THE SELECTIVE INHIBITION OF VIRAL DNA SYNTHESIS BY CHEMOTHERAPEUTIC AGENTS: AN INDICATOR OF CLINICAL USEFULNESS? *

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72533/1/j.1749-6632.1977.tb21976.x.pd

Synthesis of Imidazo[4,5‐b]quinoxaline Ribonucleosides as Linear Dimensional Analogs of Antiviral Polyhalogenated Benzimidazole Ribonucleosides

We have recently found that 2,5,6‐trichloro‐1‐(β‐D‐ribofuranosyl)benzimidazole (TCRB) and the corresponding 2‐bromo analog have better in vitro activities against HCMV than the clinically used agents ganciclovir and foscarnet. These benzimidazole nucleosides act by a unique mechanism, however, their biological target has not been completely identified. As an approach to probing the target, we have designed imidazo[4,5‐b]quinoxaline nucleosides as linear dimensional analogs of the benzimidazole nucleosides to study the spatial limitation of the binding site in the target enzyme. A convenient route was developed for the synthesis of 2‐substituted 6,7‐dichloroimidazo[4,5‐b]quinoxalines involving a reaction of 2,3,6,7‐tetrachloroquinoxaline with ammonia followed by a ring annulation as the key step. This furnished the versatile heterocycle 6,7‐dichloroimidazo[4,5‐b]quinoxalin‐2‐one. Ribosylation of 2‐substituted imidazo[4,5‐b]quinoxalines was influenced by the functional group at the 2‐position and the 2‐one compound was found to smoothly undergo ribosylation. The 2‐one group of the nucleoside was converted into specifically selected 2‐substituted compounds. Evaluation of the compounds for activity against two herpesviruses and for cytotoxicity showed they were less active and/or more cytotoxic than TCRB. We conclude therefore, that the binding pocket on the protein target of TCRB will tolerate some electronic and size changes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112195/1/199800071_ftp.pd

Some simple thin-layer chromatographic systems for the separation of purines and purine nucleosides

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/33914/1/0000179.pd

Selective inhibition of herpes simplex virus ribonucleoside diphosphate reductase by derivatives of 2-acetylpyridine thiosemicarbazone

The effects of thiosemicarbazone derivatives of 2-acetylpyridine on mammalian and viral ribonucleoside diphosphate reductases were investigated. The enzymes were partially purified from uninfected and herpes simplex virus type-1 (HSV-1)-infected KB cells by sequential salt fractionation with streptomycin sulfate and ammonium sulfate and by affinity chromatography on ATP-agarose. The five thiosemicarbazone derivatives investigated were all potent inhibitors of the virus-induced reductase. Fifty percent inhibitory concentrations (50 values) range from 2 to 13 [mu]M. Four of the five derivatives also were inhibitors of the host cell reductase . A semicarbazone was inactive against the cellular enzyme and relatively weak as an inhibitor of the viral enzyme . Four of the six compounds were preferential inhibitors of the viral reductase based on a comparison of 50 values (5- to > 85-fold difference). Kinetic experiments revealed that inhibition of the HSV-1 reductase by the thiosemicarbazones was noncompetitive with respect to CDP and dithiothreitol. A comparison of the inhibitory effects of 2-acetylpyridine thiosemicarbazone itself on viral reductase and on virus replication in vitro demonstrated a similarity in the dose-response relationships for the two parameters. This observation supports the hypothesis that the HSV-induced ribonucleoside diphosphate reductase is an important target for the design of antiviral drugs.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/26189/1/0000268.pd

Human cytomegalovirus UL27 is not required for viral replication in human tissue implanted in SCID mice

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

DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2[middle dot]106 Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 [mu]M. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 [mu]M, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 [mu]M) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24900/1/0000327.pd

A Rational Approach to Personalized Anticancer Therapy: Chemoinformatic Analysis Reveals Mechanistic Gene-Drug Associations

Purpose . To predict the response of cells to chemotherapeutic agents based on gene expression profiles, we performed a chemoinformatic study of a set of standard anticancer agents assayed for activity against a panel of 60 human tumor-derived cell lines from the Developmental Therapeutics Program (DTP) at the National Cancer Institute (NCI).Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/41497/1/11095_2004_Article_465512.pd

Resistance of Human Cytomegalovirus to Cyclopropavir Maps to a Base Pair Deletion in the Open Reading Frame of \u3cem\u3eUL97\u3c/em\u3e

Human cytomegalovirus (HCMV) is a widespread pathogen in the human population, affecting many immunologically immature and immunocompromised patients, and can result in severe complications, such as interstitial pneumonia and mental retardation. Current chemotherapies for the treatment of HCMV infections include ganciclovir (GCV), foscarnet, and cidofovir. However, the high incidences of adverse effects (neutropenia and nephrotoxicity) limit the use of these drugs. Cyclopropavir (CPV), a guanosine nucleoside analog, is 10-fold more active against HCMV than GCV (50% effective concentrations [EC50s] = 0.46 and 4.1 μM, respectively). We hypothesize that the mechanism of action of CPV is similar to that of GCV: phosphorylation to a monophosphate by viral pUL97 protein kinase with further phosphorylation to a triphosphate by endogenous kinases, re- sulting in inhibition of viral DNA synthesis. To test this hypothesis, we isolated a CPV-resistant virus, sequenced its genome, and discovered that bp 498 of UL97 was deleted. This mutation caused a frameshift in UL97 resulting in a truncated protein that lacks a kinase domain. To determine if this base pair deletion was responsible for drug resistance, the mutation was engineered into the wild-type viral genome, which was then exposed to increasing concentrations of CPV. The results demonstrate that the engineered virus was approximately 72-fold more resistant to CPV (EC50 = 25.8 ± 3.1 μM) than the wild-type virus (EC50 = 0.36 ± 0.11 μM). We conclude, therefore, that this mutation is sufficient for drug resistance and that pUL97 is involved in the mechanism of action of CPV

Design, synthesis and activity against human cytomegalovirus of non-phosphorylatable analogs of toyocamycin, sangivamycin and thiosangivamycin

A number of 7-alkyl 4-aminopyrrolo[2,3-pyrimidine derivatives related to toyocamycin, sangivamycin and thiosangivamycin have been prepared and tested for their activity against human cytomegalovirus (HCMV). Only the thioamide substituted derivatives demonstrated biological activity.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29717/1/0000051.pd

Broad-Spectrum Antiviral Activities of Neplanocin A, 3-Deazaneplanocin A, and Their 5'-Nor Derivatives

The neplanocin A analogs, 3-deazaneplanocin A, 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)adenine (DHCA), and 9-(trans-2',trans-3'-dihydroxycyclopent-4'-enyl)-3-deazaadenine (DHCDA), all potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, were studied for their broad-spectrum antiviral potential. 3-Deazaneplanocin A, DHCA, and DHCDA proved specifically effective against vesicular stomatitis virus, vaccinia virus, parainfluenza virus, reovirus, and rotavirus. Their selectivity was greater than that of neplanocin A, particularly against vesicular stomatitis virus and rotavirus. As could be expected from adenosine analogs that are directly targeted at AdoHcy hydrolase, 3-deazaneplanocin A, DHCA, and DHCDA were fully active in adenosine kinase-deficient cells, implying that their activity did not depend on phosphorylation by adenosine kinase. None of the AdoHcy hydrolase inhibitors showed selective activity against human immunodeficiency virus (type 1). 3-Deazaneplanocin A at a dose of 0.5 mg/kg per day conferred marked protection against a lethal infection of newborn mice with vesicular stomatitis virus.This work was supported by the Belgian Fonds voor Geneeskundig Wetenschappelijk Onderzoek (project no. 3.0040.83) and the Belgian Geconcerteerde Onderzoeksacties (project no. 85/90-79). We thank Anita Van Lierde, Frieda De Meyer, Ria Van Berwaer, Ann Absillis, Etsuko Nitanai, and Willy Zeegers for excellent technical assistance and Christiane Callebaut for fine editorial help