Antiviral and Kinesin Eg5 Activities of New Indomethacin Analogues
Najim A. Al-Masoudi1*and Dawood S.
American Chemical Science Journal4(4):516-536,
Aim: Synthesis, characterization, anti-HIV, anti-HCV and kinesin activities of new indomethacin analogues have been carried out.
Methodology: Arylated derivatives of indomethacin via the Suzuki-Miyaura cross-coupling reaction using palladium acetate/triphenylphophine or palladium based N-heterocyclic carbene (Pd-NHC) complexes as catalysts were synthesized and characterized by the1H and13C and 2D NMR study. Analogously, indomethacin analogues bearing thioureido and amide moieties of various L-amino acid esters were prepared via Kabbani and coupling reactions, respectively.
Results: All the new analogues were evaluated In vitro for their antiviral activity against the replication of HIV-1 and HIV-2 in MT-4 cells using MTT assay. Compounds28,31and32were evaluated In vitro for their inhibitory activity against hepatitis virus C (HCV) in the Huh 5-2 replicon system (type 1b, Con1 strain). Additionally, some analogues were screened for their inhibitory activity against the ATPase enzyme and the motor-protein Kinesin Eg5.In conclusion, Compounds31and39showed anti-HIV activity with IC50values of>1.81 and> 3.21μM (CC50of 3.31 and28.89μM), resulting in selectivity indexes (SI) of 6 and 9, respectively.
Conclusion: Compounds31and39displayed better anti-HIV activity than the other derivatives (SI = 6 and 9, respectively). Compound27showed ATPase inhibition value of48% at 100μMconcentration.
Selective and ATP‐competitive kinesin KIF18A inhibitor suppresses the replication of influenza A virus
Yong‐Bin Cho, 1 Sungguan Hong, 2 Kyung‐Won Kang, 3 Ji‐Hun Kang, 1 Sang‐Myeong Lee,corresponding author 3 and Young‐Jin Seocorresponding author 1
J Cell Mol Med. 2020 May; 24(10): 5463–5475.
The influenza virus is one of the major public health threats. However, the development of efficient vaccines and therapeutic drugs to combat this virus is greatly limited by its frequent genetic mutations. Because of this, targeting the host factors required for influenza virus replication may be a more effective strategy for inhibiting a broader spectrum of variants. Here, we demonstrated that inhibition of a motor protein kinesin family member 18A (KIF18A) suppresses the replication of the influenza A virus (IAV). The expression of KIF18A in host cells was increased following IAV infection. Intriguingly, treatment with the selective and ATP‐competitive mitotic kinesin KIF18A inhibitor BTB‐1 substantially decreased the expression of viral RNAs and proteins, and the production of infectious viral particles, while overexpression of KIF18A enhanced the replication of IAV. Importantly, BTB‐1 treatment attenuated the activation of AKT, p38 MAPK, SAPK and Ran‐binding protein 3 (RanBP3), which led to the prevention of the nuclear export of viral ribonucleoprotein complexes. Notably, administration of BTB‐1 greatly improved the viability of IAV‐infected mice. Collectively, our results unveiled a beneficial role of KIF18A in IAV replication, and thus, KIF18A could be a potential therapeutic target for the control of IAV infection.
Functional genomics highlights differential induction of antiviral pathways in the lungs of SARS-CoV-infected macaques
Anna de Lang 1, Tracey Baas, Thomas Teal, Lonneke M Leijten, Brandon Rain, Albert D Osterhaus, Bart L Haagmans, Michael G Katze
PLoS Pathog. 2007 Aug 10;3(8):e112. doi: 10.1371/journal.ppat.0030112.
The pathogenesis of severe acute respiratory syndrome coronavirus (SARS-CoV) is likely mediated by disproportional immune responses and the ability of the virus to circumvent innate immunity. Using functional genomics, we analyzed early host responses to SARS-CoV infection in the lungs of adolescent cynomolgus macaques (Macaca fascicularis) that show lung pathology similar to that observed in human adults with SARS. Analysis of gene signatures revealed induction of a strong innate immune response characterized by the stimulation of various cytokine and chemokine genes, including interleukin (IL)-6, IL-8, and IP-10, which corresponds to the host response seen in acute respiratory distress syndrome. As opposed to many in vitro experiments, SARS-CoV induced a wide range of type I interferons (IFNs) and nuclear translocation of phosphorylated signal transducer and activator of transcription 1 in the lungs of macaques. Using immunohistochemistry, we revealed that these antiviral signaling pathways were differentially regulated in distinctive subsets of cells. Our studies emphasize that the induction of early IFN signaling may be critical to confer protection against SARS-CoV infection and highlight the strength of combining functional genomics with immunohistochemistry to further unravel the pathogenesis of SARS.
Viral stop-and-go along microtubules: taking a ride with dynein and kinesins
Katinka Döhner 1, Claus-Henning Nagel, Beate Sodeik
Trends Microbiol. 2005 Jul;13(7):320-7. doi: 10.1016/j.tim.2005.05.010.
Incoming viral particles move from the cell surface to sites of viral transcription and replication. By contrast, during assembly and egress, subviral nucleoprotein complexes and virions travel back to the plasma membrane. Because diffusion of large molecules is severely restricted in the cytoplasm, viruses use ATP-hydrolyzing molecular motors of the host for propelling along the microtubules, which are the intracellular highways. Recent studies have revealed that, besides travelling inside endocytic or exocytic vesicles, viral proteins interact directly with dynein or kinesin motors. Understanding the molecular mechanisms of cytoplasmic viral transport will aid in the construction of viral vectors for human gene therapy and the search for new antiviral targets.
HIV-1 uncoating is facilitated by dynein and kinesin 1
Zana Lukic 1, Adarsh Dharan 2, Thomas Fricke 3, Felipe Diaz-Griffero 3, Edward M Campbell 4
J Virol. 2014 Dec;88(23):13613-25. doi: 10.1128/JVI.02219-14. Epub 2014 Sep 17.
Following entry into the target cell, human immunodeficiency virus type 1 (HIV-1) must reverse transcribe its RNA genome to DNA and traffic to the nuclear envelope, where the viral genome is translocated into the nucleus for subsequent integration into the host cell chromosome. During this time, the viral core, which houses the genome, undergoes a poorly understood process of disassembly, known as uncoating. Collectively, many studies suggest that uncoating is tightly regulated to allow nuclear import of the genome while minimizing the exposure of the newly synthesized DNA to cytosolic DNA sensors. However, whether host cellular proteins facilitate this process remains poorly understood. Here we report that intact microtubules facilitate HIV-1 uncoating in target cells. Disruption of microtubules with nocodazole substantially delays HIV-1 uncoating, as revealed with three different assay systems. This defect in uncoating did not correlate with defective reverse transcription at early times postinfection, demonstrating that microtubule-facilitated uncoating is distinct from the previously reported role of viral reverse transcription in the uncoating process. We also find that pharmacological or small interfering RNA (siRNA)-mediated inhibition of cytoplasmic dynein or the kinesin 1 heavy chain KIF5B delays uncoating, providing detailed insight into how microtubules facilitate the uncoating process. These studies reveal a previously unappreciated role for microtubules and microtubule motor function in HIV-1 uncoating, establishing a functional link between viral trafficking and uncoating. Targeted disruption of the capsid motor interaction may reveal novel mechanisms of inhibition of viral infection or provide opportunities to activate cytoplasmic antiviral responses directed against capsid or viral DNA.
Importance: During HIV-1 infection, fusion of viral and target cell membranes dispenses the viral ribonucleoprotein complex into the cytoplasm of target cells. During this time, the virus must reverse transcribe its RNA genome, traffic from the location of fusion to the nuclear membrane, and undergo the process of uncoating, whereby the viral capsid core disassembles to allow the subsequent nuclear import of the viral genome. Numerous cellular restriction factors target the viral capsid, suggesting that perturbation of the uncoating process represents an excellent antiviral target. However, this uncoating process, and the cellular factors that facilitate uncoating, remains poorly understood. The main observation of this study is that normal uncoating requires intact microtubules and is facilitated by dynein and kinesin motors. Targeting these factors may either directly inhibit infection or delay it enough to trigger mediators of intrinsic immunity that recognize cytoplasmic capsid or DNA and subsequently induce an antiviral state in these cells.
Tubulins interact with porcine and human S proteins of the genus Alphacoronavirus and support successful assembly and release of infectious viral particles
Anna-Theresa Rüdiger 1, Peter Mayrhofer 2, Yue Ma-Lauer 2, Gottfried Pohlentz 3, Johannes Müthing 3, Albrecht von Brunn 4, Christel Schwegmann-Weßels 5
Virology. 2016 Oct;497:185-197. doi: 10.1016/j.virol.2016.07.022. Epub 2016 Jul 30.
Coronavirus spike proteins mediate host-cell-attachment and virus entry. Virus replication takes place within the host cell cytosol, whereas assembly and budding occur at the endoplasmic reticulum-Golgi intermediate compartment. In this study we demonstrated that the last 39 amino acid stretches of Alphacoronavirus spike cytoplasmic domains of the human coronavirus 229E, NL63, and the porcine transmissible gastroenteritis virus TGEV interact with tubulin alpha and beta chains. In addition, a partial co-localization of TGEV spike proteins with authentic host cell β-tubulin was observed. Furthermore, drug-induced microtubule depolymerization led to changes in spike protein distribution, a reduction in the release of infectious virus particles and less amount of spike protein incorporated into virions. These data demonstrate that interaction of Alphacoronavirus spike proteins with tubulin supports S protein transport and incorporation into virus particles.
October 2020 Newsletter : Coronavirus And The Cytoskeleton
The Coronavirus superfamily (Coronaviridae) includes several human pathogens with large positive stranded RNA-encoded genomes, e.g. common cold, viral encephalitis and Covid-19, which are classified into alpha-, beta-, gamma- and delta-coronavirus families by phylogenetic clustering, with further division into Lineages A, B, and C. The virus that causes COVID-19 is classed as a lineage B beta-coronavirus with high similarity to SARS-CoV, thus it’s renaming to SARS-CoV-2 (1). Although the first members of the beta-coronavirus family were recorded in the 1960’s, the family’s rate of new virulent human pathogens has increased rapidly over the past 20 years, now numbering six in total with the latest ones having the familiar pseudonyms SARS (Severe Acute Respiratory Syndrome, 2002), HKU1 (HongKong, 2005), MERS (Middle East Respiratory Syndrome, 2012), and now COVID-19. Their emergence is linked to increased density of human and animal populations which has enhanced zoonotic transmission rates (2).
Here, we describe the five stages of virus “life” cycle with respect to its interaction with the cytoskeleton. The first stage of infection for Coronaviridae is Spike (S) protein-mediated attachment to the cell surface via its binding to a prevalent receptor called angiotensin-converting enzyme 2 (ACE2). The receptor is widespread in air-track and lung tissue, and is more prevalent in older males which accounts for their higher death rates.
After binding, virus particles actively rearrange the cytoskeleton by regulating the FAK/Cofilin/Rac/Cdc42 pathway (4). Lv et al. showed temporal fluctuations in filopodia, lamellipodia, and stress fiber proportions after administering coronavirus PHEV to mouse brain neuronal (N2a) cells in vitro. Interestingly, within 5 min, stress fibers were largely depleted, whereas filopodia dominated the detectable F-actin content, producing 80% of the total fluorescent phalloidin signal (4). Between 20-40 min, lamelopodia emerged as an equal co-player, and finally at 60 min, proportions returned to near normal levels, i.e., 60% of the signal was stress fibers. Although this is a macroscopic observation, it indicates on a nanometer scale that virions may create a local environment that regulates their own individual cellular entry. In another report, Owczarek et al. administered coronavirus OC43 to human colorectal adenocarcinoma (HCT-8) cells to study the method of invagination, vesical excision, and function of F-actin (5). Clathrin-dependent endocytosis, caveolae- dependent invagination, and pinocytosis were all implicated in redundant virion entry mechanisms. And dynamin, a large GTPase, was necessary for vesical entry and excision (5). Similar studies of viral entry were described by Milewska et al. (6) with active endocytosis or mass action entry of virions through other mechanisms, e.g., caveolae- dependent invagination or pinocytosis, with the infection rate being dependent on the viral loading of the cell surface. Cellular entry also packages the virion for the second stage which is transportion to the perinuclear region (5).
Several studies have implicated both actin and tubulin based systems are complementary cytoskeleton components of intracellular transport. Owczarek et al. probed the function of F-actin in intracellular localization; interestingly, jasplakinolide, a cell permeable F-actin stabilizing compound, inhibited viral entry of plasma membrane bound virions, whereas cytochalsin D, an F-actin depolymerizing compound, did not inhibit viral entry but did disrupt the normal localization of virions from peri-nuclear to cytoplasmic areas (5). In contrast, Rüdiger et al. used a spin-down format to show preferential tubulin isoform binding of virions. Coronavirus Spike protein C-terminal peptide (S-protein) bound to several beta-tubulin isoforms in a coronavirus strain-specific manner (9). A scrambled peptide showed the binding was not due to random ionic charge interaction. Thus, intracellular transport of virions utilizes multiple cytoskeletal structural proteins to navigate through and localize to specific areas within the cell (see Figure 1).
The third stage is, transcription and translation. After transport to the endoplasmic reticulum(ER)/Golgi/microtubule organizing center(MTOC)/perinuclear (PN) region (see Figure 1), coronavirus RNA exudes from the endosome vesicle and virion capsule and is translated to form two contiguous multigene polypeptides, which contains proteases that clip the peptide into functional proteins. The positive strand RNA is also reverse transcibed by an RNA polymerase dependent reverse transcriptase to form an antisense replication template. This replicon is subsequently transcribed back into positive sense RNA which is the starting point for viroid assembly and maturation. All these processes occur in the Golgi/ER/MTOC/PN region which is why drugs that disrupt ER, Golgi or MTOC also inhibit viral replication and reduce viral load (10).
The fourth stage is assembly and maturation. Initially, the nucleocapsid (N) protein binds to an RNA copy and binds to vesicle membranes (10,11), and further maturation occurs with N and E proteins which are required for assembly of the basic virus-like particle (VLP). If Spike (S) protein is co-expressed, then this is incorporated into the virus particle (10). In concert with several cytoskeleton and membrane regulator proteins, e.g., HDAC6, ubiquitin, and Rab GTPases, which aid assembly by concentrating packaging components (7). It is unknown which one of these predominates for COVID-19 / SARS-CoV-2.
The fifth stage is egress. The genetic fusion of the coronavirus nucleocapsid or spike proteins with GFP permits tracking of non-infective virus particles by fluorescent microscopy. Using this technique, Siu et al. monitored SARS-CoV egress and, found vesicles that fused into multi-particle conglomerates (10). The transport was sensitive to Brefeldin A, which indicates that the secretory pathway was being used. Other studies have found nocodazole was an effective inhibitor of virion transport to the plasma membrane (7,12), indicating that microtubules are an essential component of egress. Rab11 is implicated in binding to KHC for microtubule transport and then binds to myosin to help transverse the peripheal actin matrix and egress from the cell.
In summary, coronavirus has a highly specific mechanism of receptor binding with multiple mechanisms to enter the cell. Subsequently, both actin, tubulin, and their chemo-mechanical motors dynein, kinesin and myosin cytoskeleton components are required for intracellular transport to the correct location for translation and replication. After reverse transcription and transcription, positive strand RNA is packaged on a scaffold of Golgi/ER/microtubule complexes. The vesicle-encased virions track along microtubules before fusing with the plasma membrane and escaping the cell. Many of the methods in coronavirus research use reagents such as fluorescent phalloidins, microtubule and actin spin-down kits, dynein, kinesin proteins, and Rac and Cdc42 activation assays that are present in the Cytoskeleton catalog and noted below in the tables. There are many un-answered questions in coronavirus research; for example, how do the virions regulate the cytoskeleton through the plasma membrane interactions to coordinate their entry? What is the significance of using actin and microtubule cytoskeletons for different functions? How does the virus evade the immune system for so long compared to influenza and the common cold? Answering these questions will undoubtedly lead to greater knowledge and possible pharmacological breakthroughs in the future.
Microtubule Regulation and Function during Virus Infection
Mojgan H Naghavi 1, Derek Walsh 2
J Virol. 2017 Jul 27;91(16):e00538-17. doi: 10.1128/JVI.00538-17. Print 2017 Aug 15.
Microtubules (MTs) form a rapidly adaptable network of filaments that radiate throughout the cell. These dynamic arrays facilitate a wide range of cellular processes, including the capture, transport, and spatial organization of cargos and organelles, as well as changes in cell shape, polarity, and motility. Nucleating from MT-organizing centers, including but by no means limited to the centrosome, MTs undergo rapid transitions through phases of growth, pause, and catastrophe, continuously exploring and adapting to the intracellular environment. Subsets of MTs can become stabilized in response to environmental cues, acquiring distinguishing posttranslational modifications and performing discrete functions as specialized tracks for cargo trafficking. The dynamic behavior and organization of the MT array is regulated by MT-associated proteins (MAPs), which include a subset of highly specialized plus-end-tracking proteins (+TIPs) that respond to signaling cues to alter MT behavior. As pathogenic cargos, viruses require MTs to transport to and from their intracellular sites of replication. While interactions with and functions for MT motor proteins are well characterized and extensively reviewed for many viruses, this review focuses on MT filaments themselves. Changes in the spatial organization and dynamics of the MT array, mediated by virus- or host-induced changes to MT regulatory proteins, not only play a central role in the intracellular transport of virus particles but also regulate a wider range of processes critical to the outcome of infection.
We add in this chapter references for the vascular endothelial growth factor receptor (VEGFR) inhibitors vandetanib and cabozantinib, so as gossypol, a BCL2 inhibitor. These molecules show multiple interactions with many proteins besides kinesins. In any case, potencies for VEGFR and BCL2 are much higher than for kinesins displayed in graphs.
Growth Factor Receptor Signaling Inhibition Prevents SARS-CoV-2 Replication
Kevin Klann,1,6 Denisa Bojkova,2,6 Georg Tascher,1 Sandra Ciesek,2,3,5 Christian Münch,1,4,6,7,∗ and Jindrich Cinatl2,6,∗∗
Mol Cell. 2020 Oct 1; 80(1): 164–174.e4.
SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinders therapy development. We use a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phosphoproteomics. We identify viral protein phosphorylation and define phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways are activated. Drug-protein network analyses revealed GFR signaling as key pathways targetable by approved drugs. The inhibition of GFR downstream signaling by five compounds prevents SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as a central pathway essential for SARS-CoV-2 replication. It provides novel strategies for COVID-19 treatment.
Antiviral activities of gossypol and its derivatives against herpes simplex virus type II
R J Radloff, L M Deck, R E Royer, D L Vander Jagt
Pharmacol Res Commun. 1986 Nov;18(11):1063-73. doi: 10.1016/0031-6989(86)90023-8.
Gossypol, a disequiterpene obtained from cottonseed oil, and a series of peri-acylated gossylic nitriles were compared for their antiviral activities against HSV-II and for their toxicities to the host Vero cells. All of the peri-acylated gossylic nitriles exhibited lower cytotoxicities to the host cell than did the parent compound gossypol. Both gossypol and the series of derivatives exhibited antiviral activities against HSV-II when the virus was treated with drug at concentrations as low as 5 X 10(-7) M. Two of the derivatives, gossylic nitrile-1,1′-diacetate and gossylic nitrile-1,1′-divalerate, were capable of inhibiting viral multiplication in Vero cells that were infected with virus before administration of the drug. The results of this study indicate that modification of the aldehyde functional groups on gossypol lowers the toxicity of this drug but does not abolish its antiviral properties. Derivatives of gossypol may be useful antiviral agents.
Therapeutic potential of gossypol: An overview
Hoda Keshmiri-Neghab &Bahram Goliaei
Pharmaceutical Biology. Volume 52, 2014 – Issue 1 Pages 124-128
Context: Polyphenols are naturally occurring compounds found in fruits, vegetables, cereals, and beverages. Polyphenols occupy a unique place in biological science for their pharmacological properties. Gossypol is a polyphenolic compound that has attracted attention because of its biological effects.
Objective: Gossypol is reported to exhibit antifertility, antioxidant, anticancer, antivirus, antiparasitic, and antimicrobial properties and lower plasma cholesterol. These are summarized with attention to the mechanisms of activity.
Methods: This review summarizes the results of studies obtained in a comprehensive search of ScienceDirect, PubMed, Scirus, and Web of Science.
Results and conclusion: The results of these studies provide a comprehensive understanding of the biological action of gossypol and its potential for the prevention of and therapy for resistant tumors and chronic human diseases such as HIV, malaria, and psoriasis.