scholarly journals Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 471 ◽  
Author(s):  
Sewar Alkhashrom ◽  
Jintawee Kicuntod ◽  
Sigrun Häge ◽  
Johannes Schweininger ◽  
Yves A. Muller ◽  
...  
Keyword(s):  
Small Molecule ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Small Molecule Inhibitors ◽  
Inhibitory Effect ◽  
Drug Candidate ◽  
Chemical Library ◽  
Nuclear Egress ◽  
Complex Core ◽  
New Type

Nuclear egress is an essential process in the replication of human cytomegalovirus (HCMV), as it enables the migration of newly formed viral capsids from the nucleus into the cytoplasm. Inhibition of the HCMV core nuclear egress complex (core NEC), composed of viral proteins pUL50 and pUL53, has been proposed as a potential new target for the treatment of HCMV infection and disease. Here, we present a new type of small molecule inhibitors of HCMV core NEC formation, which inhibit the pUL50-pUL53 interaction at nanomolar concentrations. These inhibitors, i.e., verteporfin and merbromin, were identified through the screening of the Prestwick Chemical Library® of approved drug compounds. The inhibitory effect of merbromin is both compound- and target-specific, as no inhibition was seen for other mercury-organic compounds. Furthermore, merbromin does not inhibit an unrelated protein–protein interaction either. More importantly, merbromin was found to inhibit HCMV infection of cells in three different assays, as well as to disrupt HCMV NEC nuclear rim formation. Thus, while not being an ideal drug candidate by itself, merbromin may serve as a blueprint for small molecules with high HCMV core NEC inhibitory potential, as candidates for novel anti-herpesviral drugs.

scholarly journals Human Cytomegalovirus Drives WDR5 to the Virion Assembly Compartment to Facilitate Virion Assembly

2020 ◽  
Author(s):  
Bo Yang ◽  
YongXuan Yao ◽  
Hui Wu ◽  
Hong Yang ◽  
Xue-Hui Ma ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Antiviral Treatment ◽  
Critical Role ◽  
Virion Assembly ◽  
Potential Target ◽  
Nuclear Egress ◽  
Early Endosomes ◽  
Hcmv Replication ◽  
Assembly Compartment

AbstractWe previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WDR5 to facilitate capsid nuclear egress. Here, we further show that HCMV infection drives WDR5 to the perinuclear region by a mechanism that requires viral replication and intact microtubules. WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 interacted with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain the increasing WDR5 accumulation in the vAC during infection. WDR5 was then incorporated into HCMV virions and localized to the tegument layer, as demonstrated by fractionation and immune-gold electron microscopy. Thus, WDR5 is driven to the vAC and incorporated into virions, suggesting that WDR5 facilitates HCMV replication at later stage of virion assembly besides the capsid nuclear egress stage. These data highlight that WDR5 is a potential target for antiviral therapy.ImportanceHuman cytomegalovirus (HCMV) has a large (~235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are profoundly reconfigured to establish the virion assembly compartment (vAC), which is important for efficient assembly of progeny virions. We previously reported that WDR5 promotes HCMV nuclear egress. Here, we show that WDR5 is further driven to the vAC and incorporated into virions, perhaps to facilitate efficient virion maturation. This work identified potential roles for WDR5 in HCMV replication in the cytoplasmic stages of virion assembly. Taken together, WDR5 plays a critical role in HCMV capsid nuclear egress and is important for virion assembly, and thus is a potential target for antiviral treatment of HCMV-associated diseases.

scholarly journals The Human Cytomegalovirus Transmembrane Protein pUL50 Induces Loss of VCP/p97 and Is Regulated by a Small Isoform of pUL50

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Myoung Kyu Lee ◽  
Seokhwan Hyeon ◽  
Jin-Hyun Ahn
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Transmembrane Domain ◽  
Transmembrane Protein ◽  
Amino Acid Position ◽  
Mutant Virus ◽  
Early Gene ◽  
Immediate Early ◽  
Nuclear Egress ◽  
The Unfolded Protein Response

ABSTRACT The human cytomegalovirus (HCMV) UL50 gene encodes a transmembrane protein, pUL50, which acts as a core component of the nuclear egress complex (NEC) for nucleocapsids. Recently, pUL50 has been shown to have NEC-independent activities: downregulation of IRE1 to repress the unfolded protein response and degradation of UBE1L to inhibit the protein ISG15 modification pathway. Here, we demonstrate that a 26-kDa N-terminal truncated isoform of pUL50 (UL50-p26) is expressed from an internal methionine at amino acid position 199 and regulates the activity of pUL50 to induce the loss of valosin-containing protein (VCP/p97). A UL50(M199V) mutant virus expressing pUL50(M199V) but not UL50-p26 showed delayed growth at a low multiplicity of infection. There was also delayed accumulation of the viral immediate early 2 (IE2) protein in the mutant virus, and this correlated with the reduced expression of VCP/p97, which promotes IE2 expression. Infection with mutant virus did not significantly alter ISGylation levels. In transient expression assays, pUL50 induced VCP/p97 loss posttranscriptionally, and this was dependent on the presence of its transmembrane domain. In contrast, UL50-p26 did not destabilize VCP/p97 but, rather, inhibited pUL50-mediated VCP/p97 loss and the associated major IE gene suppression. Both pUL50 and UL50-p26 interacted with VCP/p97, although UL50-p26 did so more weakly than pUL50. UL50-p26 interacted with pUL50, and this interaction was much stronger than the pUL50 self-interaction. Furthermore, UL50-p26 was able to interfere with the pUL50-VCP/p97 interaction. Our study newly identifies UL50-p26 expression during HCMV infection and suggests a regulatory role for UL50-p26 in blocking pUL50-mediated VCP/p97 loss by associating with pUL50. IMPORTANCE Targeting the endoplasmic reticulum (ER) by viral proteins may affect ER-associated protein homeostasis. During human cytomegalovirus (HCMV) infection, pUL50 targets the ER through its transmembrane domain and moves to the inner nuclear membrane (INM) to form the nuclear egress complex (NEC), which facilitates capsid transport from the nucleus to the cytoplasm. Here, we demonstrate that pUL50 induces the loss of valosin-containing protein (VCP/p97), which promotes the expression of viral major immediate early gene products, in a manner dependent on its membrane targeting but that a small isoform of pUL50 is expressed to negatively regulate this pUL50 activity. This study reports a new NEC-independent function of pUL50 and highlights the fine regulation of pUL50 activity by a smaller isoform for efficient viral growth.

scholarly journals Specific Inhibition of Human Cytomegalovirus Glycoprotein B-Mediated Fusion by a Novel Thiourea Small Molecule

2004 ◽  
Vol 78 (3) ◽  
pp. 1289-1300 ◽  
Author(s):  
Thomas R. Jones ◽  
Shi-Wu Lee ◽  
Stephen V. Johann ◽  
Vladimir Razinkov ◽  
Robert J. Visalli ◽  
...  
Keyword(s):  
Small Molecule ◽  
Human Cytomegalovirus ◽  
Small Molecule Inhibitor ◽  
Glycoprotein B ◽  
Chemical Library ◽  
Cell Plasma ◽  
Molecule Inhibitor ◽  
Virion Envelope ◽  
Cell Spread ◽  
Cell Cell

ABSTRACT A novel small molecule inhibitor of human cytomegalovirus (HCMV) was identified as the result of screening a chemical library by using a whole-virus infected-cell assay. Synthetic chemistry efforts yielded the analog designated CFI02, a compound whose potency had been increased about 100-fold over an initial inhibitor. The inhibitory concentration of CFI02 in various assays is in the low nanomolar range. CFI02 is a selective and potent inhibitor of HCMV; it has no activity against other CMVs, alphaherpesviruses, or unrelated viruses. Mechanism-of-action studies indicate that CFI02 acts very early in the replication cycle, inhibiting virion envelope fusion with the cell plasma membrane. Mutants resistant to CFI02 have mutations in the abundant virion envelope glycoprotein B that are sufficient to confer resistance. Taken together, the data suggest that CFI02 inhibits glycoprotein B-mediated HCMV virion fusion. Furthermore, CFI02 inhibits the cell-cell spread of HCMV. This is the first study of a potent and selective small molecule inhibitor of CMV fusion and cell-cell spread.

scholarly journals Opportunities and Challenges of Small Molecule Induced Targeted Protein Degradation

2021 ◽  
Vol 9 ◽  
Author(s):  
Ming He ◽  
Wenxing Lv ◽  
Yu Rao
Keyword(s):  
Rna Interference ◽  
Protein Degradation ◽  
Small Molecules ◽  
Small Molecule ◽  
Gene Editing ◽  
Small Molecule Inhibitors ◽  
Tumor Resistance ◽  
Target Proteins ◽  
Enzymatic Function ◽  
New Type

Proteolysis targeting chimeras (PROTAC) represents a new type of small molecule induced protein degradation technology that has emerged in recent years. PROTAC uses bifunctional small molecules to induce ubiquitination of target proteins and utilizes intracellular proteasomes for chemical knockdown. It complements the gene editing and RNA interference for protein knockdown. Compared with small molecule inhibitors, PROTAC has shown great advantages in overcoming tumor resistance, affecting the non-enzymatic function of target proteins, degrading undruggable targets, and providing new rapid and reversible chemical knockout tools. At the same time, its challenges and problems also need to be resolved as a fast-developing newchemical biology technology.

scholarly journals Preclinical Evaluation of Orally Bioavailable Small-Molecule Inhibitors of Complement Factor D As a Potential Treatment for Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4819-4819 ◽  
Author(s):  
Jason Wiles ◽  
Steven Podos ◽  
Jane Thanassi ◽  
Avinash Phadke ◽  
Venkat Gadhachanda ◽  
...  
Keyword(s):  
Small Molecule ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Small Molecule Inhibitors ◽  
Cell Lysis ◽  
Inhibitory Effect ◽  
Complement Factor ◽  
Complement Pathway ◽  
Preclinical Evaluation ◽  
Pharmacokinetic Properties ◽  
Terminal Complement

Abstract BACKGROUND: Complement factor D, a serine protease, plays an essential role in the activation of the alternative complement pathway and provides important amplification of the classical and lectin complement pathways. Cleavage of factor B by factor D generates C3 convertase that leads to opsonization of targeted surfaces with complement activation fragments and to the formation of the terminal complement complex (TCC); both events lead to cell lysis. Complement dysregulation underlies multiple hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH), which is characterized by complement-mediated lysis of clonal populations of erythrocytes that lack glycophosphatidylinositol-anchored complement regulators. The current treatment for PNH is intravenous infusion of the anti-C5 monoclonal antibody eculizumab. Although eculizumab lessens intravascular hemolysis, it does not prevent opsonization of erythrocytes and subsequent extravascular hemolysis by immune cells. In contrast, factor D inhibitors are expected to inhibit both terminal complement pathway activation as well as opsonization and should, therefore, be well-positioned to potentially serve this unmet medical need. Herein, we present the preclinical evaluation of our small-molecule inhibitors of factor D including potency, off-target activities, metabolism, and pharmacokinetic properties. METHODS & RESULTS: Initial inhibitors were discovered through laboratory and virtual screening efforts. A high-resolution (1.5 Å) X-ray structure of an early proprietary inhibitor co-crystallized with factor D aided our optimization campaign that culminated in compounds with IC50 values below 100 nM in biochemical protease assays using natural and non-specific substrates. The potent inhibitory effect of the compounds on factor D protease activity translated to inhibition of cell lysis in an alternative pathway hemolytic assay with EC50 values as low as single-digit nM. These small molecules are highly selective for factor D and displayed no significant inhibitory effect on a panel of human serine proteases. In accordance with the high selectivity, the inhibitors showed minimal cellular toxicity, no effect on multiple human receptor-ligand interactions, and no inhibition of hERG potassium channel current. Finally, oral and intravenous administration of selected lead compounds to preclinical animal species showed pharmacokinetic properties that suggest their suitability for oral dosing in humans. CONCLUSIONS: We have discovered highly active small-molecule inhibitors of factor D that demonstrate oral bioavailability and low off-target activities. These key attributes, and the potential to inhibit both intravascular and extravascular hemolysis through inhibition of both terminal complement pathway and opsonization, position these inhibitors as promising development candidates for the oral treatment of PNH. Disclosures Wiles: Achillion Pharmaceuticals: Employment. Podos:Achillion Pharmaceuticals : Employment. Thanassi:Achillion Pharmaceuticals: Employment. Phadke:Achillion Pharmaceuticals: Employment. Gadhachanda:Achillion Pharmaceuticals: Employment. Pais:Achillion Pharmaceuticals: Employment. Hashimoto:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Chen:Achillion Pharmaceuticals: Employment. Wang:Achillion Pharmaceuticals: Employment. Agarwal:Achillion Pharmaceuticals: Employment. Rivera:Achillion Pharmaceuticals: Employment. Elliot:Achillion Pharmaceuticals: Employment. Marlor:Achillion Pharmaceuticals: Employment. Zhang:Achillion Pharmaceuticals: Employment. Deshpande:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment. Huang:Achillion Pharmaceuticals: Employment.

scholarly journals Peptide Inhibitors of Kv1.5: An Option for the Treatment of Atrial Fibrillation

2021 ◽  
Vol 14 (12) ◽  
pp. 1303
Author(s):  
Jesús Borrego ◽  
Adam Feher ◽  
Norbert Jost ◽  
Gyorgy Panyi ◽  
Zoltan Varga ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Amino Acid Sequences ◽  
Rational Drug Design ◽  
Drug Candidate ◽  
In Silico Docking ◽  
Rational Drug ◽  
Wide Range ◽  
Potential Therapeutic Application

The human voltage gated potassium channel Kv1.5 that conducts the IKur current is a key determinant of the atrial action potential. Its mutations have been linked to hereditary forms of atrial fibrillation (AF), and the channel is an attractive target for the management of AF. The development of IKur blockers to treat AF resulted in small molecule Kv1.5 inhibitors. The selectivity of the blocker for the target channel plays an important role in the potential therapeutic application of the drug candidate: the higher the selectivity, the lower the risk of side effects. In this respect, small molecule inhibitors of Kv1.5 are compromised due to their limited selectivity. A wide range of peptide toxins from venomous animals are targeting ion channels, including mammalian channels. These peptides usually have a much larger interacting surface with the ion channel compared to small molecule inhibitors and thus, generally confer higher selectivity to the peptide blockers. We found two peptides in the literature, which inhibited IKur: Ts6 and Osu1. Their affinity and selectivity for Kv1.5 can be improved by rational drug design in which their amino acid sequences could be modified in a targeted way guided by in silico docking experiments.

scholarly journals Identification of a novel small molecule that inhibits deacetylase but not defatty-acylase reaction catalysed by SIRT2

2018 ◽  
Vol 373 (1748) ◽  
pp. 20170070 ◽  
Author(s):  
Norio Kudo ◽  
Akihiro Ito ◽  
Mayumi Arata ◽  
Akiko Nakata ◽  
Minoru Yoshida
Keyword(s):  
Small Molecule ◽  
Binding Pocket ◽  
Inhibitory Effect ◽  
Crystallographic Analysis ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Human Pancreatic Cancer ◽  
Chemical Library ◽  
Eukaryotic Translation ◽  
Potent Inhibitory Effect

SIRT2 is a member of the human sirtuin family of proteins and possesses NAD + -dependent lysine deacetylase/deacylase activity. SIRT2 has been implicated in carcinogenesis in various cancers including leukaemia and is considered an attractive target for cancer therapy. Here, we identified NPD11033, a selective small-molecule SIRT2 inhibitor, by a high-throughput screen using the RIKEN NPDepo chemical library. NPD11033 was largely inactive against other sirtuins and zinc-dependent deacetylases. Crystallographic analysis revealed a unique mode of action, in which NPD11033 creates a hydrophobic cavity behind the substrate-binding pocket after a conformational change of the Zn-binding small domain of SIRT2. Furthermore, it forms a hydrogen bond to the active site histidine residue. In addition, NPD11033 inhibited cell growth of human pancreatic cancer PANC-1 cells with a concomitant increase in the acetylation of eukaryotic translation initiation factor 5A, a physiological substrate of SIRT2. Importantly, NPD11033 failed to inhibit defatty-acylase activity of SIRT2, despite its potent inhibitory effect on its deacetylase activity. Thus, NPD11033 will serve as a useful tool for both developing novel anti-cancer agents and elucidating the role of SIRT2 in various cellular biological processes. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.

scholarly journals Rho-Associated Coiled-Coil Kinase 1 Translocates to the Nucleus and Inhibits Human Cytomegalovirus Propagation

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Erez Eliyahu ◽  
Osnat Tirosh ◽  
Martina Dobesova ◽  
Aharon Nachshon ◽  
Michal Schwartz ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Coiled Coil ◽  
Inhibitory Effect ◽  
Cellular Stress Response ◽  
Cellular Polarity ◽  
Cellular Functions ◽  
Proliferation And Apoptosis ◽  
Rock Activity

ABSTRACTRho-associated coiled-coil kinase (ROCK) protein is a central kinase that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here, we demonstrate that ROCK has antiviral properties, and inhibition of its activity results in enhanced propagation of human cytomegalovirus (HCMV). We show that during HCMV infection, ROCK1 translocates to the nucleus and concentrates in the nucleolus, where it colocalizes with the stress-related chaperone heat shock cognate 71-kDa protein (Hsc70). Gene expression measurements show that inhibition of ROCK activity does not seem to affect the cellular stress response. We demonstrate that inhibition of myosin, one of the central targets of ROCK, also increases HCMV propagation, implying that the antiviral activity of ROCK might be mediated by activation of the actomyosin network. Finally, we demonstrate that inhibition of ROCK results in increased levels of the tegument protein UL32 and of viral DNA in the cytoplasm, suggesting ROCK activity hinders the efficient egress of HCMV particles out of the nucleus. Altogether, our findings illustrate ROCK activity restricts HCMV propagation and suggest this inhibitory effect may be mediated by suppression of capsid egress out of the nucleus.IMPORTANCEROCK is a central kinase in cells that regulates numerous cellular functions, including cellular polarity, motility, proliferation, and apoptosis. Here we reveal a novel antiviral activity of ROCK during infection with HCMV, a prevalent pathogen infecting most of the population worldwide. We reveal ROCK1 is translocated to the nucleus, where it mainly localizes to the nucleolus. Our findings suggest that ROCK’s antiviral activity may be related to activation of the actomyosin network and inhibition of capsid egress out of the nucleus.

scholarly journals Localization of the WD repeat-containing protein 5 to the Virion Assembly Compartment Facilitates Human Cytomegalovirus Assembly

2021 ◽  
Author(s):  
Bo Yang ◽  
YongXuan Yao ◽  
Hui Wu ◽  
Hong Yang ◽  
Xue-Hui Ma ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Late Phase ◽  
Viral Gene ◽  
Virion Assembly ◽  
Nuclear Egress ◽  
Early Endosomes ◽  
Virion Morphogenesis ◽  
Wd Repeat ◽  
Assembly Compartment

We previously reported that human cytomegalovirus (HCMV) utilizes the cellular protein WD repeat-containing protein 5 (WDR5) to facilitate capsid nuclear egress. Here, we further show that HCMV infection results in WDR5 localization in a juxtanuclear region, and that its localization to this cellular site is associated with viral replication and late viral gene expression. Furthermore, WDR5 accumulated in the virion assembly compartment (vAC) and co-localized with vAC markers of gamma-tubulin (γ-tubulin), early endosomes, and viral vAC marker proteins pp65, pp28, and glycoprotein B (gB). WDR5 co-immunoprecipitated with multiple virion proteins, including MCP, pp150, pp65, pIRS1, and pTRS1, which may explain WDR5 accumulation in the vAC during infection. WDR5 fractionated with virions either in the presence or absence of Triton X-100 and was present in purified viral particles, suggesting that WDR5 was incorporated into HCMV virions. Thus, WDR5 localized to the vAC and was incorporated into virions, raising the possibility that in addition to capsid nuclear egress, WDR5 could also participate in cytoplasmic HCMV virion morphogenesis. Importance Human cytomegalovirus (HCMV) has a large (∼235-kb) genome that contains over 170 ORFs and exploits numerous cellular factors to facilitate its replication. In the late phase of HCMV infection cytoplasmic membranes are reorganized to establish the virion assembly compartment (vAC), which has been shown to necessary for efficient assembly of progeny virions. We previously reported that WDR5 facilitates HCMV nuclear egress. Here, we show that WDR5 is localized to the vAC and incorporated into virions, perhaps contributing to efficient virion maturation. Thus, findings in this study identified a potential role for WDR5 in HCMV assembly in the cytoplasmic phase of virion morphogenesis.

scholarly journals BH3 Profiling Reveals Selectivity by Herpesviruses for Specific Bcl-2 Proteins To Mediate Survival of Latently Infected Cells

2015 ◽  
Vol 89 (10) ◽  
pp. 5739-5746 ◽  
Author(s):  
Olesea Cojohari ◽  
Christine M. Burrer ◽  
Megan A. Peppenelli ◽  
Fardokht A. Abulwerdi ◽  
Zaneta Nikolovska-Coleska ◽  
...  
Keyword(s):  
Small Molecule ◽  
Human Cytomegalovirus ◽  
Kaposi's Sarcoma ◽  
Epstein Barr Virus ◽  
Small Molecule Inhibitors ◽  
Cellular Survival ◽  
Barr Virus ◽  
Latently Infected ◽  
Infected Cells ◽  
Epstein Barr

Herpesviruses, including human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), and Kaposi’s sarcoma-associated herpesvirus, establish latency by modulating or mimicking antiapoptotic Bcl-2 proteins to promote survival of carrier cells. BH3 profiling, which assesses the contribution of Bcl-2 proteins towards cellular survival, was able to globally determine the level of dependence on individual cellular and viral Bcl-2 proteins within latently infected cells. Moreover, BH3 profiling predicted the sensitivity of infected cells to small-molecule inhibitors of Bcl-2 proteins.

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