A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase IIIβ

ABSTRACTDespite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIβ in anin vitrokinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIβ carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model.

Download Full-text

In Vitro Activity of DC-159a, a New Broad-Spectrum Fluoroquinolone, Compared with That of Other Agents against Drug-Susceptible and -Resistant Pneumococci

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01229-07 ◽

2007 ◽

Vol 52 (1) ◽

pp. 77-84 ◽

Cited By ~ 5

Author(s):

Catherine Clark ◽

Kathy Smith ◽

Lois Ednie ◽

Tatiana Bogdanovich ◽

Bonifacio Dewasse ◽

...

Keyword(s):

Broad Spectrum ◽

The Other ◽

Quinolone Resistance ◽

Vitro Activity ◽

In Vitro Activity ◽

Resistance Selection ◽

Resistant Strains

ABSTRACT DC-159a yielded MICs of ≤1 μg/ml against 316 strains of both quinolone-susceptible and -resistant pneumococci (resistance was defined as a levofloxacin MIC ≥4 μg/ml). Although the MICs for DC-159a against quinolone-susceptible pneumococci were a few dilutions higher than those of gemifloxacin, the MICs of these two compounds against 28 quinolone-resistant pneumococci were identical. The DC-159a MICs against quinolone-resistant strains did not appear to depend on the number or the type of mutations in the quinolone resistance-determining region. DC-159a, as well as the other quinolones tested, was bactericidal after 24 h at 2× MIC against 11 of 12 strains tested. Two of the strains were additionally tested at 1 and 2 h, and DC-159a at 4× MIC showed significant killing as early as 2 h. Multistep resistance selection studies showed that even after 50 consecutive subcultures of 10 strains in the presence of sub-MICs, DC-159a produced only two mutants with maximum MICs of 1 μg/ml.

Download Full-text

Phosphatidylinositol 4-Kinase III Beta Is Essential for Replication of Human Rhinovirus and Its Inhibition Causes a Lethal PhenotypeIn Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00303-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3358-3368 ◽

Cited By ~ 48

Author(s):

Catherine Spickler ◽

Julie Lippens ◽

Marie-Kristine Laberge ◽

Sophie Desmeules ◽

Édith Bellavance ◽

...

Keyword(s):

Clinical Stage ◽

Human Rhinovirus ◽

Chronic Obstructive ◽

Chronic Obstructive Pulmonary Disorder ◽

Lipid Kinase ◽

Nucleotide Mutation ◽

Pulmonary Disorder ◽

Phosphatidylinositol 4 ◽

Sirna Knockdown

ABSTRACTHuman rhinovirus (HRV) is the predominant cause of the common cold, but more importantly, infection may have serious repercussions in asthmatics and chronic obstructive pulmonary disorder (COPD) patients. A cell-based antiviral screen against HRV was performed with a subset of our proprietary compound collection, and an aminothiazole series with pan-HRV species and enteroviral activity was identified. The series was found to act at the level of replication in the HRV infectious cycle.In vitroselection and sequencing of aminothiazole series-resistant HRV variants revealed a single-nucleotide mutation leading to the amino acid change I42V in the essential HRV 3A protein. This same mutation has been previously implicated in resistance to enviroxime, a former clinical-stage antipicornavirus agent. Enviroxime-like compounds have recently been shown to target the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIβ). A good correlation between PI4KIIIβ activity and HRV antiviral potency was found when analyzing the data over 80 compounds of the aminothiazole series, covering a 750-fold potency range. The mechanism of action through PI4KIIIβ inhibition was further demonstrated by small interfering RNA (siRNA) knockdown of PI4KB, which reduced HRV replication and also increased the potency of the PI4KIIIβ inhibitors. Inhibitors from two different structural classes with promising pharmacokinetic profiles and with very good selectivity for PI4KIIIβ were used to dissociate compound-related toxicity from target-related toxicity. Mortality was seen in all dosing groups of mice treated with either compound, therefore suggesting that short-term inhibition of PI4KIIIβ is deleterious.

Download Full-text

Septin function tunes lipid kinase activity and phosphatidylinositol 4,5 bisphosphate turnover during G-protein coupled PLC signaling in vivo

10.1101/2021.05.14.444211 ◽

2021 ◽

Author(s):

Aastha Kumari ◽

Avishek Ghosh ◽

Sourav Kolay ◽

RAGHU PADINJAT

Keyword(s):

Plasma Membrane ◽

Kinase Activity ◽

Electrical Response ◽

Receptor Activation ◽

Sensory Transduction ◽

Photon Absorption ◽

Lipid Kinase ◽

Phosphatidylinositol 4

The hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane by receptor activated phospholipase C (PLC) activity is a conserved mechanism of signal transduction. Given the low abundance of PI(4,5)P2 at the plasma membrane, its hydrolysis needs to be coupled to lipid resynthesis to ensure continued PLC activity during receptor activation. However, the mechanism by which PI(4,5)P2 depletion during signalling is coupled to its resynthesis remains unknown. PI(4,5)P2 synthesis is catalyzed by lipid kinase activity and the phosphorylation of phosphatidylinositol 4 phosphate (PI4P) by phosphatidylinositol 4 phosphate 5 kinase (PIP5K) is the final step in this process. In Drosophila photoreceptors, sensory transduction of photon absorption is transduced into PLC activity leading to an electrical response to light. During this process, PI(4,5)P2 is resynthesized by a PIP5K activity but the mechanism by which the activity of this enzyme is coupled to PLC signalling is not known. In this study, we identify a unique protein isoform of dPIP5K, dPIP5KL that is both necessary and sufficient to mediate PI(4,5)P2 synthesis during phototransduction. The activity of dPIP5KL in vitro is enhanced by depletion of PNUT, a non-redundant subunit of the septin family of GTP binding proteins and in vivo, depletion of pnut rescues the effect of dPIP5KL depletion on the light response and PI(4,5)P2 resynthesis during PLC signalling. Lastly we find that depletion of Septin Interacting Protein 1 (Sip1),previously shown to bind PNUT, phenocopies the effect of dPIP5KL depletion in vivo. Thus, our work defines a septin 7 and Sip1 mediated mechanism through which PIP5K activity is coupled to ongoing PLC mediated PI(4,5)P2 depletion.

Download Full-text

Activities of Two Novel Macrolides, GW 773546 and GW 708408, Compared with Those of Telithromycin, Erythromycin, Azithromycin, and Clarithromycin against Haemophilus influenzae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.11.4113-4119.2004 ◽

2004 ◽

Vol 48 (11) ◽

pp. 4113-4119 ◽

Cited By ~ 14

Author(s):

Klaudia Kosowska ◽

Kim Credito ◽

Glenn A. Pankuch ◽

Dianne Hoellman ◽

Gengrong Lin ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Ribosomal Proteins ◽

Selection Process ◽

Single Step ◽

23S Rrna ◽

Resistance Selection ◽

Resistant Strains ◽

Step Selection ◽

Better Than

ABSTRACT The MIC at which 50% of strains are inhibited (MIC50) and the MIC90 of GW 773546, a novel macrolide, were 1.0 and 2.0 μg/ml, respectively, for 223 β-lactamase-positive, β-lactamase-negative, and β-lactamase-negative ampicillin-resistant Haemophilus influenzae strains. The MIC50s and MIC90s of GW 708408, a second novel macrolide, and telithromycin, an established ketolide, were 2.0 and 4.0 μg/ml, respectively, while the MIC50 and MIC90 of azithromycin were 1.0 and 2.0 μg/ml, respectively. The MIC50 and MIC90 of erythromycin were 4.0 and 8.0 μg/ml, respectively; and those of clarithromycin were 4.0 and 16.0 μg/ml, respectively. All compounds except telithromycin were bactericidal (99.9% killing) against nine strains at two times the MIC after 24 h. Telithromycin was bactericidal against eight of the nine strains. In addition, both novel macrolides and telithromycin at two times the MIC showed 99% killing of all nine strains after 12 h and 90% killing of all strains after 6 h. After 24 h, all drugs were bactericidal against four to seven strains when they were tested at the MIC. Ten of 11 strains tested by multistep selection analysis yielded resistant clones after 14 to 43 passages with erythromycin. Azithromycin gave resistant clones of all strains after 20 to 50 passages, and clarithromycin gave resistant clones of 9 of 11 strains after 14 to 41 passages. By comparison, GW 708408 gave resistant clones of 9 of 11 strains after 14 to 44 passages, and GW 773546 gave resistant clones of 10 of 11 strains after 14 to 45 passages. Telithromycin gave resistant clones of 7 of 11 strains after 18 to 45 passages. Mutations mostly in the L22 and L4 ribosomal proteins and 23S rRNA were detected in resistant strains selected with all compounds, with alterations in the L22 protein predominating. Single-step resistance selection studies at the MIC yielded spontaneous resistant mutants at frequencies of 1.5 × 10−9 to 2.2 × 10−6 with GW 773546, 1.5 × 10−9 to 6.0 × 10−4 with GW 708408, and 7.1 × 10−9 to 3.8 × 10−4 with telithromycin, whereas the frequencies were 1.3 × 10−9 to 6.0 × 10−4 with erythromycin and azithromycin and 2.0 × 10−9 to 2.0 × 10−3 with clarithromycin. Alterations in the L22 protein (which were predominant) and the L4 protein were present in mutants selected by the single-step selection process. The postantibiotic effects of GW 773546, GW 708408, and telithromycin for seven H. influenzae strains were 6.6 h (range, 5.2 to 8.8 h), 4.7 h (range, 2.6 to 6.9 h), and 6.4 h (range, 3.8 to 9.7 h), respectively. The results of in vitro studies obtained with both novel macrolides were similar to those obtained with telithromycin and better than those obtained with older macrolides.

Download Full-text

Increased Protein Synthesis by Human Platelets during Phagocytosis of Latex Particles in Vitro

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647928 ◽

1976 ◽

Vol 35 (02) ◽

pp. 350-357 ◽

Cited By ~ 8

Author(s):

Hana Bessler ◽

Galila Agam ◽

Meir Djaldetti

Keyword(s):

Protein Synthesis ◽

Fold Increase ◽

Human Platelets ◽

Polystyrene Latex ◽

Latex Particles ◽

The Third ◽

Platelet Protein ◽

Dose Dependent ◽

Phagocytotic Activity

SummaryA three-fold increase of protein synthesis by human platelets during in vitro phagocytosis of polystyrene latex particles was detected. During the first two hours of incubation, the percentage of phagocytizing platelets and the number of latex particles per platelet increased; by the end of the third hour, the first parameter remained stable, while the number of latex particles per cell had decreased.Vincristine (20 μg/ml of cell suspension) inhibited platelet protein synthesis. This effect was both time- and dose-dependent. The drug also caused a decrease in the number of phagocytizing cells, as well as in their phagocytotic activity.

Download Full-text

The Effect of Adrenaline Infusions on Blood Coagulation in Normal and Haemophilia B Dogs

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649437 ◽

1966 ◽

Vol 15 (03/04) ◽

pp. 349-364 ◽

Cited By ~ 5

Author(s):

A.H Özge ◽

H.C Rowsell ◽

H.G Downie ◽

J.F Mustard

Keyword(s):

Body Weight ◽

Factor Viii ◽

Factor Ix ◽

Clotting Factor ◽

Blood Ph ◽

Order Of Magnitude ◽

Dose Dependent ◽

Generation Test ◽

Plasma Activity

SummaryThe addition of trace amounts of adrenaline to whole blood in plasma in vitro increased factor VIII, factor IX and whole plasma activity in the thromboplastin generation test. This was dose dependent.Adrenaline infusions less than 22 (μg/kg body weight in normal dogs accelerated clotting, increased factor IX, factor VIII and whole plasma activity in the thromboplastin generation test and caused a fall in blood pH. In a factor IX deficient dog, there was no increase in factor IX activity. After adrenaline infusions, however, the other changes occurred and were of the same order of magnitude as in the normal. Adrenaline in doses greater than 22 μg/kg body weight did not produce as great an effect on clotting in normal or factor IX deficient dogs. The platelet count in the peripheral blood was increased following the infusion of all doses of adrenaline. These observations suggest that the accelerating effect of adrenaline on clotting is not mediated through increase in activity of a specific clotting factor.

Download Full-text

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653872 ◽

1995 ◽

Vol 73 (05) ◽

pp. 805-811 ◽

Cited By ~ 12

Author(s):

Yasuo Takahashi ◽

Yoshitaka Hosaka ◽

Hiromi Niina ◽

Katsuaki Nagasawa ◽

Masaaki Naotsuka ◽

...

Keyword(s):

Human Urine ◽

Specific Activity ◽

Anticoagulant Activity ◽

Rabbit Lung ◽

Soluble Thrombomodulin ◽

Molecular Weights ◽

Dose Dependent Fashion ◽

Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Download Full-text

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

Acta Endocrinologica ◽

10.1530/acta.0.1070395 ◽

1984 ◽

Vol 107 (3) ◽

pp. 395-400 ◽

Cited By ~ 9

Author(s):

Itaru Kojima ◽

Etsuro Ogata ◽

Hiroshi Inano ◽

Bun-ichi Tamaoki

Keyword(s):

Carbon Monoxide ◽

Hydroxysteroid Dehydrogenase ◽

Dependent Manner ◽

In Vitro Production ◽

Mitochondrial Preparation ◽

Final Reaction ◽

Vitro Production ◽

Dose Dependent ◽

Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

Download Full-text

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽

10.1530/reprod/119.1.15 ◽

2000 ◽

pp. 15-23 ◽

Cited By ~ 15

Author(s):

K Jewgenow ◽

M Rohleder ◽

I Wegner

Keyword(s):

In Vitro Fertilization ◽

Zona Pellucida ◽

Antigenic Determinants ◽

Vitro Fertilization ◽

Contraceptive Vaccine ◽

Sperm Binding ◽

Dependent Inhibition ◽

Dose Dependent Inhibition ◽

Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

Download Full-text

Preliminary in Vitro Investigations on The Inhibitory Activity of The Original Dietary Supplement Oxidal® on Pathogenic Bacterial Strains

JOURNAL OF ADVANCES IN AGRICULTURE ◽

10.24297/jaa.v11i.8693 ◽

2020 ◽

Vol 11 ◽

pp. 37-43

Author(s):

Prof. Teodora P. Popova ◽

Toshka Petrova ◽

Ignat Ignatov ◽

Stoil Karadzhov

Keyword(s):

Dietary Supplement ◽

Broad Spectrum ◽

Pathogenic Bacteria ◽

Antimicrobial Agents ◽

Clinical Effectiveness ◽

Inhibitory Effect ◽

Diffusion Method ◽

Bacterial Strains ◽

Microbial Strains

The antimicrobial action of the dietary supplement Oxidal® was tested using the classic Bauer and Kirby agar-gel diffusion method. Clinical and reference strains of Staphylococcus aureus and Escherichia coli were used in the studies. The tested dietary supplement showed a well-pronounced inhibitory effect against the microbial strains commensurable with that of the broad-spectrum chemotherapeutic agent Enrofloxacin and showed even higher activity than the broad spectrum antibiotic Thiamphenicol. The proven inhibitory effect of the tested dietary supplement against the examined pathogenic bacteria is in accordance with the established clinical effectiveness standards for antimicrobial agents.

Download Full-text