480

  Abstract

  Targeted protein degradation (TPD) has emerged as a new modality in drug discovery in which small molecules are used to drive degradation of the target protein of interest. Whereas most direct-acting antivirals have occupancy-driven pharmacology, the pharmacology of degrader molecules is event-driven. These contrasting mechanisms can result in significant differences in drug efficacy and pharmacodynamics that may be useful in development of new classes of antivirals. While now being widely pursued in cancer biology and autoimmune disease, TPD has not yet been widely applied in antivirals development. Here briefly review TPD pharmacology along with the current status of tools available for developing small molecules that achieve antiviral activity through a TPD mechanism while also highlighting aspects of TPD that may be especially useful in the development of antivirals.

  View details for DOI 10.1016/j.antiviral.2022.105480

  View details for PubMedID 36567024

• Anti-SARS-CoV-2 Activity of Targeted Kinase Inhibitors: Repurposing Clinically Available Drugs for COVID-19 Therapy. Journal of medical virology Boytz, R., Slabicki, M., Ramaswamy, S., Patten, J. J., Zou, C., Meng, C., Hurst, B. L., Wang, J., Nowak, R. P., Yang, P. L., Sattler, M., Stone, R. M., Griffin, J. D., Gray, N. S., Gummuluru, S., Davey, R. A., Weisberg, E. 2022

  Abstract

  PURPOSE: Coronavirus disease 2019 (COVID-19) remains a major public health concern, and vaccine unavailability, hesitancy, or failure underscore the need for discovery of efficacious antiviral drug therapies. Numerous approved drugs target protein kinases associated with viral life cycle and symptoms of infection. Repurposing of kinase inhibitors is appealing as they have been vetted for safety and are more accessible for COVID-19 treatment. However, an understanding of drug mechanism is needed to improve our understanding of the factors involved in pathogenesis.METHODS: We tested the in vitro activity of three kinase inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including inhibitors of AXL kinase, a host cell factor that contributes to successful SARS-CoV-2 infection. Using multiple cell-based assays and approaches, gilteritinib, nintedanib, and imatinib were thoroughly evaluated for activity against SARS-CoV-2 variants.RESULTS: Each drug exhibited antiviral activity, but with stark differences in potency, suggesting differences in host dependency for kinase targets. Importantly, for gilteritinib, the amount of compound needed to achieve 90% infection inhibition, at least in part involving blockade of spike protein-mediated viral entry and at concentrations not inducing phospholipidosis (PLD), approached a clinically achievable concentration. Knockout of AXL, a target of gilteritinib and nintedanib, impaired SARS-CoV-2 variant infectivity, supporting a role for AXL in SARS-CoV-2 infection and supporting further investigation of drug-mediated AXL inhibition as a COVID-19 treatment.CONCLUSIONS: This work supports further evaluation of AXL-targeting kinase inhibitors as potential antiviral agents and treatments for COVID-19. Additional mechanistic studies are needed to determine underlying differences in virus response. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/jmv.28157

  View details for PubMedID 36117402

• Small-Molecule Inhibition of Viral Fusion Glycoproteins. Annual review of virology Liu, H., Yang, P. L. 2021

  Abstract

  Viral fusion glycoproteins catalyze membrane fusion during viral entry. Unlike most enzymes, however, they lack a conventional active site in which formation or scission of a specific covalent bond is catalyzed. Instead, they drive the membrane fusion reaction by cojoining highly regulated changes in conformation to membrane deformation. Despite the challenges in applying inhibitor design approaches to these proteins, recent advances in knowledge of the structures and mechanisms of viral fusogens have enabled the development of small-molecule inhibitors of both class I and class II viral fusion proteins. Here, we review well-validated inhibitors, including their discovery, targets, and mechanism(s) of action, while highlighting mechanistic similarities and differences. Together, these examples make a compelling case for small-molecule inhibitors as tools for probing the mechanisms of viral glycoprotein-mediated fusion and for viral glycoproteins as druggable targets. Expected final online publication date for the Annual Review of Virology, Volume 8 is September 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  View details for DOI 10.1146/annurev-virology-022221-063725

  View details for PubMedID 34197186

• Antiviral Therapeutics. ACS infectious diseases Yang, P. L. 2021; 7 (6): 1297

  View details for DOI 10.1021/acsinfecdis.1c00271

  View details for PubMedID 34061494

• Hepatitis C virus NS3-4A protease regulates the lipid environment for RNA replication by cleaving host enzyme 24-dehydrocholesterol reductase JOURNAL OF BIOLOGICAL CHEMISTRY Tallorin, L., Villareal, V. A., Hsia, C., Rodgers, M. A., Burri, D. J., Pfeil, M., Llopis, P., Lindenbach, B. D., Yang, P. L. 2020; 295 (35): 12426-12436

  Abstract

  Many RNA viruses create specialized membranes for genome replication by manipulating host lipid metabolism and trafficking, but in most cases, we do not know the molecular mechanisms responsible or how specific lipids may impact the associated membrane and viral process. For example, hepatitis C virus (HCV) causes a specific, large-fold increase in the steady-state abundance of intracellular desmosterol, an immediate precursor of cholesterol, resulting in increased fluidity of the membrane where HCV RNA replication occurs. Here, we establish the mechanism responsible for HCV's effect on intracellular desmosterol, whereby the HCV NS3-4A protease controls activity of 24-dehydrocholesterol reductase (DHCR24), the enzyme that catalyzes conversion of desmosterol to cholesterol. Our cumulative evidence for the proposed mechanism includes immunofluorescence microscopy experiments showing co-occurrence of DHCR24 and HCV NS3-4A protease; formation of an additional, faster-migrating DHCR24 species (DHCR24*) in cells harboring a HCV subgenomic replicon RNA or ectopically expressing NS3-4A; and biochemical evidence that NS3-4A cleaves DHCR24 to produce DHCR24* in vitro and in vivo We further demonstrate that NS3-4A cleaves DHCR24 between residues Cys91 and Thr92 and show that this reduces the intracellular conversion of desmosterol to cholesterol. Together, these studies demonstrate that NS3-4A directly cleaves DHCR24 and that this results in the enrichment of desmosterol in the membranes where NS3-4A and DHCR24 co-occur. Overall, this suggests a model in which HCV directly regulates the lipid environment for RNA replication through direct effects on the host lipid metabolism.

  View details for DOI 10.1074/jbc.RA120.013455

  View details for Web of Science ID 000567571100009

  View details for PubMedID 32641492

  View details for PubMedCentralID PMC7458815

• A Sensitive Yellow Fever Virus Entry Reporter Identifies Valosin-Containing Protein (VCP/p97) as an Essential Host Factor for Flavivirus Uncoating MBIO Ramanathan, H. N., Zhang, S., Douam, F., Mar, K. B., Chang, J., Yang, P. L., Schoggins, J. W., Ploss, A., Lindenbach, B. D. 2020; 11 (2)

  Abstract

  While the basic mechanisms of flavivirus entry and fusion are understood, little is known about the postfusion events that precede RNA replication, such as nucleocapsid disassembly. We describe here a sensitive, conditionally replication-defective yellow fever virus (YFV) entry reporter, YFVΔSK/Nluc, to quantitively monitor the translation of incoming, virus particle-delivered genomes. We validated that YFVΔSK/Nluc gene expression can be neutralized by YFV-specific antisera and requires known flavivirus entry pathways and cellular factors, including clathrin- and dynamin-mediated endocytosis, endosomal acidification, YFV E glycoprotein-mediated fusion, and cellular LY6E and RPLP1 expression. The initial round of YFV translation was shown to require cellular ubiquitylation, consistent with recent findings that dengue virus capsid protein must be ubiquitylated in order for nucleocapsid uncoating to occur. Importantly, translation of incoming YFV genomes also required valosin-containing protein (VCP)/p97, a cellular ATPase that unfolds and extracts ubiquitylated client proteins from large complexes. RNA transfection and washout experiments showed that VCP/p97 functions at a postfusion, pretranslation step in YFV entry. Finally, VCP/p97 activity was required by other flaviviruses in mammalian cells and by YFV in mosquito cells. Together, these data support a critical role for VCP/p97 in the disassembly of incoming flavivirus nucleocapsids during a postfusion step in virus entry.IMPORTANCE Flaviviruses are an important group of RNA viruses that cause significant human disease. The mechanisms by which flavivirus nucleocapsids are disassembled during virus entry remain unclear. Here, we used a yellow fever virus entry reporter, which expresses a sensitive reporter enzyme but does not replicate, to show that nucleocapsid disassembly requires the cellular protein-disaggregating enzyme valosin-containing protein, also known as p97.

  View details for DOI 10.1128/mBio.00467-20

  View details for Web of Science ID 000531071300100

  View details for PubMedID 32291299

  View details for PubMedCentralID PMC7157815

• A broad-spectrum antiviral molecule, QL47, selectively inhibits eukaryotic translation. The Journal of biological chemistry de Wispelaere, M., Carocci, M., Burri, D. J., Neidermyer, W. J., Olson, C. M., Roggenbach, I., Liang, Y., Wang, J., Whelan, S. P., Gray, N. S., Yang, P. L. 2020; 295 (6): 1694-1703

  Abstract

  Small-molecule inhibitors of translation are critical tools to study the molecular mechanisms of protein synthesis. In this study, we sought to characterize how QL47, a host-targeted, small-molecule antiviral agent, inhibits steady-state viral protein expression. We demonstrate that this small molecule broadly inhibits both viral and host protein synthesis and targets a translation step specific to eukaryotic cells. We show that QL47 inhibits protein neosynthesis initiated by both canonical cap-driven and noncanonical initiation strategies, most likely by targeting an early step in translation elongation. Our findings thus establish QL47 as a new small-molecule inhibitor that can be utilized to probe the eukaryotic translation machinery and that can be further developed as a new therapeutic agent.

  View details for DOI 10.1074/jbc.RA119.011132

  View details for PubMedID 31914414

• Discovery of Immunologically Inspired Small Molecules That Target the Viral Envelope Protein (vol 4, pg 1395, 2018) ACS INFECTIOUS DISEASES Lian, W., Jang, J., Potisopon, S., Li, P., Rahmeh, A., Wang, J., Kwiatkowski, N. P., Gray, N. S., Yang, P. L. 2019; 5 (12): 2176

  View details for DOI 10.1021/acsinfecdis.9b00401

  View details for Web of Science ID 000503114600025

  View details for PubMedID 31675213

• Small molecule degraders of the hepatitis C virus protease reduce susceptibility to resistance mutations NATURE COMMUNICATIONS de Wispelaere, M., Du, G., Donovan, K. A., Zhang, T., Eleuteri, N. A., Yuan, J. C., Kalabathula, J., Nowak, R. P., Fischer, E. S., Gray, N. S., Yang, P. L. 2019; 10: 3468

  Abstract

  Targeted protein degradation is a promising drug development paradigm. Here we leverage this strategy to develop a new class of small molecule antivirals that induce proteasomal degradation of viral proteins. Telaprevir, a reversible-covalent inhibitor that binds to the hepatitis C virus (HCV) protease active site is conjugated to ligands that recruit the CRL4CRBN ligase complex, yielding compounds that can both inhibit and induce the degradation of the HCV NS3/4A protease. An optimized degrader, DGY-08-097, potently inhibits HCV in a cellular infection model, and we demonstrate that protein degradation contributes to its antiviral activity. Finally, we show that this new class of antiviral agents can overcome viral variants that confer resistance to traditional enzymatic inhibitors such as telaprevir. Overall, our work provides proof-of-concept that targeted protein degradation may provide a new paradigm for the development of antivirals with superior resistance profiles.

  View details for DOI 10.1038/s41467-019-11429-w

  View details for Web of Science ID 000478014000010

  View details for PubMedID 31371704

  View details for PubMedCentralID PMC6672008

• Identification of small molecule inhibitors targeting the Zika virus envelope protein ANTIVIRAL RESEARCH Pitts, J., Hsia, C., Lian, W., Wang, J., Pfeil, M., Kwiatkowski, N., Li, Z., Jang, J., Gray, N. S., Yang, P. L. 2019; 164: 147-153

  Abstract

  The recent emergence of Zika virus, a mosquito-borne flavivirus, in the Americas has shed light on the severe neurological diseases associated with infection, notably congenital microcephaly in newborns and Guillain-Barré syndrome in adults. Despite the recent focus on Zika virus, there are currently no approved vaccines or antiviral therapies available to treat or prevent infection. In this study we established a competitive amplified luminescent proximity homogeneous assay (ALPHAscreen) to identify small molecule inhibitors targeting the envelope protein of Zika virus (Zika E). We utilized this assay to screen two libraries of nearly 27,000 compounds and identified seven novel inhibitors of Zika E. Characterization of these primary screening leads demonstrated that inhibition of Zika virus occurs at non-cytotoxic concentrations for all seven lead compounds. In addition, we found that all seven lead compounds have potent activity against the closely related dengue virus 2 but not vesicular stomatitis virus, an unrelated enveloped virus. Biochemical experiments indicate that these compounds act by preventing E-mediated membrane fusion. This work highlights a new method for the discovery and optimization of direct-acting antivirals targeting the E protein of Zika and other flaviviruses.

  View details for DOI 10.1016/j.antiviral.2019.02.008

  View details for Web of Science ID 000463303700014

  View details for PubMedID 30771406

  View details for PubMedCentralID PMC6675404

• Small Molecules Targeting the Flavivirus E Protein with Broad-Spectrum Activity and Antiviral Efficacy in Vivo ACS INFECTIOUS DISEASES Li, P., Jang, J., Hsia, C., Groomes, P. V., Lian, W., de Wispelaere, M., Pitts, J. D., Wang, J., Kwiatkowski, N., Gray, N. S., Yang, P. L. 2019; 5 (3): 460-472

  Abstract

  Vaccines and antivirals to combat dengue, Zika, and other flavivirus pathogens present a major, unmet medical need. Vaccine development has been severely challenged by the antigenic diversity of these viruses and the propensity of non-neutralizing, cross-reactive antibodies to facilitate cellular infection and increase disease severity. As an alternative, direct-acting antivirals targeting the flavivirus envelope protein, E, have the potential to act via an analogous mode of action without the risk of antibody-dependent enhancement of infection and disease. We previously discovered that structurally diverse small molecule inhibitors of the dengue virus E protein exhibit varying levels of antiviral activity against other flaviviruses in cell culture. Here, we demonstrate that the broad-spectrum activity of several cyanohydrazones against dengue, Zika, and Japanese encephalitis viruses is due to specific inhibition of E-mediated membrane fusion during viral entry and provide proof of concept for pharmacological inhibition of E as an antiviral strategy in vivo.

  View details for DOI 10.1021/acsinfecdis.8b00322

  View details for Web of Science ID 000461130800014

  View details for PubMedID 30608640

  View details for PubMedCentralID PMC6548335

• A call to arms: Unifying the fight against resistance SCIENCE SIGNALING Kaushansky, A., Hedstrom, L., Goldman, A., Singh, J., Yang, P. L., Rathod, P. K., Cynamon, M., Wodarz, D., Mahadevan, D., Tomaras, A., Navia, M. A., Schiffer, C. A. 2018; 11 (553)

  Abstract

  This Editorial discusses the state of research on drug resistance in the fields of cancer, infectious disease, and agriculture. Reaching across the aisle for a more cross-collaborative approach may lead to exciting breakthroughs toward tackling the challenges of drug resistance in each field.

  View details for DOI 10.1126/scisignal.aav0442

  View details for Web of Science ID 000448044800004

  View details for PubMedID 30352947

  View details for PubMedCentralID PMC6464114

• Discovery of Immunologically Inspired Small Molecules That Target the Viral Envelope Protein ACS INFECTIOUS DISEASES Lian, W., Jang, J., Potisopon, S., Li, P., Rahrneh, A., Wang, J., Kwiatkowski, N. P., Gray, N. S., Yang, P. L. 2018; 4 (9): 1395-1406

  Abstract

  Dengue virus is a major human pathogen that infects over 390 million people annually leading to approximately 500 000 hospitalizations due to severe dengue. Since the only marketed vaccine, Dengvaxia, has recently been shown to increase disease severity in those lacking natural immunity, antivirals to prevent or treat dengue  infection represent a large, unmet medical need. Small molecules that target the dengue virus envelope protein, E, on the surface of the virion could act analogously to antibodies by engaging E extracellularly to block infection; however, a shortage of target-based assays suitable for screening and medicinal chemistry studies has limited efforts in this area. Here we demonstrate that the dengue E protein offers a tractable drug target for preventing dengue infection by developing a target-based assay using a recombinantly expressed dengue serotype 2 E protein. We performed a high-throughput screen of ∼20 000 compounds followed by secondary assays to confirm target-binding and antiviral activity and counter-screens to exclude compounds with nonspecific activities. These efforts yielded eight distinct chemical leads that inhibit dengue infection by binding to E and preventing E-mediated membrane fusion with potencies equal to or greater than previously described small molecule inhibitors of E. We show that a subset of these compounds inhibit viruses representative of the other three dengue serotypes and Zika virus. This work provides tools for discovery and optimization of direct-acting antivirals against dengue E and shows that this approach may be useful in developing antivirals with broad-spectrum activity against other flavivirus pathogens.

  View details for DOI 10.1021/acsinfecdis.8b00127

  View details for Web of Science ID 000445053200014

  View details for PubMedID 30027735

  View details for PubMedCentralID PMC6392429

• Inhibition of Flaviviruses by Targeting a Conserved Pocket on the Viral Envelope Protein CELL CHEMICAL BIOLOGY de Wispelaere, M., Lian, W., Potisopon, S., Li, P., Jang, J., Ficarro, S. B., Clark, M. J., Zhu, X., Kaplan, J. B., Pitts, J. D., Wales, T. E., Wang, J., Engen, J. R., Marto, J. A., Gray, N. S., Yang, P. L. 2018; 25 (8): 1006-+

  Abstract

  Viral envelope proteins are required for productive viral entry and initiation of infection. Although the humoral immune system provides ample evidence for targeting envelope proteins as an antiviral strategy, there are few pharmacological interventions that have this mode of action. In contrast to classical antiviral targets such as viral proteases and polymerases, viral envelope proteins as a class do not have a well-conserved active site that can be rationally targeted with small molecules. We previously identified compounds that inhibit dengue virus by binding to its envelope protein, E. Here, we show that these small molecules inhibit dengue virus fusion and map the binding site of these compounds to a specific pocket on E. We further demonstrate inhibition of Zika, West Nile, and Japanese encephalitis viruses by these compounds, providing pharmacological evidence for the pocket as a target for developing broad-spectrum antivirals against multiple, mosquito-borne flavivirus pathogens.

  View details for DOI 10.1016/j.chembiol.2018.05.011

  View details for Web of Science ID 000441791200010

  View details for PubMedID 29937406

  View details for PubMedCentralID PMC6097893

• How small-molecule inhibitors of dengue-virus infection interfere with viral membrane fusion ELIFE Chao, L. H., Jang, J., Johnson, A., Nguyen, A., Gray, N. S., Yang, P. L., Harrison, S. C. 2018; 7

  Abstract

  Dengue virus (DV) is a compact, icosahedrally symmetric, enveloped particle, covered by 90 dimers of envelope protein (E), which mediates viral attachment and membrane fusion. Fusion requires a dimer-to-trimer transition and membrane engagement of hydrophobic 'fusion loops'. We previously characterized the steps in membrane fusion for the related West Nile virus (WNV), using recombinant, WNV virus-like particles (VLPs) for single-particle experiments (Chao et al., 2014). Trimerization and membrane engagement are rate-limiting; fusion requires at least two adjacent trimers; availability of competent monomers within the contact zone between virus and target membrane creates a trimerization bottleneck. We now report an extension of that work to dengue VLPs, from all four serotypes, finding an essentially similar mechanism. Small-molecule inhibitors of dengue virus infection that target E block its fusion-inducing conformational change. We show that ~12-14 bound molecules per particle (~20-25% occupancy) completely prevent fusion, consistent with the proposed mechanism.

  View details for DOI 10.7554/eLife.36461

  View details for Web of Science ID 000439413700001

  View details for PubMedID 29999491

  View details for PubMedCentralID PMC6056230

• Antiviral activity of N-(4-hydroxyphenyl) retinamide (4-HPR) against Zika virus ANTIVIRAL RESEARCH Pitts, J. D., Li, P., de Wispelaere, M., Yang, P. L. 2017; 147: 124-130

  Abstract

  The rapid spread of Zika virus (ZIKV) in recent years has highlighted the severe diseases associated with ZIKV infection, such as Guillain-Barré syndrome in adults and microcephaly in newborns; yet no vaccines or antivirals currently exist to prevent or treat ZIKV infection. We and others have previously identified N-(4-hydroxyphenyl) retinamide (fenretinide or 4-HPR) as an antiviral compound that inhibits dengue virus 2 (DV2) and other flaviviruses by limiting the steady-state accumulation of viral RNA. Here we show that 4-HPR potently inhibits ZIKV in mammalian cell culture and significantly reduces both serum viremia and brain viral burden in a murine model of ZIKV infection. Consistent with previous observations with dengue virus, this antiviral activity is associated with a significant reduction in the steady-state abundance of viral genomic RNA. We show this reduction is due to a major decrease in the rate of viral RNA synthesis, though not via direct inhibition of the activity of the viral replicase. These results establish 4-HPR's mode of action against DV and ZIKV and, taken with previous clinical trials that established 4-HPR's safety and tolerability, illustrate the potential utility of 4-HPR as an agent for treatment of ZIKV infection.

  View details for DOI 10.1016/j.antiviral.2017.10.014

  View details for Web of Science ID 000416394900015

  View details for PubMedID 29051080

  View details for PubMedCentralID PMC5703041

• Structure-Activity Relationship Study of QL47: A Broad-Spectrum Antiviral Agent ACS MEDICINAL CHEMISTRY LETTERS Liang, Y., de Wispelaere, M., Carocci, M., Liu, Q., Wang, J., Yang, P. L., Gray, N. S. 2017; 8 (3): 344-349

  Abstract

  Here we report the structure-activity relationship (SAR) investigations of QL-XII-47 (QL47), a compound that possesses broad-spectrum antiviral activity against dengue virus and other RNA viruses. A medicinal chemistry campaign initiated from QL47, a previously reported covalent BTK inhibitor, to derive YKL-04-085, which is devoid of any kinase activity when screened against a panel of 468 kinases and with improved pharmacokinetic properties. Both QL47 and YKL-04-085 are potent inhibitors of viral translation and exhibit cellular antiviral activity at 35-fold lower concentrations relative to inhibition of host-cell proliferation.

  View details for DOI 10.1021/acsmedchemlett.7b00008

  View details for Web of Science ID 000396297700016

  View details for PubMedID 28337328

• Discovery of host-targeted covalent inhibitors of dengue virus ANTIVIRAL RESEARCH de Wispelaere, M., Carocci, M., Liang, Y., Liu, Q., Sun, E., Vetter, M. L., Wang, J., Gray, N. S., Yang, P. L. 2017; 139: 171-179

  Abstract

  We report here on an approach targeting the host reactive cysteinome to identify inhibitors of host factors required for the infectious cycle of Flaviviruses and other viruses. We used two parallel cellular phenotypic screens to identify a series of covalent inhibitors, exemplified by QL-XII-47, that are active against dengue virus. We show that the compounds effectively block viral protein expression and that this inhibition is associated with repression of downstream processes of the infectious cycle, and thus significantly contributes to the potent antiviral activity of these compounds. We demonstrate that QL-XII-47's antiviral activity requires selective, covalent modification of a host target by showing that the compound's antiviral activity is recapitulated when cells are preincubated with QL-XII-47 and then washed prior to viral infection and by showing that QL-XII-47R, a non-reactive analog, lacks antiviral activity at concentrations more than 20-fold higher than QL-XII-47's IC90. QL-XII-47's inhibition of Zika virus, West Nile virus, hepatitis C virus, and poliovirus further suggests that it acts via a target mediating inhibition of these other medically relevant viruses. These results demonstrate the utility of screens targeting the host reactive cysteinome for rapid identification of compounds with potent antiviral activity.

  View details for DOI 10.1016/j.antiviral.2016.12.017

  View details for Web of Science ID 000394397100021

  View details for PubMedID 28034743

• Desmosterol Increases Lipid Bilayer Fluidity during Hepatitis C Virus Infection ACS INFECTIOUS DISEASES Costello, D. A., Villareal, V. A., Yang, P. L. 2016; 2 (11): 852-862

  Abstract

  Hepatitis C virus (HCV) uniquely affects desmosterol homeostasis by increasing its intracellular abundance and affecting its localization. These effects are important for productive viral replication because the inhibition of desmosterol synthesis has an antiviral effect that can be rescued by the addition of exogenous desmosterol. Here, we use subgenomic replicons to show that desmosterol has a major effect on the replication of HCV JFH1 RNA. Fluorescence recovery after photobleaching (FRAP) experiments performed with synthetic supported lipid bilayers demonstrate that the substitution of desmosterol for cholesterol significantly increases the lipid bilayer fluidity, especially in the presence of saturated phospholipids and ceramides. We demonstrate using LC-MS that desmosterol is abundant in the membranes upon which genome replication takes place and that supported lipid bilayers derived from these specialized membranes also exhibit significantly higher fluidity compared to that of negative control membranes isolated from cells lacking HCV. Together, these data suggest a model in which the fluidity-promoting effects of desmosterol on lipid bilayers play a crucial role in the extensive membrane remodeling that takes place in the endoplasmic reticulum during HCV infection. We anticipate that the supported lipid bilayer system described can provide a useful model system in which to interrogate the effects of lipid structure and composition on the biophysical properties of lipid membranes as well as their function in viral processes such as genome replication.

  View details for DOI 10.1021/acsinfecdis.6b00086

  View details for Web of Science ID 000388161300012

  View details for PubMedID 27933788

  View details for PubMedCentralID PMC5161114

• Hepatitis C Virus Selectively Alters the Intracellular Localization of Desmosterol ACS CHEMICAL BIOLOGY Villareal, V. A., Fu, D., Costello, D. A., Xie, X. S., Yang, P. L. 2016; 11 (7): 1827-1833

  Abstract

  Hepatitis C virus (HCV) increases intracellular desmosterol without affecting the steady-state abundance of other sterols, and the antiviral activity of inhibitors of desmosterol synthesis is suppressed by the addition of exogenous desmosterol. These observations suggest a model in which desmosterol has a specific function, direct or indirect, in HCV replication and that HCV alters desmosterol homeostasis to promote viral replication. Here, we use stimulated Raman scattering (SRS) microscopy in combination with isotopically labeled sterols to show that HCV causes desmosterol to accumulate in lipid droplets that are closely associated with the viral NS5A protein and that are visually distinct from the broad distribution of desmosterol in mock-infected cells and the more heterogeneous and disperse lipid droplets to which cholesterol traffics. Localization of desmosterol in NS5A-associated lipid droplets suggests that desmosterol may affect HCV replication via a direct mechanism. We anticipate that SRS microscopy and similar approaches can provide much needed tools to study the localization of specific lipid molecules in cellulo and in vivo.

  View details for DOI 10.1021/acschembio.6b00324

  View details for Web of Science ID 000380181900009

  View details for PubMedID 27128812

  View details for PubMedCentralID PMC5025300

• Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor JOURNAL OF VIROLOGY Chou, Y., Cuevas, C., Carocci, M., Stubbs, S. H., Ma, M., Cureton, D. K., Chao, L., Evesson, F., He, K., Yang, P. L., Whelan, S. P., Ross, S. R., Kirchhausen, T., Gaudina, R. 2016; 90 (9): 4494-4510

  Abstract

  Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens.The Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential.

  View details for DOI 10.1128/JVI.00103-16

  View details for Web of Science ID 000375125400022

  View details for PubMedID 26912630

  View details for PubMedCentralID PMC4836360

• GNF-2 Inhibits Dengue Virus by Targeting Abl Kinases and the Viral E Protein CELL CHEMICAL BIOLOGY Clark, M. J., Miduturu, C., Schmidt, A. G., Zhu, X., Pitts, J. D., Wang, J., Potisopon, S., Zhang, J., Wojciechowski, A., Chu, J., Gray, N. S., Yang, P. L. 2016; 23 (4): 443-452

  Abstract

  Dengue virus infects more than 300 million people annually, yet there is no widely protective vaccine or drugs against the virus. Efforts to develop antivirals against classical targets such as the viral protease and polymerase have not yielded drugs that have advanced to the clinic. Here, we show that the allosteric Abl kinase inhibitor GNF-2 interferes with dengue virus replication via activity mediated by cellular Abl kinases but additionally blocks viral entry via an Abl-independent mechanism. To characterize this newly discovered antiviral activity, we developed disubstituted pyrimidines that block dengue virus entry with structure-activity relationships distinct from those driving kinase inhibition. We demonstrate that biotin- and fluorophore-conjugated derivatives of GNF-2 interact with the dengue glycoprotein, E, in the pre-fusion conformation that exists on the virion surface, and that this interaction inhibits viral entry. This study establishes GNF-2 as an antiviral compound with polypharmacological activity and provides "lead" compounds for further optimization efforts.

  View details for DOI 10.1016/j.chembiol.2016.03.010

  View details for Web of Science ID 000381508400005

  View details for PubMedID 27105280

  View details for PubMedCentralID PMC4865888

• Lactimidomycin is a broad-spectrum inhibitor of dengue and other RNA viruses ANTIVIRAL RESEARCH Carocci, M., Yang, P. L. 2016; 128: 57-62

  Abstract

  Dengue virus, a member of the Flaviviridae family, is a mosquito-borne pathogen and the causative agent of dengue fever. Despite the nearly 400 million new infections estimated annually, no vaccines or specific antiviral therapeutics are currently available. We identified lactimidomycin (LTM), a recently established inhibitor of translation elongation, as a potent inhibitor of dengue virus 2 infection in cell culture. The antiviral activity is observed at concentrations that do not affect cell viability. We show that Kunjin virus and Modoc virus, two other members of the Flavivirus genus, as well as vesicular stomatitis virus and poliovirus 1, are also sensitive to LTM. Our findings suggest that inhibition of translation elongation, an obligate step in the viral replication cycle, may provide a general antiviral strategy against fast-replicating RNA viruses.

  View details for DOI 10.1016/j.antiviral.2016.02.005

  View details for Web of Science ID 000372765000008

  View details for PubMedID 26872864

  View details for PubMedCentralID PMC4850914

• Targeting host lipid synthesis and metabolism to inhibit dengue and hepatitis C viruses ANTIVIRAL RESEARCH Villareal, V. A., Rodgers, M. A., Costello, D. A., Yang, P. L. 2015; 124: 110-121

  Abstract

  Lipids are necessary for every step in the replication cycle of hepatitis C virus (HCV) and dengue virus (DENV), members of the family Flaviviridae. Recent studies have demonstrated that discrete steps in the replication cycles of these viruses can be inhibited by pharmacological agents that target host factors mediating lipid synthesis, metabolism, trafficking, and signal transduction. Despite this, targeting host lipid metabolism and trafficking as an antiviral strategy by blockade of entire pathways may be limited due to host toxicity. Knowledge of the molecular details of lipid structure and function in replication and the mechanisms whereby specific lipids are generated and trafficked to the relevant sites may enable more targeted antiviral strategies without global effects on the host cell. In this review, we discuss lipids demonstrated to be critical to the replication cycles of HCV and DENV and highlight potential areas for anti-viral development. This review article forms part of a symposium on flavivirus drug discovery in Antiviral Research.

  View details for DOI 10.1016/j.antiviral.2015.10.013

  View details for Web of Science ID 000370463200013

  View details for PubMedID 26526588

  View details for PubMedCentralID PMC4699661

• Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics. Proceedings of the National Academy of Sciences of the United States of America Inci, F., Filippini, C., Baday, M., Ozen, M. O., Calamak, S., Durmus, N. G., Wang, S., Hanhauser, E., Hobbs, K. S., Juillard, F., Kuang, P. P., Vetter, M. L., Carocci, M., Yamamoto, H. S., Takagi, Y., Yildiz, U. H., Akin, D., Wesemann, D. R., Singhal, A., Yang, P. L., Nibert, M. L., Fichorova, R. N., Lau, D. T., Henrich, T. J., Kaye, K. M., Schachter, S. C., Kuritzkes, D. R., Steinmetz, L. M., Gambhir, S. S., Davis, R. W., Demirci, U. 2015; 112 (32): E4354-63

  Abstract

  Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

  View details for DOI 10.1073/pnas.1510824112

  View details for PubMedID 26195743

  View details for PubMedCentralID PMC4538635

• Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device ((NERD)-R-2) for diagnostics PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Inci, F., Filippini, C., Baday, M., Ozen, M. O., Calamak, S., Durmus, N. G., Wang, S., Hanhauser, E., Hobbs, K. S., Juillard, F., Kuang, P. P., Vetter, M. L., Carocci, M., Yamamoto, H. S., Takagi, Y., Yildiz, U. H., Akin, D., Wesemann, D. R., Singhal, A., Yang, P. L., Nibert, M. L., Fichorova, R. N., Lau, D. T., Henrich, T. J., Kaye, K. M., Schachter, S. C., Kuritzkes, D. R., Steinmetz, L. M., Gambhir, S. S., Davis, R. W., Demirci, U. 2015; 112 (32): E4354-E4363

  Abstract

  Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

  View details for DOI 10.1073/pnas.1510824112

  View details for Web of Science ID 000359285100006

  View details for PubMedID 26195743

  View details for PubMedCentralID PMC4538635

• The Bioactive Lipid 4-Hydroxyphenyl Retinamide Inhibits Flavivirus Replication ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Carocci, M., Hinshaw, S. M., Rodgers, M. A., Villareal, V. A., Burri, D. J., Pilankatta, R., Maharaj, N. P., Gack, M. U., Stavale, E. J., Warfield, K. L., Yang, P. L. 2015; 59 (1): 85–95

  Abstract

  Dengue virus (DENV), a member of the Flaviviridae family, is a mosquito-borne pathogen and the cause of dengue fever. The increasing prevalence of DENV worldwide heightens the need for an effective vaccine and specific antivirals. Due to the dependence of DENV upon the lipid biosynthetic machinery of the host cell, lipid signaling and metabolism present unique opportunities for inhibiting viral replication. We screened a library of bioactive lipids and modulators of lipid metabolism and identified 4-hydroxyphenyl retinamide (4-HPR) (fenretinide) as an inhibitor of DENV in cell culture. 4-HPR inhibits the steady-state accumulation of viral genomic RNA and reduces viremia when orally administered in a murine model of DENV infection. The molecular target responsible for this antiviral activity is distinct from other known inhibitors of DENV but appears to affect other members of the Flaviviridae, including the West Nile, Modoc, and hepatitis C viruses. Although long-chain ceramides have been implicated in DENV replication, we demonstrate that DENV is insensitive to the perturbation of long-chain ceramides in mammalian cell culture and that the effect of 4-HPR on dihydroceramide homeostasis is separable from its antiviral activity. Likewise, the induction of reactive oxygen species by 4-HPR is not required for the inhibition of DENV. The inhibition of DENV in vivo by 4-HPR, combined with its well-established safety and tolerability in humans, suggests that it may be repurposed as a pan-Flaviviridae antiviral agent. This work also illustrates the utility of bioactive lipid screens for identifying critical interactions of DENV and other viral pathogens with host lipid biosynthesis, metabolism, and signal transduction.

  View details for DOI 10.1128/AAC.04177-14

  View details for Web of Science ID 000348609500010

  View details for PubMedID 25313218

  View details for PubMedCentralID PMC4291433

• Fluorescent Visualization of Src by Using Dasatinib-BODIPY CHEMBIOCHEM Vetter, M. L., Zhang, Z., Liu, S., Wang, J., Cho, H., Zhang, J., Zhang, W., Gray, N. S., Yang, P. L. 2014; 15 (9): 1317-1324

  Abstract

  Many biological experiments are not compatible with the use of immunofluorescence, genetically encoded fluorescent tags, or FRET-based reporters. Conjugation of existing kinase inhibitors to cell-permeable fluorophores can provide a generalized approach to develop fluorescent probes of intracellular kinases. Here, we report the development of a small molecule probe of Src through conjugation of BODIPY to two well-established dual Src-Abl kinase inhibitors, dasatinib and saracatinib. We show that this approach is not successful for saracatinib but that dasatinib-BODIPY largely retains the biological activity of its parent compound and can be used to monitor the presence of Src kinase in individual cells by flow cytometry. It can also be used to track the localization of Src by fixed and live-cell fluorescence microscopy. This strategy could enable generation of additional kinase-specific probes useful in systems not amenable to genetic manipulation or could be used together with fluorescent proteins to enable a multiplexed assay readout.

  View details for DOI 10.1002/cbic.201402010

  View details for Web of Science ID 000337638400014

  View details for PubMedID 24828915

• The Small Molecules AZD0530 and Dasatinib Inhibit Dengue Virus RNA Replication via Fyn Kinase JOURNAL OF VIROLOGY de Wispelaere, M., LaCroix, A. J., Yang, P. L. 2013; 87 (13): 7367-7381

  Abstract

  In this study, we characterized the antiviral mechanism of action of AZD0530 and dasatinib, two pharmacological inhibitors of host kinases, that also inhibit dengue virus (DV) infection. Using Northern blot and reporter replicon assays, we demonstrated that both small molecules inhibit the DV2 infectious cycle at the step of steady-state RNA replication. In order to identify the cellular target of AZD0530 and dasatinib mediating this anti-DV2 activity, we examined the effects of RNA interference (RNAi)-mediated depletion of the major kinases known to be inhibited by these small molecules. We determined that Fyn kinase, a target of both AZD0530 and dasatinib, is involved in DV2 RNA replication and is probably a major mediator of the anti-DV activity of these compounds. Furthermore, serial passaging of DV2 in the presence of dasatinib led to the identification of a mutation in the transmembrane domain 3 of the NS4B protein that overcomes the inhibition of RNA replication by AZD0530, dasatinib, and Fyn RNAi. Although we observed that dasatinib also inhibits DV2 particle assembly and/or secretion, this activity does not appear to be mediated by Src-family kinases. Together, our results suggest that AZD0530 and dasatinib inhibit DV at the step of viral RNA replication and demonstrate a critical role for Fyn kinase in this viral process. The antiviral activity of these compounds in vitro makes them useful pharmacological tools to validate Fyn or other host kinases as anti-DV targets in vivo.

  View details for DOI 10.1128/JVI.00632-13

  View details for Web of Science ID 000320116500016

  View details for PubMedID 23616652

  View details for PubMedCentralID PMC3700292

• Chemoproteomic Profiling Identifies Changes in DNA-PK as Markers of Early Dengue Virus Infection ACS CHEMICAL BIOLOGY Vetter, M. L., Rodgers, M. A., Patricelli, M. P., Yang, P. L. 2012; 7 (12): 2019-2026

  Abstract

  Many cellular factors are regulated via mechanisms affecting protein conformation, localization, and function that may be undetected by most commonly used RNA- and protein-based profiling methods that monitor steady-state gene expression. Mass-spectrometry-based chemoproteomic profiling provides alternatives for interrogating changes in the functional properties of proteins that occur in response to biological stimuli, such as viral infection. Taking dengue virus 2 (DV2) infection as a model system, we utilized reactive ATP- and ADP-acyl phosphates as chemical proteomic probes to detect changes in host kinase function that occur within the first hour of infection. The DNA-dependent protein kinase (DNA-PK) was discovered as a host enzyme with significantly elevated probe labeling within 60 min of DV2 infection. Increased probe labeling was associated with increased DNA-PK activity in nuclear lysates and localization of DNA-PK in nucleoli. These effects on DNA-PK were found to require a postfusion step of DV2 entry and were recapitulated by transfection of cells with RNA corresponding to stem loop B of the DV2 5' untranslated region. Upon investigation of the potential downstream consequences of these phenomena, we detected a modest but significant reduction in the interferon response induced by DV2 in cells partially depleted of the Ku80 subunit of DNA-PK. These findings identify changes in DNA-PK localization and activity as very early markers of DV2 infection. More broadly, these results highlight the utility of chemoproteomic profiling as a tool to detect changes in protein function associated with different cell states and that may occur on very short time scales.

  View details for DOI 10.1021/cb300420z

  View details for Web of Science ID 000312564000011

  View details for PubMedID 22999307

  View details for PubMedCentralID PMC3528803

• Mutagenesis of the DI/DIII Linker in Dengue Virus Envelope Protein Impairs Viral Particle Assembly JOURNAL OF VIROLOGY de Wispelaere, M., Yang, P. L. 2012; 86 (13): 7072-7083

  Abstract

  The dengue virus (DV) envelope (E) protein is important in mediating viral entry and assembly of progeny virus during cellular infection. Domains I and III (DI and DIII, respectively) of the DV E protein are connected by a highly conserved but poorly ordered region, the DI/DIII linker. Although the flexibility of the DI/DIII linker is thought to be important for accommodating the structural rearrangements undergone by the E protein during viral entry, the function of the linker in the DV infectious cycle is not well understood. In this study, we performed site-directed mutagenesis on conserved residues in the DI/DIII linker of the DV2 E protein and showed that the resulting mutations had little or no effect on the entry process but greatly affected virus assembly. Biochemical fractionation and immunofluorescence microscopy experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the DI/DIII linker mutants express the DV structural proteins at the sites of particle assembly near the ER but fail to form infectious particles. This defect is not due to disruption of E's interaction with prM and pr in immature and mature virions, respectively. Serial passaging of the DV2 mutant E-Y299F led to the identification of a mutation in the membrane-proximal stem region of E that fully compensates for the assembly defect of this DI/DIII linker mutant. Together, our results suggest a critical and previously unidentified role for the E protein DI/DIII linker region during the DV2 assembly process.

  View details for DOI 10.1128/JVI.00224-12

  View details for Web of Science ID 000305501600006

  View details for PubMedID 22532681

  View details for PubMedCentralID PMC3416339

• Lipid Metabolite Profiling Identifies Desmosterol Metabolism as a New Antiviral Target for Hepatitis C Virus JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rodgers, M. A., Villareal, V. A., Schaefer, E. A., Peng, L. F., Corey, K. E., Chung, R. T., Yang, P. L. 2012; 134 (16): 6896-6899

  Abstract

  Hepatitis C virus (HCV) infection has been clinically associated with serum lipid abnormalities, yet our understanding of the effects of HCV on host lipid metabolism and conversely the function of individual lipids in HCV replication remains incomplete. Using liquid chromatography-mass spectrometry metabolite profiling of the HCV JFH1 cell culture infection model, we identified a significant steady-state accumulation of desmosterol, an immediate precursor to cholesterol. Pharmacological inhibition or RNAi-mediated depletion of DHCR7 significantly reduced steady-state HCV protein expression and viral genomic RNA. Moreover, this effect was reversed when cultures were supplemented with exogenous desmosterol. Together, these observations suggest an intimate connection between HCV replication and desmosterol homeostasis and that the enzymes responsible for synthesis of desmosterol may be novel targets for antiviral design.

  View details for DOI 10.1021/ja207391q

  View details for Web of Science ID 000303139800003

  View details for PubMedID 22480142

  View details for PubMedCentralID PMC3375380

• Small-Molecule Inhibitors of Dengue-Virus Entry PLOS PATHOGENS Schmidt, A. G., Lee, K., Yang, P. L., Harrison, S. C. 2012; 8 (4)

  Abstract

  Flavivirus envelope protein (E) mediates membrane fusion and viral entry from endosomes. A low-pH induced, dimer-to-trimer rearrangement and reconfiguration of the membrane-proximal "stem" of the E ectodomain draw together the viral and cellular membranes. We found stem-derived peptides from dengue virus (DV) bind stem-less E trimer and mimic the stem-reconfiguration step in the fusion pathway. We adapted this experiment as a high-throughput screen for small molecules that block peptide binding and thus may inhibit viral entry. A compound identified in this screen, 1662G07, and a number of its analogs reversibly inhibit DV infectivity. They do so by binding the prefusion, dimeric E on the virion surface, before adsorption to a cell. They also block viral fusion with liposomes. Structure-activity relationship studies have led to analogs with submicromolar IC₉₀s against DV2, and certain analogs are active against DV serotypes 1,2, and 4. The compounds do not inhibit the closely related Kunjin virus. We propose that they bind in a previously identified, E-protein pocket, exposed on the virion surface and although this pocket is closed in the postfusion trimer, its mouth is fully accessible. Examination of the E-trimer coordinates (PDB 1OK8) shows that conformational fluctuations around the hinge could open the pocket without dissociating the trimer or otherwise generating molecular collisions. We propose that compounds such as 1662G07 trap the sE trimer in a "pocket-open" state, which has lost affinity for the stem peptide and cannot support the final "zipping up" of the stem.

  View details for DOI 10.1371/journal.ppat.1002627

  View details for Web of Science ID 000303444200023

  View details for PubMedID 22496653

  View details for PubMedCentralID PMC3320583

• Anti-HCV drugs in the pipeline CURRENT OPINION IN VIROLOGY Yang, P. L., Gao, M., Lin, K., Liu, Q., Villareal, V. A. 2011; 1 (6): 607-616

  Abstract

  Several directly acting and host targeting antivirals that inhibit hepatitis C virus replication have entered clinical trials. Among the most advanced of these are RG7128, an inhibitor of the NS5B polymerase; BMS-790052, an inhibitor of NS5A; and alisporivir, an inhibitor of human cyclophilins. These agents have potent antiviral activity in chronic HCV patients, act additively or synergistically with inhibitors of the HCV NS3/4A protease, and improve the rate of virologic response produced by traditional pegylated interferon plus ribavirin therapy. No cross resistance has been observed; moreover, nucleoside NS5B and cyclophilin inhibitors appear to suppress resistance to non-nucleoside NS5B and NS3/4A inhibitors. Several recent reports of virologic responses produced by combinations of agents that inhibit HCV replication in the absence of interferon provide optimism that eradication of HCV will be possible without interferon in the future.

  View details for DOI 10.1016/j.coviro.2011.10.019

  View details for Web of Science ID 000312112500020

  View details for PubMedCentralID PMC3775341

• Peptide Inhibitors of Flavivirus Entry Derived from the E Protein Stem JOURNAL OF VIROLOGY Schmidt, A. G., Yang, P. L., Harrison, S. C. 2010; 84 (24): 12549-12554

  Abstract

  Peptides derived from the "stem" of dengue virus (DV) type 2 (DV2) envelope (E) protein inhibit DV2 infectivity, targeting a late-stage fusion intermediate. We show here that stem peptides from all DV serotypes cross-inhibit DV1 to DV4 but that corresponding peptides derived from related flaviviruses do not. This failure to inhibit infection is not due to poor interaction with the E protein but rather to loss of association with the virion membrane. Residues 442 to 444 of the stem are determinants of inhibition; increasing hydrophobicity in this region increases inhibitory strength. These results support a two-step model of how stem-derived peptides inhibit viral entry.

  View details for DOI 10.1128/JVI.01440-10

  View details for Web of Science ID 000284469600008

  View details for PubMedID 20881042

  View details for PubMedCentralID PMC3004314

• Peptide Inhibitors of Dengue-Virus Entry Target a Late-Stage Fusion Intermediate PLOS PATHOGENS Schmidt, A. G., Yang, P. L., Harrison, S. C. 2010; 6 (4): e1000851

  Abstract

  The mechanism of membrane fusion by "class II" viral fusion proteins follows a pathway that involves large-scale domain rearrangements of the envelope glycoprotein (E) and a transition from dimers to trimers. The rearrangement is believed to proceed by an outward rotation of the E ectodomain after loss of the dimer interface, followed by a reassociation into extended trimers. The approximately 55-aa-residue, membrane proximal "stem" can then zip up along domain II, bringing together the transmembrane segments of the C-terminus and the fusion loops at the tip of domain II. We find that peptides derived from the stem of dengue-virus E bind stem-less E trimer, which models a conformational intermediate. In vitro assays demonstrate that these peptides specifically block viral fusion. The peptides inhibit infectivity with potency proportional to their affinity for the conformational intermediate, even when free peptide is removed from a preincubated inoculum before infecting cells. We conclude that peptides bind virions before attachment and are carried with virions into endosomes, the compartment in which acidification initiates fusion. Binding depends on particle dynamics, as there is no inhibition of infectivity if preincubation and separation are at 4 degrees C rather than 37 degrees C. We propose a two-step model for the mechanism of fusion inhibition. Targeting a viral entry pathway can be an effective way to block infection. Our data, which support and extend proposed mechanisms for how the E conformational change promotes membrane fusion, suggest strategies for inhibiting flavivirus entry.

  View details for DOI 10.1371/journal.ppat.1000851

  View details for Web of Science ID 000277722400023

  View details for PubMedID 20386713

  View details for PubMedCentralID PMC2851732

• Identification of an Overabundant Cholesterol Precursor in Hepatitis B Virus Replicating Cells by Untargeted Lipid Metabolite Profiling JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rodgers, M. A., Saghatelian, A., Yang, P. L. 2009; 131 (14): 5030-+

  Abstract

  Viruses rely upon host lipid metabolic pathways for successful replication, and there is increasing interest in these pathways as novel therapeutic targets for antiviral drug discovery. Despite this, relatively little is known about the impact of viral infection on cellular lipid metabolism, and the specific lipid metabolites utilized by viruses have not yet been examined. We have applied liquid chromatography-mass spectroscopy (LC-MS) based untargeted metabolite profiling to identify lipid metabolites whose steady-state abundance is significantly altered by replication of hepatitis B virus (HBV), a major human pathogen. Untargeted metabolite profiling indicated that although major lipid classes were unaffected by HBV, an ion of 367 m/z was overabundant in HBV+ cells by 18-fold. As shown by ion fragmentation mass spectrometry and coinjection with standard, the identity of this ion is 7-dehydrocholesterol (7-DHC), an immediate dehydrogenated precursor to cholesterol. While cholesterol has previously been demonstrated to be essential in the replication of many viruses, this is the first to show that viral replication is associated with the selective accumulation of 7-DHC. Most virological studies to date have relied upon methods that deplete all sterols and preclude the observation of any selectivity in sterol utilization by viral pathogens. Our study suggests that HBV may selectively utilize 7-DHC versus other sterols and prompts experiments investigating the functional significance of this enrichment and the elucidation of the mechanism by which it is achieved. The results also highlight the value of untargeted metabolite profiling as a method for identifying critical metabolites for viral infection.

  View details for DOI 10.1021/ja809949r

  View details for Web of Science ID 000265039000010

  View details for PubMedID 19301856

  View details for PubMedCentralID PMC4166558

• Targeting cancer with small molecule kinase inhibitors NATURE REVIEWS CANCER Zhang, J., Yang, P. L., Gray, N. S. 2009; 9 (1): 28-39

  Abstract

  Deregulation of kinase activity has emerged as a major mechanism by which cancer cells evade normal physiological constraints on growth and survival. To date, 11 kinase inhibitors have received US Food and Drug Administration approval as cancer treatments, and there are considerable efforts to develop selective small molecule inhibitors for a host of other kinases that are implicated in cancer and other diseases. Herein we discuss the current challenges in the field, such as designing selective inhibitors and developing strategies to overcome resistance mutations. This Review provides a broad overview of some of the approaches currently used to discover and characterize new kinase inhibitors.

  View details for DOI 10.1038/nrc2559

  View details for Web of Science ID 000261929700017

  View details for PubMedID 19104514

• c-Src protein kinase inhibitors block assembly and maturation of dengue virus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chu, J. H., Yang, P. L. 2007; 104 (9): 3520-3525

  Abstract

  Dengue virus is a mosquito-borne flavivirus that represents an important emerging infectious disease and is an international health concern. Currently, there is no vaccine or effective antiviral therapy to prevent or to treat dengue virus infection. The slow progress in developing antiviral agents might be alleviated by the availability of efficient high-throughput anti-dengue virus screening assays. In this study, we report an immunofluorescence image-based assay suitable for identification of small molecule inhibitors of dengue virus infection and replication. Using this assay, we have discovered that inhibitors of the c-Src protein kinase exhibit a potent inhibitory effect on dengue virus (serotypes 1-4) and murine flavivirus Modoc. Mechanism of action studies demonstrated that the c-Src protein kinase inhibitor dasatinib prevents the assembly of dengue virions within the virus-induced membranous replication complex. These results demonstrate that this cell-based screen may provide a powerful means to identify new potential targets for anti-dengue drug development while simultaneously providing pharmacological probes to investigate dengue virus-host cell interactions at the biochemical level. Given the simplicity and excellent reproducibility of the assay, it should be useful in high-throughput screens of both small molecule and RNAi libraries when implemented on a robotic image-based high-throughput screen (HTS) platform. Given the reasonable clinical safety of inhibitors such as dasatinib and AZD0530, inhibitors of c-Src protein kinase may have the potential to become a new class of anti-dengue viral therapeutic agents.

  View details for DOI 10.1073/pnas.0611681104

  View details for Web of Science ID 000244661400088

  View details for PubMedID 17360676

  View details for PubMedCentralID PMC1805510

• The immunological evolution of catalysis SCIENCE Patten, P. A., Gray, N. S., Yang, P. L., Marks, C. B., Wedemayer, G. J., Boniface, J. J., Stevens, R. C., Schultz, P. G. 1996; 271 (5252): 1086–91

  Abstract

  The germline genes used by the mouse to generate the esterolytic antibody 48G7 were cloned and expressed in an effort to increase our understanding of the detailed molecular mechanisms by which the immune system evolves catalytic function. The nine replacement mutations that were fixed during affinity maturation increased affinity for the transition state analogue by a factor of 10(4), primarily the result of a decrease in the dissociation rate of the hapten-antibody complex. There was a corresponding increase in the rate of reaction of antibody with substrate, k(cat)/k(m), from 1.7 x 10(2)M(-1) min(-1) to 1.4 x 10(4)M(-1) min(-1). The three-dimensional crystal structure of the 48G7-transition state analogue complex at 2.0 angstroms resolution indicates that one of the nine residues in which somatic mutations have been fixed directly contact the hapten. Thus, in the case of 48G7, affinity maturation appears to play a conformational role, either in reorganizing the active site geometry of limiting side-chain and backbone flexibility of the germline antibody. The crystal structure and analysis of somatic and directed active site mutants underscore the role of transition state stabilization in the evolution of this catalytic antibody.

  View details for DOI 10.1126/science.271.5252.1086

  View details for Web of Science ID A1996TW70100029

  View details for PubMedID 8599084