The Experts below are selected from a list of 453 Experts worldwide ranked by ideXlab platform
Sunwen Chou - One of the best experts on this subject based on the ideXlab platform.
-
Drug resistance mutations and associated phenotypes detected in clinical trials of Maribavir for treatment of cytomegalovirus infection.
The Journal of infectious diseases, 2020Co-Authors: Sunwen Chou, Kening Song, Clyde S. CrumpackerAbstract:BACKGROUND In separate Phase 2 trials, 120 patients received Maribavir for cytomegalovirus (CMV) infection failing conventional therapy (trial 202) and 119 received Maribavir for asymptomatic infection (trial 203). Overall, 172 cleared their CMV infection (CMV DNA 14 days of therapy, 9 showed T409M or H411Y and 4 others showed C480F alone. CONCLUSIONS After Maribavir therapy (400-1200 mg twice daily), UL97 mutations T409M, H411Y or C480F emerge to confer Maribavir resistance in patients with recurrent CMV infection while on therapy, or no response to therapy.
-
Advances in the genotypic diagnosis of cytomegalovirus antiviral drug resistance.
Antiviral research, 2020Co-Authors: Sunwen ChouAbstract:Abstract Cytomegalovirus (CMV) drug resistance mutation maps are updated with recent information for polymerase inhibitors, the terminase inhibitor letermovir and the UL97 kinase inhibitor Maribavir. Newly mapped mutations and their phenotypes provide more detail on cross-resistance properties and suggest the need to expand the CMV gene regions covered in diagnostic testing. Next-generation deep sequencing technology offers a more sensitive, higher resolution view of emerging antiviral resistance and is recommended for use in clinical trials. Issues of standardization and diagnostic utility in comparison with traditional Sanger sequencing remain unresolved. Quality control is important for the accurate and reproducible detection of mutant viral populations in clinical specimens.
-
Novel UL97 drug resistance mutations identified at baseline in a clinical trial of Maribavir for resistant or refractory cytomegalovirus infection.
Antiviral research, 2019Co-Authors: Sunwen Chou, Kening SongAbstract:In a Phase 2 clinical trial, 120 subjects with cytomegalovirus (CMV) infection refractory or resistant to standard therapy were randomized equally to 3 doses of oral Maribavir treatment, and 70% achieved undetectable plasma CMV DNA within 12 weeks. At study entry, standard diagnostic UL97 genotyping was available for 71 subjects, with 60 (85%) revealing well-characterized ganciclovir resistance mutations that did not preclude a therapeutic response to Maribavir. Central laboratory testing of a range of UL97 codons (288-468) not fully covered by standard genotyping was done on 93 subjects at baseline. This detected no previously known Maribavir resistance mutations, but identified atypical mutations in 3 subjects, including a P-loop substitution F342Y, and ATP-binding region substitutions K359E/Q. By recombinant phenotyping, K359E and K359Q each conferred a nearly 4-fold increased ganciclovir 50% inhibitory concentration (EC50) without Maribavir resistance, whereas F342Y conferred a 6-fold increased ganciclovir EC50 and a 4.5-fold increased Maribavir EC50. The subject with F342Y detected at baseline did not achieve plasma CMV DNA clearance after 12 weeks of Maribavir therapy and later developed an additional UL97 substitution H411Y known to confer 12- to 20-fold increased MBV EC50 by itself. The combination of F342Y and H411Y was shown to increase the Maribavir EC50 by 56-fold. Diagnostic genotyping of UL97 should be expanded to cover the ATP-binding region beginning at codon 335 to enable the detection of atypical resistance mutations and further correlation of their clinical significance.
-
Antiviral activity of Maribavir in combination with other drugs active against human cytomegalovirus.
Antiviral Research, 2018Co-Authors: Sunwen Chou, Ronald J Ercolani, Katayoun DerakhchanAbstract:Abstract The human cytomegalovirus (CMV) UL97 kinase inhibitor Maribavir is in Phase III clinical trials as antiviral therapy, including use for infections refractory or resistant to standard therapy. To assess its activity in combination with approved and experimental CMV antivirals, and with the mTor inhibitor rapamycin (sirolimus), drug effects were tested by in vitro checkerboard assays and the data were analyzed using a three dimensional model based on an independent effects definition of additive interactions. Baseline virus and representative drug-resistant mutants were tested. According to the volume of synergy at 95% confidence, Maribavir showed additive interactions with foscarnet, cidofovir, letermovir and GW275175X when tested against wild type and mutant viruses, strong antagonism with ganciclovir, and strong synergy with rapamycin, the latter suggesting a potentially useful therapeutic combination.
-
Virologic Characterization
2016Co-Authors: Sunwen ChouAbstract:The experimental cytomegalovirus UL97 kinase inhibitor Maribavir was used to treat 2 cases of infection in which viral mutations that conferred ganciclovir and foscarnet re-sistance had evolved sequentially. In one case, viral shedding was cleared without evidence of Maribavir resistance in an isolate obtained after therapy. In the other case, a high-grade viremia was initially reduced 50-fold but rebounded 2 months later, coincident with the emergence of viral UL97 mutations T409M and H411Y, which confer Maribavir re-sistance. The relatively rapid onset of Maribavir resistance probably resulted from incomplete viral suppression in an immunosuppressed host with a high viral load. Antiviral treatment with ganciclovir (GCV) or its oral prodrug valganciclovir (VGCV), which is routinely used to prevent and treat post-transplant cytomegalovirus (CMV) infection, some
Stephen Villano - One of the best experts on this subject based on the ideXlab platform.
-
Maribavir for Preemptive Treatment of Cytomegalovirus Reactivation.
The New England journal of medicine, 2019Co-Authors: Johan Maertens, Catherine Cordonnier, Peter Jaksch, Xavier Poiré, Marc E. Uknis, Anna Wijatyk, Faouzi Saliba, Oliver Witzke, Stephen VillanoAbstract:Abstract Background Maribavir is a benzimidazole riboside with activity against cytomegalovirus (CMV). The safety and efficacy of Maribavir for preemptive treatment of CMV infection in transplant r...
-
Maribavir for refractory or resistant cytomegalovirus infections in hematopoietic cell or solid organ transplant recipients a randomized dose ranging double blind phase 2 study
Clinical Infectious Diseases, 2019Co-Authors: Genovefa A. Papanicolaou, Francisco M Marty, Robin K. Avery, Michael Boeckh, Marc E. Uknis, Anna Wijatyk, Fernanda P. Silveira, Amelia Langston, Marcus R. Pereira, Stephen VillanoAbstract:BACKGROUND Cytomegalovirus (CMV) infections that are refractory or resistant (RR) to available antivirals ([val]ganciclovir, foscarnet, cidofovir) are associated with higher mortality in transplant patients. Maribavir is active against RR CMV strains. METHODS Hematopoietic-cell or solid-organ transplant recipients ≥12 years old with RR CMV infections and plasma CMV deoxyribonucleic acid (DNA) ≥1000 copies/mL were randomized (1:1:1) to twice-daily dose-blinded Maribavir 400, 800, or 1200 mg for up to 24 weeks. The primary efficacy endpoint was the proportion of patients with confirmed undetectable plasma CMV DNA within 6 weeks of treatment. Safety analyses included the frequency and severity of treatment-emergent adverse events (TEAEs). RESULTS From July 2012 to December 2014, 120 patients were randomized and treated (40 per dose group): 80/120 (67%) patients achieved undetectable CMV DNA within 6 weeks of treatment (95% confidence interval, 57-75%), with rates of 70%, 63%, and 68%, respectively, for Maribavir 400, 800, and 1200 mg twice daily. Recurrent on-treatment CMV infections occurred in 25 patients; 13 developed mutations conferring Maribavir resistance. Maribavir was discontinued due to adverse events in 41/120 (34%) patients, and 17/41 discontinued due to CMV infections. During the study, 32 (27%) patients died, 4 due to CMV disease. Dysgeusia was the most common TEAE (78/120; 65%) and led to Maribavir discontinuation in 1 patient. Absolute neutrophil counts <1000/µL were noted in 12/106 (11%) evaluable patients, with rates similar across doses. CONCLUSIONS Maribavir ≥400 mg twice daily was active against RR CMV infections in transplant recipients; no new safety signals were identified. CLINICAL TRIALS REGISTRATION NCT01611974.
-
Effects on Maribavir susceptibility of cytomegalovirus UL97 kinase ATP binding region mutations detected after drug exposure in vitro and in vivo.
Antiviral research, 2012Co-Authors: Sunwen Chou, Morgan Hakki, Stephen VillanoAbstract:Abstract Resistance to the experimental human cytomegalovirus (CMV) UL97 kinase inhibitor Maribavir has been mapped to UL97 mutations at codons 353, 397, 409 and 411, in the kinase ATP-binding region, and to mutations in the UL27 gene. We studied the Maribavir susceptibility phenotypes of additional UL97 mutations observed in vitro and in clinical trials, and the effect of simultaneous mutation in both UL97 and UL27. In vitro selection under Maribavir identified a new locus of UL97 mutation within the conserved kinase p-loop (L337M), which conferred low grade Maribavir resistance (3.5-fold increased EC50) without ganciclovir cross-resistance. During Maribavir Phase III CMV prevention clinical trials, three previously unknown UL97 sequence variants were detected in plasma samples after 27–98 days of drug exposure (I324V, S334G and S386L). These variants did not confer any drug resistance despite proximity to mutations that confer Maribavir resistance. The UL27 resistance mutation R233S, when added to strains containing UL97 mutations L337M or V353A, doubled their Maribavir EC50s. These results expand the range of UL97 Maribavir-resistance mutations into another part of the kinase ATP-binding region, but offer no genotypic evidence that development of drug resistance affected the outcomes of Phase III Maribavir clinical trials after drug exposure of up to 14 weeks. There is a potential for increased Maribavir resistance in UL27–UL97 double mutants.
-
Oral Maribavir for treatment of refractory or resistant cytomegalovirus infections in transplant recipients
Transplant infectious disease : an official journal of the Transplantation Society, 2010Co-Authors: Robin K. Avery, Lynne Strasfeld, Francisco M Marty, Ingi Lee, A. Arrieta, S. Chou, W. Tatarowicz, Stephen VillanoAbstract:R.K. Avery, F.M. Marty, L. Strasfeld, I. Lee, A. Arrieta, S. Chou, W. Tatarowicz, S. Villano. Oral Maribavir for treatment of refractory or resistant cytomegalovirus infections in transplant recipients. Transpl Infect Dis 2010: 12: 489–496. All rights reserved Background. Despite advances in cytomegalovirus (CMV) prophylaxis and therapy, some transplant recipients still develop refractory CMV infections. Maribavir (MBV), an investigational benzimidazole antiviral agent, acts by a mechanism different from that of existing anti-CMV drugs. Previous Phase I and II studies have demonstrated a favorable safety profile for MBV, but its utility in treatment of complex CMV syndromes is unknown. Methods. Between June and December 2008, MBV was released for use under individual emergency investigational new drug applications requested by treating physicians and approved by the US Food and Drug Administration and local institutional review boards. Six patients (5 solid organ transplant recipients and 1 hematopoietic stem cell transplant recipient) who had failed to respond to other therapies and/or had known ganciclovir-resistant CMV were treated with MBV at a starting oral dose of 400 mg twice daily. Results. Patients were treated for a median of 207 days (range, 15–376). Four of 6 patients had no detectable CMV DNAemia within 6 weeks of starting MBV therapy. One patient, who had an initial viral load of 1.8 million copies/mL, developed MBV resistance mutations. One patient, who had low serum levels of MBV, had persistent CMV DNAemia and viruria without developing genotypic or phenotypic resistance to MBV. One patient cleared CMV DNAemia, but died of pneumonia and multiorgan failure. No significant adverse effects attributable to MBV were observed. Conclusions. MBV deserves further systematic evaluation as treatment for CMV infection that is resistant and/or refractory to standard therapies, but its optimal dose, duration of therapy, and use in combinations versus as a single agent have yet to be determined.
-
Effect of Ketoconazole on the Pharmacokinetics of Maribavir in Healthy Adults
Antimicrobial agents and chemotherapy, 2008Co-Authors: D. Ronald Goldwater, Carolyn Dougherty, Mary Schumacher, Stephen VillanoAbstract:Maribavir, an oral antiviral drug with activity against cytomegalovirus, is currently undergoing studies to assess its efficacy and safety as cytomegalovirus prophylaxis following stem cell or solid organ transplantation. The main objective of this study was to assess the effects of oral ketoconazole, a potent inhibitor of the cytochrome P450 3A4 (CYP3A4) isoenzyme, on the pharmacokinetics of Maribavir. This was an open-label crossover study with 20 healthy adults. Subjects were administered a single dose of Maribavir at 400 mg. After a washout period, subjects received a single dose of ketoconazole at 400 mg followed by a single dose of Maribavir. Blood samples were collected for each drug sequence, and pharmacokinetic parameters for Maribavir and its principal metabolite, VP 44469, were determined. Safety was evaluated by physical examination, clinical laboratory testing, 12-lead electrocardiogram, and monitoring for adverse events. Ketoconazole moderately reduced the clearance of both Maribavir and VP 44469; oral clearance values were 35% and 13% lower, respectively, for Maribavir-plus-ketoconazole treatment than for Maribavir alone. Based on the assumption of complete inhibition of CYP3A4 activity, CYP3A4 is responsible for 35% of the overall clearance of Maribavir. Treatment was generally well tolerated. The most-common adverse event was dysgeusia (taste disturbance), reported by nine (47%) and seven (35%) subjects in the Maribavir alone and Maribavir-plus-ketoconazole groups, respectively. The pharmacokinetic findings, in combination with the acceptable tolerability within the Maribavir and Maribavir-plus-ketoconazole treatment groups, suggest that no dose adjustment of Maribavir is necessary when coadministered with CYP3A4 inhibitors or substrates.
Mark N. Prichard - One of the best experts on this subject based on the ideXlab platform.
-
Differential Properties of Cytomegalovirus pUL97 Kinase Isoforms Affect Viral Replication and Maribavir Susceptibility
Journal of virology, 2014Co-Authors: Rike Webel, Morgan Hakki, William D. Rawlinson, Mark N. Prichard, Manfred Marschall, Sunwen ChouAbstract:The human cytomegalovirus (HCMV)-encoded kinase pUL97 is required for efficient viral replication. Previous studies described two isoforms of pUL97, the full-length isoform (M1) and a smaller isoform likely resulting from translation initiation at codon 74 (M74). Here, we report the detection of a third pUL97 isoform during viral infection resulting from translation initiation at codon 157 (isoform M157). The consistent expression of isoform M157 as a minor component of pUL97 during infection with clinical and laboratory-adapted HCMV strains was suppressed when codon 157 was mutagenized. Viral mutants expressing specific isoforms were generated to compare their growth and drug susceptibility phenotypes, as well as pUL97 intracellular localization patterns and kinase activities. The exclusive expression of isoform M157 resulted in substantially reduced viral growth and resistance to the pUL97 inhibitor Maribavir while retaining susceptibility to ganciclovir. Confocal imaging demonstrated reduced nuclear import of amino-terminal deletion isoforms compared to isoform M1. Isoform M157 showed reduced efficiency of various substrate protein interactions and autophosphorylation, whereas Rb phosphorylation was preserved. These results reveal differential properties of pUL97 isoforms that affect viral replication, with implications for the antiviral efficacy of Maribavir. IMPORTANCE The HCMV UL97 kinase performs important functions in viral replication that are targeted by the antiviral drug Maribavir. Here, we describe a naturally occurring short isoform of the kinase that when expressed by itself in a recombinant virus results in altered intracellular localization, impaired growth, and high-level resistance to Maribavir compared to those of the predominant full-length counterpart. This is another factor to consider in explaining why Maribavir appears to have variable antiviral activity in cell culture and in vivo.
-
Benzimidazole Analogs Inhibit Human Herpesvirus 6
Antimicrobial agents and chemotherapy, 2011Co-Authors: Mark N. Prichard, John C. Drach, Leroy B Townsend, Samuel L. Frederick, Shannon Daily, Katherine Z. Borysko, Earl R. KernAbstract:Several benzimidazole nucleoside analogs, including 1H-β-D-ribofuranosyl-2-bromo-5,6-dichlorobenzimidazole (BDCRB) and 1H-β-L-ribofuranosyl-2-isopropylamino-5,6-dichlorobenzimidazole (Maribavir [MBV]), inhibit the replication of human cytomegalovirus. Neither analog inhibited the related betaherpesvirus human herpesvirus 6 (HHV-6). Additional analogs of these compounds were evaluated against both variants of HHV-6, and two L -analogs of BDCRB had good antiviral activity against HHV-6A, as well as more modest inhibition of HHV-6B replication.
-
Progress in the development of new therapies for herpesvirus infections.
Current opinion in virology, 2011Co-Authors: Nathan B. Price, Mark N. PrichardAbstract:Resurgent interest in antiviral drugs for the treatment of herpesvirus has led to the development of new compounds that are progressing through clinical trials. This is important because there are few therapeutic options for resistant infections and some viruses such as human cytomegalovirus remain underserved. New compounds include conventional DNA polymerase inhibitors such as valomaciclovir and cyclopropavir, as well as CMX001 that has a broad spectrum of antiviral activity that includes all the herpesviruses. It also includes compounds with new molecular targets such as Maribavir (MBV), FV-100, AIC361, and AIC246. Recent advances with each of these compounds will be reviewed including their virus specificity, mechanism of action, and stage of development. The potential of these new compounds to improve clinical outcome will also be discussed.
-
Conserved retinoblastoma protein-binding motif in human cytomegalovirus UL97 kinase minimally impacts viral replication but affects susceptibility to Maribavir
Virology Journal, 2009Co-Authors: Rachel B Gill, Sunwen Chou, Samuel L. Frederick, Caroll B. Hartline, Mark N. PrichardAbstract:The UL97 kinase has been shown to phosphorylate and inactivate the retinoblastoma protein (Rb) and has three consensus Rb-binding motifs that might contribute to this activity. Recombinant viruses containing mutations in the Rb-binding motifs generally replicated well in human foreskin fibroblasts with only a slight delay in replication kinetics. Their susceptibility to the specific UL97 kinase inhibitor, Maribavir, was also examined. Mutation of the amino terminal motif, which is involved in the inactivation of Rb, also renders the virus hypersensitive to the drug and suggests that the motif may play a role in its mechanism of action.
-
Human cytomegalovirus UL27 is not required for viral replication in human tissue implanted in SCID mice
Virology Journal, 2006Co-Authors: Mark N. Prichard, Sunwen Chou, John C. Drach, Gloria Komazin, Debra C Quenelle, Deborah J Bidanset, Earl R. KernAbstract:Inhibition of the human cytomegalovirus UL97 kinase by Maribavir is thought to be responsible for the antiviral activity of this compound. Some mutations that confer resistance to Maribavir map to UL97 , however additional mutations that also confer resistance to the drug were mapped to UL27 . These open reading frames share a low level of homology, yet the function of pUL27 remains unknown. A recombinant virus with a deletion in the UL27 open reading frame was reported previously to exhibit a slight replication deficit, but a more important function in vivo was hypothesized given its homology to the UL97 kinase. The potential for an important function in vivo was investigated by determining if these knockout viruses could replicate in human tissue implanted in SCID mice. None of the AD169 derived viruses replicated well in the implanted thymus/liver tissue, and is consistent with previous observations, although all of the viruses replicated to some degree in retinal tissue implants. Replication of the parent viruses was observed at 7 days post inoculation, whereas no replication was detected with any of the recombinant viruses with deletions in UL27. By day 14, replication was detected in two of the three knockout viruses and in all of the viruses by day 42. These data are consistent with minimal defects observed in cell culture, but are not consistent with an important role for UL27 in vivo. We conclude that UL27 is not required for viral replication in vivo.
Joseph S. Pagano - One of the best experts on this subject based on the ideXlab platform.
-
Maribavir Inhibits Epstein-Barr Virus Transcription through the EBV Protein Kinase
Journal of virology, 2013Co-Authors: Christopher B. Whitehurst, Marcia K. Sanders, Mankit Law, Fu-zhang Wang, Jie Xiong, Dirk P. Dittmer, Joseph S. PaganoAbstract:ABSTRACT Maribavir (MBV) inhibits Epstein-Barr virus (EBV) replication and the enzymatic activity of the viral protein kinase BGLF4. MBV also inhibits expression of multiple EBV transcripts during EBV lytic infection. Here we demonstrate, with the use of a BGLF4 knockout virus, that effects of MBV on transcription take place primarily through inhibition of BGLF4. MBV inhibits viral genome copy numbers and infectivity to levels similar to and exceeding levels produced by BGLF4 knockout virus.
-
Maribavir Inhibits Epstein-Barr Virus Transcription in Addition to Viral DNA Replication
Journal of virology, 2009Co-Authors: Fu-zhang Wang, Edward Gershburg, Christopher B. Whitehurst, Dirk P. Dittmer, Debasmita Roy, Joseph S. PaganoAbstract:Although many drugs inhibit the replication of Epstein-Barr virus (EBV) in cell culture systems, there is still no drug that is effective and approved for use in primary EBV infection. More recently, Maribavir (MBV), an l-ribofuranoside benzimidazole, has been shown to be a potent and nontoxic inhibitor of EBV replication and to have a mode of action quite distinct from that of acyclic nucleoside analogs such as acyclovir (ACV) that is based primarily on MBV's ability to block the phosphorylation of target proteins by EBV and human cytomegalovirus protein kinases. However, since the antiviral mechanisms of the drug are complex, we have carried out a comprehensive analysis of the effects of MBV on the RNA expression levels of all EBV genes with a quantitative real-time reverse transcription-PCR-based array. We show that in comparisons with ACV, the RNA expression profiles produced by the two drugs are entirely different, with MBV causing a pronounced inhibition of multiple viral mRNAs and with ACV causing virtually none. The results emphasize the different modes of action of the two drugs and suggest that the action of MBV may be linked to indirect effects on the transcription of EBV genes through the interaction of BGLF4 with multiple viral proteins.
-
Maribavir Inhibits Epstein-Barr Virus Transcription in Addition to
2009Co-Authors: Viral Dna Replication, Edward Gershburg, Christopher B. Whitehurst, Fu-zhang Wang, Dirk P. Dittmer, Debasmita Roy, Joseph S. PaganoAbstract:Although many drugs inhibit the replication of Epstein-Barr virus (EBV) in cell culture systems, there is still no drug that is effective and approved for use in primary EBV infection. More recently, Maribavir (MBV), an L-ribofuranoside benzimidazole, has been shown to be a potent and nontoxic inhibitor of EBV replication and to have a mode of action quite distinct from that of acyclic nucleoside analogs such as acyclovir (ACV) that is based primarily on MBV’s ability to block the phosphorylation of target proteins by EBV and human cyto-megalovirus protein kinases. However, since the antiviral mechanisms of the drug are complex, we have carried out a comprehensive analysis of the effects of MBV on the RNA expression levels of all EBV genes with a quantitative real-time reverse transcription-PCR-based array. We show that in comparisons with ACV, the RNA expression profiles produced by the two drugs are entirely different, with MBV causing a pronounced inhibition of multiple viral mRNAs and with ACV causing virtually none. The results emphasize the different modes of action of the two drugs and suggest that the action of MBV may be linked to indirect effects on the transcription of EBV genes through the interaction of BGLF4 with multiple viral proteins. After nearly three decades of study, there is still no drug approved for use in primary Epstein-Barr virus (EBV) infec-tion despite the fact that many drugs inhibit the replication o
-
Epstein–Barr virus infections: prospects for treatment
The Journal of antimicrobial chemotherapy, 2005Co-Authors: Edward Gershburg, Joseph S. PaganoAbstract:Epstein–Barr virus (EBV) causes infectious mononucleosis and oral hairy leucoplakia, and is associated with a number of malignancies. There are, however, no regulatory agency-approved treatments for EBV-related diseases. Several antiviral drugs inhibit replication of EBV in cell culture including acyclic nucleoside and nucleotide analogues and pyrophosphate analogues, all of which inhibit the EBV DNA polymerase. Despite their potency in vitro, these drugs have limited use in vivo for treatment of acute primary EBV infection as well as EBV-associated malignancies for several reasons. Here we discuss novel anti-EBV compounds, including Maribavir, potentially useful for the treatment of acute EBV infections. A number of experimental approaches for treatment of EBV-related malignancies that are not susceptible to conventional antiviral drug treatment are also discussed.
-
Effects of Maribavir and Selected Indolocarbazoles on Epstein-Barr Virus Protein Kinase BGLF4 and on Viral Lytic Replication
Antimicrobial agents and chemotherapy, 2004Co-Authors: Edward Gershburg, Ke Hong, Joseph S. PaganoAbstract:The human cytomegalovirus (HCMV) homolog of the Epstein-Barr virus (EBV) protein kinase (PK), UL97, is inhibited by Maribavir (1263W94) and selected indolocarbazoles. Here we show that only one of these indolocarbazoles (K252a), but not Maribavir, inhibits autophosphorylation of the EBV PK, BGLF4. However, Maribavir and another indolocarbazole, NGIC-I, do inhibit EBV DNA synthesis, suggesting that although these last compounds inhibit both HCMV and EBV, they seem to operate through differ-ent pathways.
Donald M. Coen - One of the best experts on this subject based on the ideXlab platform.
-
Stereoselective Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir
Antimicrobial Agents and Chemotherapy, 2010Co-Authors: Brian G. Gentry, Jeremy P. Kamil, Donald M. Coen, Jiri Zemlicka, John C. DrachAbstract:Human cytomegalovirus (HCMV) is a widespread pathogen that can cause severe disease in immunologically immature and immunocompromised individuals. Cyclopropavir (CPV) is a guanine nucleoside analog active against human and murine cytomegaloviruses in cell culture and efficacious in mice by oral administration. Previous studies established that the mechanism of action of CPV involves inhibition of viral DNA synthesis. Based upon this action and the structural similarity of CPV to ganciclovir (GCV), we hypothesized that CPV must be phosphorylated to a triphosphate to inhibit HCMV DNA synthesis and that pUL97 is the enzyme responsible for the initial phosphorylation of CPV to a monophosphate (CPV-MP). We found that purified pUL97 phosphorylated CPV 45-fold more extensively than GCV, a known pUL97 substrate and the current standard of treatment for HCMV infections. Kinetic studies with CPV as the substrate for pUL97 demonstrated a Km of 1,750 ± 210 μM. Introduction of 1.0 or 10 nM Maribavir, a known pUL97 inhibitor, and subsequent Lineweaver-Burk analysis demonstrated competitive inhibition of CPV phosphorylation, with a Ki of 3.0 ± 0.3 nM. Incubation of CPV with pUL97 combined with GMP kinase [known to preferentially phosphorylate the (+)-enantiomer of CPV-MP] established that pUL97 stereoselectively phosphorylates CPV to its (+)-monophosphate. These results elucidate the mechanism of CPV phosphorylation and help explain its selective antiviral action.
-
The Human Cytomegalovirus UL44 Protein Is a Substrate for the UL97 Protein Kinase
Journal of virology, 2003Co-Authors: Paula M Krosky, Donald M. Coen, Moonchang Baek, Karen K. Biron, Wan Jin Jahng, Imma Barrera, Robert J. Harvey, Phiroze SethnaAbstract:Protein kinases play a crucial role in the regulation of protein function through phosphorylation of targeted proteins. Human cytomegalovirus (HCMV) encodes many phosphorylated proteins and at least one protein kinase, the product of the UL97 open reading frame (ORF) (6, 13). UL97 is most closely related in sequence to certain protein kinases encoded by various herpesviruses, which collectively form the HvUL protein kinase family (18). These enzymes share many motifs typical of protein kinases but are relatively divergent. The protein kinase motifs of UL97 are even more divergent than those of other family members (6). Biochemically, UL97 is rather unusual in that it can phosphorylate nucleoside analogs such as the antiviral drugs acyclovir and ganciclovir (30). Indeed, phosphorylation of ganciclovir by UL97 is responsible for much of the antiviral selectivity of this drug (29). In vitro, UL97 can autophosphorylate (1, 13). It can also phosphorylate certain exogenous protein and peptide substrates (1, 2, 8, 13; P. B. Sethna, M. G. Davis, C. L. Talarico, W. H. Miller, K. Blackburn, W. A. Burhart, M. B. Moyer, K. K. Biron, and R. J. Harvey, submitted for publication). With these more conventional substrates, it is rather specific. For example, it phosphorylates histones only on particular serine residues (2), but it does not phosphorylate various other proteins, for example, glutathione S-transferase (GST) (8) or various serine/threonine containing peptides (2). Its specificity appears to be due in part to an unusual preference for basic residues five positions downstream of the phosphorylation site (1, 2). UL97 is important, but not completely essential, for viral replication (25). It has been reported to have roles at the stages of DNA replication, DNA encapsidation, and/or nuclear egress (3, 15, 33). An understanding of how UL97 functions during viral replication would be greatly aided by identifying its natural substrates. An initial step in that direction made use of Maribavir (also known as 1263W94), a potent inhibitor of HCMV replication in tissue culture that acts via inhibition of UL97 (3). When lysates from wild-type (wt) HCMV-infected cells were incubated with radiolabeled ATP, the inclusion of Maribavir reduced the phosphorylation of several proteins; the phosphorylation of these proteins was not reduced by Maribavir in lysates from cells infected with a Maribavir-resistant mutant (Sethna et al., submitted). One of these proteins was UL44, a phosphoprotein encoded by the HCMV UL44 ORF, also known as pp52 and ICP36 (5, 11, 16). UL44 is essential for HCMV DNA replication and appears to serve as the processivity subunit of the viral DNA polymerase (10, 24, 28, 32, 35), which might be relevant to the role of UL97 in viral infection. However, it was not shown that UL97 directly phosphorylates UL44, nor has there been evidence that UL97 is required for the normal phosphorylation of any viral protein in infected cells. We therefore sought to determine if UL44 is a substrate for UL97 in vitro and in infected cells.
-
the human cytomegalovirus ul97 protein kinase an antiviral drug target is required at the stage of nuclear egress
Journal of Virology, 2003Co-Authors: Paula M Krosky, Moonchang Baek, Donald M. CoenAbstract:Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, Maribavir. We used Maribavir and a UL97 null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or Maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNA-containing capsids, in the nuclei of wild-type- and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or Maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and Maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.
-
Specific phosphorylation of exogenous protein and peptide substrates by the human cytomegalovirus UL97 protein kinase. Importance of the P+5 position.
The Journal of biological chemistry, 2002Co-Authors: Moonchang Baek, Paula M Krosky, Donald M. CoenAbstract:Abstract Human cytomegalovirus UL97 is an unusual protein kinase that can phosphorylate nucleoside analogs such as ganciclovir but whose specificity for exogenous protein substrates has remained unknown. We found that purified, recombinant glutathioneS-transferase-UL97 fusion protein can phosphorylate histone H2B. Phosphorylation was abrogated by substitution of glutamine for a conserved lysine in subdomain II and inhibited by a new antiviral drug, Maribavir. Sequencing and mass spectrometric analyses of purified32P-labeled tryptic peptides of H2B revealed that the sites of phosphorylation were, in order of extent, Ser-38, Ser-87, Ser-6, Ser-112, and Ser-124. Phosphorylation of synthetic peptides containing these sites, analyzed using a new, chimeric gel system, correlated with their phosphorylation in H2B. Phosphorylation of the Ser-38 peptide by UL97 occurred on Ser-38 and was specifically sensitive to Maribavir, whereas phosphorylation of this peptide by cAMP-dependent protein kinase occurred on Ser-36. The extent of phosphorylation was greatest with peptides containing an Arg or Lys residue 5 positions downstream (P+5) from the Ser. Substitution with Ala at this position essentially eliminated activity. These results identify exogenous protein and peptide substrates of UL97, reveal an unusual dependence on the P+5 position, and may abet discovery of new inhibitors of UL97 and human cytomegalovirus replication.