Non-specific antiviral activity of antisense molecules targeted to the E1 region of human papillomavirus.

Antisense phosphorothioate oligonucleotides (ODN1 0x5 OMe) directed against the E1 start region of human papillomavirus 11 (HPV11) can inhibit papillomavirus induced growth of implanted human foreskin in a mouse xenograft model. Administration of a mismatch control oligonucleotide (ODN9 0x5 OMe), in which guanine was replaced with adenine in the same model, had no effect on papilloma induced growth. However, the apparent antiviral activity of ODN1 0x5 OMe was also shown in a lethal mouse cytomegalovirus (CMV) model, in which the oligonucleotides are not expected to have antisense activity. To understand the mechanisms of action of these oligonucleotides, a mismatch oligonucleotide (ODN61 0x5 OMe) was prepared which retained the CpG motifs of ODN1 0x5 OMe. This was tested in the mouse xenograft model and shown to have moderate inhibitory activity. As a definitive experiment, a comparison was made between the efficacy of the active oligonucleotide ODN1 0x5 OMe against two papilloma viruses HPV11 and HPV40. Both these viruses cause benign genital warts, but differ by four bases in their E1 sequence that was the target for ODN1 0x5 OMe. Papillomavirus induced growth in the mouse xenograft model was inhibited by ODN1 0x5 OMe in both cases, suggesting that oligonucleotide molecules have a non-specific antiviral activity that is not directly related to their antisense sequence.

Human cytomegalovirus: challenges, opportunities and new drug development.

In the age of highly active antiretroviral therapy, the incidence of human cytomegalovirus (HCMV) retinitis in AIDS patients has decreased substantially. However, this change does not indicate that HCMV disease in AIDS patients and other immunocompromised patients has abated and is no longer a concern. On the contrary, HCMV disease in graft recipients, newborns, and even in AIDS patients still accounts for considerable morbidity, and drug resistance to the anti-HCMV compounds is a major problem. Furthermore, HCMV may have a role in metabolic diseases, such as atherosclerosis. Fortunately there are novel and potentially very effective new compounds undergoing pre-clinical and clinical evaluation. These developments point the way toward new therapies and also to a clearer understanding of the biology of HCMV replication, infection and disease.

Oligonucleotides as inhibitors of human immunodeficiency virus.

Inhibition of human immunodeficiency virus (HIV) replication by oligonucleotides is a complex process and may be implemented by an array of antiviral mechanisms. These include inhibition of virus adsorption to the host cell, inhibition of transcription via antisense or as the result of triple helix formation, and inhibition of viral encoded enzymes such as reverse transcriptase and integrase. Since the particular mechanism of HIV inhibition depends on the oligonucleotide (ON) sequence and the ON chemical modifications, the design and synthesis of potent HIV inhibitors has been an important and rewarding focus of ON research. In this era of great concern that HIV may rapidly display resistance to any antiviral compound with one mechanism of viral inhibition, oligonucleotides are potentially attractive alternatives for therapy. Several ONs have entered clinical evaluation in AIDS patients. At present Zintevir, which inhibits both HIV adsorption and HIV integrase, is in phase I/II clinical trials.

Inhibition of respiratory syncytial virus replication by antisense oligodeoxyribonucleotides.

Oligodeoxyribonucleotides targeted against respiratory syncytial virus (RSV) genomic RNA inhibited RSV replication in cell culture by an apparent antisense mechanism. HEp-2 cells were infected with RSV strain A2 and incubated in the presence of oligonucleotides. Virus replication was measured by enzyme-linked immunosorbent assay (ELISA), virus yield assay, or production of specific RSV mRNAs. Using ELISA, 50% effective concentration (EC50) values were about 0.5-1 microM for an antisense oligonucleotide targeted to the start of the NS2 gene. All oligonucleotides inhibited virus antigen production as measured by ELISA. In all assays, this antisense oligonucleotide was more potent than: (1) a control oligonucleotide containing the reverse sequence; (2) oligonucleotides targeted at RSV mRNA; (3) a random sequence oligonucleotide; and (4) ribavirin. Reverse transcriptase polymerase chain reaction (PT-PCR) showed sequence specific depletion of the genomic RNA target following treatment of cells with the antisense oligonucleotide. Specific cleavage of the genomic target RNA has been detected at the antisense oligonucleotide binding site, suggesting that cellular Rnase H participates in the reaction. These results indicate that antisense oligonucleotides targeted against RSV genomic RNA can effectively inhibit RSV replication and may have therapeutic value.

Inhibition of the multiple antibiotic resistance (mar) operon in Escherichia coli by antisense DNA analogs.

The multiple antibiotic resistance operon (marORAB) in Escherichia coli controls intrinsic susceptibility and resistance to multiple, structurally different antibiotics and other noxious agents. A plasmid construct with marA cloned in the antisense direction reduced LacZ expression from a constitutively expressed marA::lacZ translational fusion and inhibited the induced expression of LacZ in cells bearing the wild-type repressed fusion. The marA antisense construction also decreased the multiple antibiotic resistance of a Mar mutant. Two antisense phosphorothioate oligonucleotides, one targeted to marO and the other targeted to marA of the mar operon, introduced by heat shock or electroporation reduced LacZ expression in the strain having the marA::lacZ fusion. One antisense oligonucleotide, tested against a Mar mutant of E. coli ML308-225, increased the bactericidal activity of norfloxacin. These studies demonstrate the efficacy of exogenously delivered antisense oligonucleotides targeted to the marRAB operon in inhibiting expression of this chromosomal regulatory locus.

Antisense oligodeoxynucleotides against the BZLF1 transcript inhibit induction of productive Epstein-Barr virus replication.

Expression of the Epstein-Barr virus (EBV) BZLF1 gene product, ZEBRA, in latently infected cells is sufficient to induce the viral lytic cycle. The use of oligodeoxynucleotides complementary to the BZLF1 transcript was studied to inhibit this induction of productive viral replication. For this purpose, we employed oligodeoxynucleotides complementary to the translation initiation codons and their flanking sequences. Incubation of Akata cells with the 25-mer phosphodiester (PO)- or phosphorothioate (PS)-antisense oligodeoxynucleotides for 3 h before stimulation with anti-immunoglobulin G antibodies (anti-IgG) partially inhibited the anti-IgG-mediated induction of ZEBRA synthesis. Both the PO- and PS-antisense oligodeoxynucleotide treatments also suppressed the productive EBV replication (as measured by linear DNA production) in a dose-dependent manner, with much greater efficiency than did PO and PS-oligodeoxynucleotides with sense, reverse or random sequences of the same length. Another 20-mer antisense oligodeoxynucleotide complementary to sequences downstream of the translation initiation codons showed a similar inhibitory effect on EBV replication. However, the inhibition was considerably lower when the cells were treated with oligodeoxynucleotides complementary to sequences upstream of the start codons. These results indicate that BZLF1 antisense oligodeoxynucleotides inhibit the viral activation in a sequence-specific fashion. In the virus-producer cell line P3HR-1, the same PS-antisense oligodeoxynucleotides also partially suppressed the spontaneous viral replication after 6-10 days, substantially more than the PS-random oligodeoxynucleotides. Inhibition of BZLF1 appears to be sufficient to suppress the induction of EBV replication.

Potent antiviral activity of an antisense oligonucleotide complementary to the intron-exon boundary of human cytomegalovirus genes UL36 and UL37.

An antisense phosphorothioate oligonucleotide complementary to the intron-exon boundary of human cytomegalovirus genes UL36 and UL37 (UL36ANTI) reduced the yield of infectious virus by 99% and inhibited human cytomegalovirus DNA replication at a concentration of 0.08 microM. In addition, oligonucleotides with base substitutions which resulted in base pair mismatches showed lesser degrees of activity, indicating a sequence-specific antisense mechanism. UL36ANTI was also shown to inhibit DNA replication of ganciclovir-resistant strains and human cytomegalovirus clinical isolates.

"The end of innocence" revisited: resistance of herpesviruses to antiviral drugs.

In the past 4 years, interest in drug-resistant herpesviruses has evolved from the realm of academic laboratory studies to that of great clinical importance. Recurrent and persistent infections due to the herpes simplex viruses, varicella-zoster virus, and human cytomegalovirus have been an unwelcome consequence of immunosuppression in graft recipients, cancer patients, and those suffering from AIDS. Treatment of these infections with the available antiviral drugs, such as acyclovir, ganciclovir, and foscarnet, has resulted in both clinical benefit and the emergence of drug-resistant variants. In addition, the role of Epstein-Barr virus is being clarified for an array of disease syndromes, and therapeutic approaches are beginning to emerge. In the present review, the emergence and clinical importance of drug resistance among the herpesviruses have been explored. Furthermore, particular attention has been focused on our understanding of the mechanisms of drug resistance and how that understanding will guide us in the development of more effective antiviral drugs and drug usage.

Synthesis and antiviral activity of novel isonucleoside analogs.

A series of branched-chain sugar isonucleosides was synthesized and evaluated for antiviral activity against herpesviruses. The preparation of homochiral [3S-(3 alpha, 4 beta, 5 alpha)]-2-amino-1, 9-dihydro-9-[tetrahydro-4,5-bis(hydroxymethyl)-3-furanyl]-6H-purin-6-one (7, BMS-181,164) and related compounds was stereospecifically achieved starting from 1,2-isopropylidene-D-xylofuranose (10). An efficient two-step reduction of the anomeric center of bis-acetate 18 involved formation of the chloride intermediate 19, followed by diisobutylaluminum hydride reduction. Tosylation of the resulting alcohol 20 provided the key intermediate 21, which was coupled with a variety of nucleobase anions. Several members of this new class of compounds possess activity against herpes simplex virus types 1 and 2 (HSV-1 and -2), varicella-zoster virus (VZV), and human cytomegalovirus (HCMV). Compound 7 exhibits potent and selective activity against thymidine kinase encoding herpesviruses, in particular, HSV-1 and HSV-2. Evaluation of compound 7 for inhibition of WI-38 cell growth indicated an ID50 of > 700 microM. Although the antiherpetic activity in vitro of 7 is less than that of acyclovir (1), compound 7 displays superior efficacy in mouse model infections. The (bromovinyl)uridine analog 8 (BMS-181,165) also exhibits selective activity against HSV-1 and VZV, with no cytostatic effect on WI-38 cell growth at > 800 microM. Compound 8 is active against simian varicella virus and is efficacious in the corresponding monkey model.

The structure and function of the HSV DNA replication proteins: defining novel antiviral targets.

The absolute dependence of herpes simplex virus (HSV) replication on HSV DNA polymerase and six other viral-encoded replication proteins implies that specific inhibitors of these proteins' functions would be potent antiviral agents. The only currently licensed anti-herpes simplex drug, acyclovir, is an inhibitor of HSV DNA polymerase and is widely held to block viral replication primarily by specifically inhibiting viral DNA replication. In spite of the substantial advance in HSV therapy in recent years through the introduction of acyclovir, this anti-HSV compound and most of the other compounds under pharmaceutical development are substrate analogs. Since antiviral drug resistance has become an issue of increasing clinical importance, the need for structurally unrelated agents which incorporate novel mechanisms of viral inhibition is apparent. Understanding the structure and function of herpesvirus DNA polymerase and its interaction with the other six essential replication proteins at the replication origin should assist us in designing the next generation of therapeutic agents. The sequences of these proteins have been deduced and the proteins themselves have been expressed and purified in a variety of systems. The current challenge, therefore, is to use the available information about these proteins to identify and develop new, exquisitely specific antiviral therapeutics. In this review, we have summarized the current approaches and the results of structure/function studies of the herpes virus proteins essential for DNA replication, with the goal of more precisely defining novel antiviral targets.

Synthesis and antiviral activity of 1-cyclobutyl-5-(2-bromovinyl)uracil nucleoside analogues and related compounds.

A series of racemic (1 alpha (E), 2 beta, 3 alpha)-1-[2,3-bis(hydroxymethyl)cyclobutyl]-5-(2-halovinyl)uracils was synthesized and evaluated in cell culture. The bromovinyl, iodovinyl, and chlorovinyl analogues, 13, 15, and 16, respectively, are all potent inhibitors of varicella zoster virus (VZV), but are less inhibitory to the replication of human cytomegalovirus (HCMV) and herpes simplex viruses 1 and 2 (HSV-1, HSV-2). The excellent anti-VZV activities of 13, 15, and 16 coupled with their virtual inability to inhibit WI-38 cell growth indicate high in vitro therapeutic indices. VZV thymidine kinase readily converts these compounds to their respective monophosphates but not to their corresponding diphosphates. Compound 13a, the (1'R) enantiomer of the bromovinyl analogue 13, was also synthesized, and its potency is comparable to that of the racemate. A lower homologue 14, (1 alpha (E),2 beta, 3 alpha)-1-[2-hydroxy-3-(hydroxymethyl)cyclobutyl]-5- (2-bromovinyl)uracil, was found to be inactive against VZV, HCMV, HSV-1, and HSV-2.

Oral bioavailability and anti-simian varicella virus efficacy of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil (BV-araU) in monkeys.

BV-araU (1-beta-D-arabinofuranosyl-E-5-[2-bromovinyl]uracil) has potent antiviral activity against varicella zoster virus in cell culture and is undergoing clinical evaluation. In the present study, pharmacokinetic parameters and the efficacy of BV-araU against infection with simian varicella virus (SVV) were evaluated in African green monkeys. Pharmacokinetic parameters were determined by analysis of the BV-araU content of sera obtained after oral and intravenous administration to normal monkeys. Peak serum concentrations showed dose proportionality, with the 0.1 mg/kg dose resulting in a peak serum concentration of 0.05 micrograms/ml, the approximately ED50 value for the SVV inoculum in cell culture. BV-araU administered orally twice daily at 0.1 mg/kg for 10 days starting 48 h after intratracheal SVV infection prevented vesicular rash development and suppressed viremia. Effective therapy could be initiated 96 h after infection. Taken together, these results indicate that BV-araU is effective oral therapy at doses that achieve peak serum levels equivalent to the ED50 for SVV in cell culture.

Selective activity and cellular pharmacology of (1R-1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine in herpesvirus-infected cells.

The cycloburtane nucleoside analog (1R-1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine [(R)-BHCG or SQ 34,514] was recently synthesized and shown to be the active enantiomer of (+/-)-BHCG (SQ 33,054), a potent inhibitor of several strains of herpesviruses [J. Med. Chem 34:1415-1421 (1991); Antiviral Res. 13:41-52 (1990)]. In plaque reduction assays, (R)-BHCG was about 1000 times more active than its S-enantiomer on herpes simplex virus type I (HSV-1) and over 200 times more active against a thymidine kinase-deficient mutant HSV-1 and human cytomegalovirus (HCMV). We now show that both (R)-BHCG and (S)-BHCG are efficiently phosphorylated to their mono-, di-, and triphosphates by HSV-1-infected cells, in a manner similar to that of acyclovir [Proc. Natl. Acad. Sci. USA 74:5716-5720 (1977)]. The uptake of both enantiomers was greatly increased upon infection; however, (S)-BHCG was taken up to about twice the extent of (R)-BHCG and accumulated primarily as the mono- and diphosphates. (R)-BHCG accumulated primarily as the triphosphate, and accumulation was linear with both time and added drug concentration. The triphosphate had an apparent half-life of about 10 hr. Metabolic studies using HCMV-infected cells showed only a small degree of phosphorylation of (R)-BHCG and none of (S)-BHCG. When cells were labeled with 25 microM (R)-BHCG, the amount of (R)-BHCG triphosphate formed was less than 0.5 pmol/10(6) cells. Interestingly, the ED50 value of (R)-BHCG is about 100-fold higher against HCMV than against HSV-1, and the relative levels of (R)-BHCG triphosphate formed in cells infected by the two viruses are roughly proportional to the antiviral activities.

Evaluation of SQ 34,514: pharmacokinetics and efficacy in experimental herpesvirus infections in mice.

The new antiviral nucleoside SQ 34,514 [(1R-1 alpha, 2 beta, 3 alpha)-2-amino-9- [2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one], the active R isomer of racemic SQ 33,054 (cyclobut-G), was evaluated for efficacy in the treatment of herpesvirus infections in mice. SQ 34,514 was orally efficacious in a herpes simplex virus type 1 (HSV-1) systemic infection, an intracerebral HSV-2 infection, a vaginally induced HSV-2 infection in ovariectomized mice, and in a systemic murine cytomegalovirus infection. SQ 34,514 compared favorably with acyclovir and ganciclovir in the treatment of these experimental infections. In mice, SQ 34,514 had an oral bioavailability of 80% based on urinary excretion. SQ 34,514 may have potential value in the therapy of HSV and cytomegalovirus infections in humans.

Synthesis and antiviral activity of enantiomeric forms of cyclobutyl nucleoside analogues.

The syntheses of the enantiomeric cyclobutyl guanine nucleoside analogues [1R-1 alpha, 2 beta, 3 alpha]- and [1S-1 alpha, 2 beta, 3 alpha]-2- amino-9-[2,3-bis(hydroxymethyl)cyclobutyl]-6H-purin-6-one (7 and 8, respectively) and the enantiomeric cyclobutyl adenine analogues [1R-1 alpha, 2 beta, 3 alpha]- and [1S-1 alpha, 2 beta, 3 alpha]-6-amino-9-[2,3-bis(hydroxymethyl) cyclobutyl]purine (9 and 10, respectively) are described. trans-3,3-Diethoxy-1,2-cyclobutanedicarboxylic acid (14) was coupled with R-(-)-2-phenylglycinol to provide a mixture of diastereomeric bis-amides, 15a and 15b, which was readily separated by crystallization. Conversion of each bis-amide to the corresponding diol enantiomer, 16a and 16b, respectively, was effected by a facile three-step sequence in high overall yield. Homochiral diol 16a was converted in a straightforward manner to 7 and 9, and homochiral diol 16b was similarly converted to the corresponding optical isomers 8 and 10. Compounds 7 and 9, which mimic the absolute configuration of natural nucleosides, are highly active against a range of herpesviruses in vitro while the isomers of opposite configuration, 8 and 10, are devoid of antiherpes activity. The corresponding triphosphates of 7 and 8 (7-TP and 8-TP) were prepared enzymatically. Compound 7-TP selectively inhibits HSV-1 DNA polymerase, compared to human (HeLa) DNA polymerase, while 8-TP is much less inhibitory than 7-TP against both types of enzymes. Compounds 7 and 9 are efficacious in a mouse cytomegalovirus model infection.

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)-cyclobutyl] guanine [(+-)-BHCG or SQ 33,054]: a potent and selective inhibitor of herpesviruses.

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl] guanine [(+-)-BHCG or SQ 33,054] is a newly synthesized nucleoside analog with potent and selective antiviral activity against members of the herpesvirus group, including human cytomegalovirus. The activity against a thymidine kinase deficient HSV-2 mutant was 25-fold poorer than against the parent virus, suggesting that phosphorylation is an important prerequisite for antiviral activity against HSV-2. (+-)-BHCG is readily phosphorylated by purified HSV-1 thymidine kinase, and BHCG triphosphate synthesized enzymatically is a selective inhibitor of HSV-1 DNA polymerase. (+-)-BHCG did not inhibit host cell growth at concentrations at least 1000-fold higher than HSV-2 inhibitory concentrations. Subcutaneous administration of (+-)-BHCG was protective against HSV-1 systemic infections in mice. BHCG is an exciting antiviral agent and represents a new class of nucleoside analogs.

Synthesis and antiherpetic activity of (+/-)-9-[[(Z)-2-(hydroxymethyl)cyclopropyl]methyl]guanine and related compounds.

A series of analogues of acyclovir and ganciclovir were prepared in which conformational constraints were imposed by incorporation of a cyclopropane ring or unsaturation into the side chain. In addition, several related base-modified compounds were synthesized. These acyclonucleosides were evaluated for enzymatic phosphorylation and DNA polymerase inhibition in a staggered assay and for inhibitory activity against herpes simplex virus types 1 and 2 in vitro. Certain of the guanine or 8-azaguanine derivatives were good substrates for the viral thymidine kinase and were further converted to triphosphate, but none was a potent inhibitor of the viral DNA polymerase. Nevertheless, one member of this group, (+/-)-9-[[(Z)-2-(hydroxymethyl)cyclopropyl]methyl]guanine (3a), displayed significant antiherpetic activity in vitro, superior to that of the corresponding cis olefin 4a. Another group, typified by (+/-)-9-[[(E)-2-(hydroxymethyl)cyclopropyl]methyl]adenine (17b), possessed modest antiviral activity despite an apparent inability to be enzymatically phosphorylated. The relationship of side-chain conformation and flexibility to biological activity in this series is discussed.

Broad-spectrum antiviral activity of the acyclic guanosine phosphonate (R,S)-HPMPG.

(R,S)-9-(3-hydroxy-2-phosphonomethoxypropyl)guanine [(R,S)-HPMPG] exhibits broad spectrum antiviral activity with an ED50 of less than 1 microM against herpes simplex virus (HSV) types 1 and 2, varicella zoster virus, human cytomegalovirus (HCMV) and vaccinia in plaque reduction assays. Wild type HSV-2 and its thymidine kinase deficient variant are equally sensitive to (R,S)-HPMPG. (R,S)-HPMPG is 100-fold more potent than acyclovir (ED50 = 0.45 microM vs. 44 microM, respectively) against HCMV in cell culture, and 10-fold more active than acyclovir in extending survival time in mice intraperitoneally infected with 70 LD50 HSV-1. However, (R,S)-HPMPG is toxic when administered repeatedly at 44 mg/kg/day in uninfected adult mice. The diphosphoryl derivative of HPMPG was enzymatically synthesized and is a competitive inhibitor of HSV-1 DNA polymerase relative to dGTP (K1 = 0.03 microM). HPMPG-PP is 70-fold less active at inhibiting HeLa DNA polymerase alpha than HSV-1 DNA polymerase. At concentrations between 0.3 and 1.5 microM (R,S)-HPMPG inhibited HSV-1 DNA replication greater than or equal to 50% in infected cells as measured by nucleic acid hybridization. Consistent with inhibition of viral DNA synthesis, 6 to 30 microM (R,S)-HPMPG reduces late viral polypeptide synthesis in HSV-1 infected cells. These data indicate that (R,S)-HPMPG is a thymidine kinase independent broad spectrum antiviral drug which is capable of inhibiting viral DNA polymerase.