Review The quest for an efficacious antiviral for respiratory syncytial virus

20 Pages·2003·0.33 MB·English

Antiviral Chemistry & Chemotherapy 13:325–344
Review
The quest for an efficacious antiviral for respiratory
syncytial virus
Paul F Torrence* and Linda D Powell
Department of Chemistry, Northern Arizona University, Flagstaff, Ariz., USA
*Corresponding author: Tel: [phone]; Fax: [phone]; E-mail: [email protected]
Respiratory syncytial virus (RSV) continues as an hypothesis for the mechanism of action of
emerging infectious disease not only among ribavirin, and a promising antisense strategy
infants and children, but also for the immune- combining the 2′-5′ oligoadenylate antisense
suppressed, hospitalized and the elderly. To date, (2-5A-antisense) approach and RSV genomics.
ribavirin (Virazole) remains the only therapeutic
agent approved for the treatment of RSV. The Keywords:2-5A-antisense, 2-5A, RNase L, inter-
prophylactic administration of palivizumab is feron, ribavirin, nucleosides, membrane fusion,
problematic and costly. The quest for an error threshold, lethal mutageneis, peptide
efficacious RSV antiviral has produced a greater nucleic acids, morpholino nucleic acids
understanding of the viral fusion process, a new
Introduction
Respiratory syncytial virus (RSV) bronchiolitis was the nonetheless, logistical problems and cost associated with
leading primary diagnosis annually for all infants hospital- palivizumab use have been considered problematic
ized for any reason between 1997 and 1999 (Collins & (Greenough,2002;Kimpen,2001).RSV vaccine develop-
Pollard, 2002; Hall, 2000); yet it may be underdiagnosed ment continues with considerable promise (Collins &
(Ruef,2002).RSV infections remain a significant problem Murphy,2002;Kahn,2000;Murphy & Collins,2002).
in adults,particularly the elderly and the immunocompro- Some clinical observations on the use of palivizumab in
mised (Collins & Pollard, 2002; Falsey & Walsh, 2000; immunocompromised adults have been reported (Boeckh
Hall, 2000). RSV is the most important cause of severe et al.,2001;Khushalani et al.,2001);however,no evidence-
viral disease among transplant recipients (Billings et al., based conclusions are possible at this time.
2002;Billings et al.,2001;Ison & Hayden,2002),is a cause The value of ribavirin in RSV infections of infants,
of exacerbation of of chronic obstructive pulmonary disease children and adults has been increasingly questioned
(COPD) (Xue et al.,2002),is associated with later reactive (Vujovic & Mills, 2001; Whimbey & Ghosh, 2000), and
airway disease (Barends et al., 2002; Piedimonte, 2002; the need for an effective antiviral is generally acknowl-
Sigurs, 2002), exacerbates asthma (Message & Johnston, edged. Thus, numerous authorities have questioned and
2002),and contributes to mortality in patients undergoing challenged the therapeutic value of ribavirin for RSV infec-
chemotherapy (Field et al., 2002). There is a correlation tions in all but very restricted applications. For instance,
between RSV genotype and severity of illness (Martinello Greenough (Greenough, 2002) states: ‘Whether ribavirin
et al.,2002). or corticosteroids reduce respiratory morbidity after RSV
On the other hand, early ribavirin treatment of RSV infection remains controversial.’ Similarly, Kimpen
bronchiolitis in previously healthy infants resulted in reduc- (Kimpen, 2001) opined:‘The antiviral agent ribavirin has
tion of incidence and severity of reactive airway disease,as not lived up to expectations and should be reserved for
well as respiratory illness-related hospitalization (Edell et selected cases.’Vujovic and Mills (Vujovic & Mills,2001)
al., 2002). Palivizumab was shown to significantly reduce expressed the view that ‘the value of the only therapeutic
RSV-related infant hospitalizations in North America and agent available to treat established infection, the antiviral
Europe with few adverse effects (Garzon & Wiles, 2002; ribavirin, is now seriously doubted and the agent is little
Groothuis & Nishida,2002;Simoes & Groothuis,2002); used’. The Swedish Consensus Group (2001) concluded
©2002 International Medical Press 0956-3202/02/$17.00 325
PF Torrence & LD Powell
that ‘ribavirin inhalation treatment may be considered in a domain which anchors the protein in the viral cell mem-
high-risk infants with clinical symptoms indicating a seri- brane and also contains a domain which anchors the pro-
ous course of an RSV infection.Treatment with ribavirin in tein in the host cell membrane (Lambert et al.,1996).It has
combination with intravenous polyclonal immunoglobulin been suggested that this protein can independently facili-
should be considered in patients who have received an allo- tate attachment and infection of cells via an interaction
genic stem cell transplantation or organ transplantation with cellular heparan sulfate (Feldman et al.,2000).Zhao
with >1 episode of rejection treatment and who have mild and colleagues (Zhao et al.,2000) propose that the model
or moderate RSV pneumonia.Evidence-based documenta- of viral membrane fusion for the HIV-1 fusion protein
tion for treatment of other groups of patients is lacking’. gp41 (Chan & Kim,1998) is applicable to the RSV-F pro-
Likewise,Van Woensel,Kimpen and Brand (Van Woensel, tein. This mechanism involves three different conforma-
2001) state that ‘based on the current available evidence tional changes:a native,nonfusogenic form;a pre-hairpin
there is no place of ribavirin in the routine treatment of intermediate; and a fusion active form.They also showed
RSV-LRTI [lower respiratory tract infection (sic)]’.Prince that peptides, which correspond to heptad, repeat regions
(Prince,2001) concurs in his review:‘…ribavirin usage has of the C-terminus for gp41 efficiently inhibited infection,
declined precipitously in recent years due to concerns over ostensibly by prohibiting conformational changes to the
efficacy,safety,ease of use and cost.’ fusogenic form. The homology between gp41 and the
This review represents a selective reading of the litera- RSV-F protein further suggests commonality in their
ture for what, in the authors’ opinions, represents a sam- fusion processes (Lambert et al.,1996).
pling of the most promising approaches to new antivirals The F protein may also interact with cellular RhoA to
for RSV infections.Three separate areas of endeavour are facilitate fusion to cell membranes (Pastey et al., 2000).
presented:progress with fusion inhibitors;a new hypothe- However, since RhoA inserts itself into the cytoplasmic
sis for the mechanism of ribavirin antiviral action;and tar- side of the cell membrane,it is unlikely that this protein is
geting RNase L to RSV RNA using antisense. a membrane receptor for the virus (Feldman et al.,2000).
The fusion process is essential in the life cycle of the
Targeting the RSV F protein: fusion
virus. It is necessary for the virus to deposit its genetic
inhibitors
material into the target cell and thus induce the production
of new virions.If this process can be prevented,the repro-
Respiratory syncytial virus contains three surface mem- duction,spread,and devastation of the virus can be halted.
brane glycoproteins;namely,F,G,and SH.All proteins are Several agents have been synthesized which purport to
essential to efficient fusion of cell membranes and conse- inhibit the fusion process of RSV.This account will cover
quent syncytia formation (Heminway et al.,1994).Karron those fusion inhibitors, which have reported advances in
and colleagues discovered that cold-passaged subgroup B approximately the past five years.
mutant RSV,cp-52,lacked the G and SH membrane gly- RFI-641 (4,4′-bis-{4,6-bis-[3-(bis-carbamoylmethyl-
coproteins (Karron et al.,1997).This mutant strain could sulfamoyl)-phenylamino]-(1,3,5)-triazin-2-ylamino}-
reproduce, infect, and illicit antibody responses in cotton biphenyl-2,2′-disulfonic acid) is a dendrimer-like com-
rats, African green monkeys and chimpanzees (Crowe et pound resulting from optimization of CL387626
al., 1996). While it showed promise in vitro, cp-52 was (Nikitenko et al., 2001). CL387626 resulted from high
found to be over attenuated when tested in infants and throughput screening of a diverse 20000 compound library
children (Karron et al.,1997) Thus,although the F protein at Wyeth-Ayerst Research Laboratories and showed
alone can account for attachment and infectivity,it is clear remarkable activity against RSV in tissue culture and in
that the assistance of the G and SH proteins may be cotton rats (Wyde et al.,1998).RFI-641 is a biphenyl ana-
required for full virus infectivity. logue of CL387626.Figure 1 shows the chemical structures
The importance of the F protein should not be underes- of these two compounds. The biphenyl analogue has an
timated. This glycoprotein is the only surface membrane even greater activity with an IC (mean cell inhibitory
50
protein that has emerged as a target for therapeutic inter- concentration) that is one-third of that of the parent.
vention. RSV-F is a target for small-molecule antiviral Wyeth-Ayerst Research Laboratories has shown using a
agents,an epitope for monoclonal antibodies,and the focus fluorescence-quenching assay that RFI-641 inhibits both
for vaccine candidates (Meanwell & Krystal, 2000). RSV-cell binding and fusion events (Razinkov et al.,2001).
Perhaps this is due to the fact that the F protein is highly The activity is specific for RSV. Little, if any, effects are
conserved and plays a crucial role in viral entry (Lambert et shown against influenza A or B,human parainfluenza virus
al.,1996). type 3, human cytomegalovirus or herpes simplex virus.
The F protein is composed of two subunits,F1 and F2, The actions of RFI-641 are not virucidal and can only be
linked by a single disulfide bond.The F1 subunit contains seen in the context of virus-cell interactions.
326 ©2002 International Medical Press
RSV antivirals
Figure 1.Wyeth-Ayerst’s inhibitors of respiratory syncytial virus fusion with host cells
O
H2N O H2N
H2N O NOSN NH HO2N N NOH2 H2ONO N OSO NH O NOH2
HN N N HO3S H OO S HN N N NaO3S O SN NH2
O N N N H O
H N N N
H2NO N S O SO3H HNN NONH H2N NS OO H SO3Na N NN NH
H2N O OS N O H2ON O HN OS N ONH2
O
NH O
O 2 NH
2
CL387626 NH RFI-641
2
RFI-641 (IC =0.15 µM) is the result of the optimization of CL387626 (IC =0.05 µM) (Nikitenko et al., 2001).
50 50
RFI-641 has been evaluated in three different animal (Meanwell & Krystal,2000).Sudo and colleagues have also
models including mice,cotton rats and African green mon- studied the pharmacokinetics of aerosol and oral treatment
keys (Huntley et al., 2002). Mice and rats which were using 14C-RD3-0028 (Sudo et al.,2002).
infected 2 h post-treatment with RFI-641 showed a signif- In immunosuppresed mice, aerosol administration of
icant decrease in viral lung titres compared with the RD3-0028 for 2 h twice daily beginning 24 h after viral
untreated control group (Huntley et al.,2002).In the mon- challenge resulted in reductions in pulmonary viral titres
key model, the nasal titres and throat samples showed a depending upon the dosage (Sudo et al.,1999).Drug con-
reduction with prophylactic treatment and therapeutic centrations in the reservoir of 1.25,2.5,and 7 µg/ml result-
treatment (Huntley et al.,2002).Huntley et al.reported a ed in 50%, 64% and 64% reduction in viral titres, respec-
reduction in lung titres with prophylactic treatment, but tively (Sudo et al.,1999).The control,ribavirin,produced a
did not mention lung titres of therapeutic treatments. 60% reduction in viral titres at a concentration of 60 mg/ml
Rational Drug Design Laboratories’ RD3-0028 (1,4- (Sudo et al., 1999).The histological examination of lung
dihydro-2,3-benzodithiin) is a structurally simple small- tissues in control animals showed evidence of interstitial
molecule inhibitor of RSV fusion with host cells (Sudo et pulmonary infiltrates, whereas the examination of tissues
al.,2001;Sudo et al.,1999;Sudo et al.,2002).Its chemical from drug treated mice did not (Sudo et al.,1999).
structure is shown in Figure 2.The efficacy of this com- ViroPharma’s VP-14637 (a 5-methyl substituted di-
pound has been evaluated extensively in vivo with tetrazole-benzhydrylphenol) is a recently identified low
immunosuppressed mice (Sudo et al.,1999) and rats (Sudo molecular weight viral replication inhibitor.VP-14637 was
et al.,2002).It was shown to inhibit laboratory and clinical actually the result of a minor impurity in one of the reac-
isolates of RSV A and B strains in cell culture, but not tants used during a high-throughput screening for antiviral
inhibit the replication of herpes simplex virus types 1 and compounds at ViroPharma’s labs (McKimm-Breschkin,
2, influenza A virus, measles virus or human 2000).The structure is shown in Figure 3.
cytomegalovirus (Watanabe et al., 1998). When viral iso-
lates were selected for a resistance to RD3-0028,these iso-
lates were found to contain at least one mutation in the F Figure 2.Rational Drug Design Laboratories’ small
gene segment (Sudo et al., 1999). These results indicate molecule inhibitor of RSV-F protein
that RD3-0028 interferes with the intracellular processing
of the RSV fusion protein.However,Meanwell and Krysal
report that virus which is incubated with RD3-0028 for S
one hour remains infectious,which would not indicate that
the changes to the F protein are irreversible, or that the
S
interference occurs late in the replication cycle of the virus
Antiviral Chemistry & Chemotherapy 13:6 327
PF Torrence & LD Powell
Figure 3.ViroPharma’s small molecule viral replica- Figure 4.Metkinen Oy’s FDA-approved agent for
tion inhibitor lowering cholesterol
N N N N
H
O
N N
H C CH HO
3 N N 3
N N
O
H
H3C O
H
H
CH
CH 3
3
OH OH H C
3
Lovastatin
OH
VP-14637
Lovastatin is reported to reduce viral replication in mice via an
interference with HMG-CoA reductase (Gower & Graham, 2001).
Similar to RFI-641 and RD3-0028,VP-14637 exhibits Pastey et al.have shown that RhoA binds to RSV F protein
activity against both strains A and B of RSV, but not and inhibits syncytium formation (Pastey et al., 1999).
against other members of the Paramyxoviridae, including RhoA cycles between an active form and an inactive form.
parainfluenza type 3, measles and mumps (McKimm- In its active form, RhoA is bound by GTP and becomes
Breschkin,2000).It was also active against strains obtained post-translationally modified at the C-terminus (Pastey et
from patients in North America and Europe over an 11 al., 1999). One of the modifications is isoprenylation.
year time period,illustrating a high degree of conservation Lovastatin is believed to inhibit RSV replication via inter-
for the target (McKimm-Breschkin,2000).As with RD3- ference with the virus-target cell fusion process (Gower &
0028, resistant strains exhibited mutations in the virus F Graham,2001).Gower and Graham reported an attenua-
protein, indicating that VP-14637 interferes with viral tion of virus replication and a decrease in virus-induced ill-
fusion activity (McKimm-Breschkin, 2000). Time-of- ness in mice (Gower & Graham,2001).
addition studies indicate that this compound acts early in
Ribavirin: a new theory about a long-
the replication cycle of RSV (McKimm-Breschkin, 2000;
established antiviral active against RSV
Meanwell & Krystal, 2000). ViroPharma reports VP-
14637 to be currently in Phase I clinical trials (McKimm-
Classical modes of action
Breschkin,2000).
Lovastatin is Metkinin Oy’s FDA-approved drug for The structural geometry of ribavirin (Figure 5) has been
the treatment of hypercholesterolaemia. Gower and associated with one of the accepted mechanisms of antivi-
Graham report an interesting antiviral activity of ral activity; namely, inhibition of inosine 5′-monophos-
Lovastatin against RSV in vivo and in vitro (Gower & phate (IMP) dehydrogenase by the intracellular metabolite
Graham, 2001). Its chemical structure is represented in ribavirin 5′-monophosphate (Fernandez et al., 1986;
Figure 4.Lovastatin is described as a fungal metabolite iso- Patterson & Fernandez-Larsson,1990).The shutdown of
lated from cultures of Monascuc ruber and Aspergillus terreus. conversion of IMP to xanthosine 5′-monophosphate
It inhibits hydroxymethylglutaryl coenzyme A (HMG- (XMP), the precursor of guanosine 5′-monophosphate,
CoA) reductase,a key enzyme in the biosynthesis of cho- results in a two-fold reduction in guanosine 5′-triphos-
lesterol. phate (GTP) cellular concentrations. According to this
A branch in the cholesterol biosynthesis pathway leads long-accepted model, such a reduction would account for
to the formation of isoprene groups, and HMG-CoA the inhibition of RNA viruses which are critically depend-
reductase inhibitors have been shown to also inhibit ger- ent on GTP pools for replication (Fernandez et al.,1986;
anylgeranylation of RhoA GTPase (Park & Galper,1999). Sidwell et al.,1979).
328 ©2002 International Medical Press
RSV antivirals
Figure 5.Structures of ribavirin and analogues
Cinader,1980;Sintchak & Nimmesgern,2000).
In the past several years, another model for ribavirin
NO2 O
H2N action has been forwarded (Cameron & Castro, 2001;
Crotty et al., 2002; Fang et al., 2001; Graci & Cameron,
N
S
O N 2002; Harki et al., 2002).This derives from the ability of
HO OH HO O cell-generated ribavirin 5′-triphosphate to act as a substrate
OH OH for poliovirus RNA-dependent RNA polymerase,and the
OH
3-NPN consequent incorporation of ribavirin into poliovirus RNA
Tiazofurin
at a rate of less than three molecules per genome per repli-
O O O
H2N H2N H2N cation cycle.The freely rotating carboxamido side chain of
N N N ribavirin leads to promiscuous base-pairing with either
F N HO
N N N cytidine or uridine.Thus,ribavirin can be incorporated into
O O O
HO HO HO poliovirus RNA in place of uridine or cytidine,and in sub-
OH OH OH
sequent replication cycles can template for either uridine or
OH OH OH
cytidine, thereby promoting G-to-A and A-to-G transi-
FICAR Ribavirin Bredinin
tion mutations as replication continues.It could be that the
O NH earlier observations (Toltzis & Huang, 1986) on the pro-
H2N H2N
N duction of non-functional VSV mRNA under ribavirin’s
Se N influence was an expression of incorporation into VSV
N N
O O RNA.
HO HO
OH OH The lack of a proof-reading activity in the RNA-
OH OH dependent RNA polymerases of RNA viruses leads to the
Selenazole Ribamidine existence of a quasispecies – a heterogenous RNA popula-
tion with resultant phenotypic variants that can readily
respond to an environmental challenge such as that pre-
Preying on promiscuity?
sented by the immune response,or during antiviral therapy.
An alternative mechanism is based upon the inhibition of Yet according to this scenario,there ought to be some level
methylation (Sharma et al.,1982) of uncapped viral RNA of mutation, the so-called error threshold, beyond which
and a resultant drop in efficiency of translation.Yet a third viability could not be maintained. Beyond this threshold,
mechanism revolves about the experimental observation even a small increase in mutation rate would lead to error
that the RNA-dependent RNA polymerase of influenza catastrophe, a massive decrease is virus viability. This
virus was inhibited by ribavirin 5′-triphosphate, readily hypothesis further holds that since RNA viruses have infor-
formed from ribavirin in intact cells (Eriksson et al.,1977). mation-rich genomes,and since they exist on the edge of
Effects on primer generation also have been witnessed the error threshold to maintain maximum adaptability,they
(Wray et al., 1985). Observations on the inhibition of should be exquisitely sensitive to factors that increase the
reovirus replication by ribavirin led to another model that mutation rate.
posits binding of ribavirin triphosphate to a site close to the In fact,Crotty et al.have shown that while the natural
catalytic site of the transcriptase (Rankin et al.,1989).This error rate for poliovirus mutation was 1.5 mutations per
binding would inhibit the helicase function of the tran- progeny genome, 400 µM ribavirin increased that rate to
scriptase and lower its affinity for template RNA,causing 6.9 mutations per progeny genome, with a concomitant
premature termination of transcription. Most recently, an 95% reduction in viral fitness as compared with wild-type
inhibitory effect of ribavirin triphosphate on the helicase virus (Crotty et al.,2002).
activity of hepatitis C virus NTPase/helicase has been For the hepatitis C virus (HCV), a similar story has
reported (Borowski et al.,2001).Conversely,host cell RNA been reported (Maag et al.,2001).HCV RNA-dependent
and DNA polymerases were significantly less susceptible to RNA polymerase also can incorporate ribavirin (as triphos-
ribavirin triphosphate. Inhibition of vesicular stomatitis phate) opposite both uridine and cytidine in the RNA tem-
virus by ribavirin may be mediated through the ribavirin- plate.In addition,the viability of GB virus B,a close rela-
induced production of vesicular stomatitis virus (VSV) tive of HCV,is dramatically decreased by infection in the
mRNA (Toltzis & Huang, 1986). Other explanations of presence of ribavirin (Lanford et al., 2001). This finding
ribavirin activity invoke immunological mechanisms may not be so relevant for HCV therapy.HCV treatment
(Cameron & Castro, 2001; Clumeck & Hermans, 1988; presently involves administration of both interferon and
Crotty et al., 2002; Fang et al., 2001; Graci & Cameron, ribavirin,with the latter mode of action involving a strong
2002;Harki et al.,2002;Marquardt et al.,1987;Nakano & immunological component (Souvignet,2000;Tam,2001).
Antiviral Chemistry & Chemotherapy 13:6 329
PF Torrence & LD Powell
Thus,the hypothesis has been advanced that ribavirin’s the 5 position of the triazole ring of ribavirin.
mechanism of antiviral action is due to its ability to act as To test further the lethal mutagenesis hypothesis, an
an RNA mutagen. A posteriori, Harki et al. (Harki et al., alternative approach might employ the substantial knowl-
2002) surmised that such agents that accelerate viral muta- edge gathered from earlier SAR studies.For instance,the
genesis represent a ‘promising new class of antiviral thera- ribavirin analogues 1-(β-D-ribofuranosyl)-5-fluoro-imida-
peutics’.A fresh as this idea is,it is subject to caveats,some zole-4-carboxamide, 1-(β-D-ribofuranosyl)-5-hydroxy-
of which have been delineated by Graci and Cameron 1,2,3-triazole-4-carboxamide, 2-(β-D-ribofuranosyl)-thia-
(Graci & Cameron,2002):ribavirin has not been demon- zole-4-carboxamide (tiazofurin), 2-(β-D-ribofuranosyl)-
strated to lead to extinction of the virus population;extinc- selenazole-4-carboxamide(selenazofurin), and 1-(β-D-
tion of FMDV (foot and mouth disease virus) in the pres- ribofuranosyl)-1,2,4-triazole-3-carboxamidine (ribami-
ence of the RNA mutagen 5-fluorouracil and the inhibitor dine) all show activity against RNA viruses. A rigorous
guanidine has been reported. examination of the antimetabolic spectrum of these agents
The complexities of moving the idea of lethal mutagen- and others,together with an assessment of their ability to
esis to the discovery of an RNA virus antiviral is illustrated induce lethal mutagenesis, could provide key information
by a recent paper by Harki et al.(Harki et al.,2002) who to assess the overall role of the error catastrophe hypothesis
tested the hypothesis that a ribonucleoside more mutagenic in relation to previously demonstrated modes of ribavirin
than ribavirin should provide a more potent antiviral.They action. Additionally, as observed by Graci and Cameron
synthesized a riboside congener (Figure 5) of 1-(2′- (Graci & Cameron,2002) the ability of ribavirin to reduce
deoxyribofuranosyl)-3-nitropyrrole (3-NPN). Bergstrom the GTP pool may help drive the lethal mutagenic effect.
and colleagues (Bergstrom et al.,1997) introduced the lat- The challenge then may be to preserve the IMP dehydro-
ter as a highly effective (and now widely used) ‘universal genase inhibitory activity of ribavirin while enhancing the
base’ that can hybridize with all four RNA bases.The 3- RNA mis-incorporation property.
nitropyrrole moiety disposes of the usual specific hydrogen
2-5A-antisense stratagem and its
bonding patterns of the pyrimidine and purine bases,and
application to RSV
instead relies upon the nitro group for weak hydrogen
bonding interactions with all four bases. In fact, the 3-
Preliminary considerations
NPN was devoid of antipoliovirus activity at 1000 µM,
even though it was 5′-phosphorylated by adenosine kinase Viruses and interferons: attack and counterattack.
as efficiently as was ribavirin. In addition, 3-NPN 5′- Viruses have evolved numerous strategies to evade the
triphosphate was incorporated into poliovirus RNA by the interferon defence systems (Katze,2002).For instance,in
RNA-dependent RNA polymerase at a rate 100-fold less the case of adenoviruses, the ‘stealth’ factor, E1A, blocks
than that for ribavirin 5′-triphosphate (Harki et al.,2002). interferon-induced gene expression and the VA-RNA
Moreover, incorporation occurred only opposite template inhibits interferon-induced PKR activity (Burgert, 2002).
uridine and adenosine, not opposite template guanosine In the hepatitis C virus,the 2-5A system’s ribonuclease L
and cytidine. cleaved HCV mRNA predominately at UA and UU dinu-
cleotides;however,HCV mRNAs from relatively interfer-
Ribavirin and the error threshold
on-resistant genotypes had fewer UA and UU dinu-
hypothesis: ruminations from the past
cleotides than HCV mRNAs from more interferon-sensi-
tive genotypes (Han, 2002). In patients, HCV mRNAs,
The results of the foregoing study with 3-NPN were con- sensitive to interferon accumulated silent mutations at UA
sonant with the massive volume of earlier work on the and UU dinucleotides during interferon therapy (Han,
structure–activity relationships (SAR) of ribavirin and its 2002). Kaposi’s sarcoma-associated herpesvirus blocked
analogues (Cook et al., 1978; De Clercq et al., 1978; virus-mediated induction of type I interferon through a
Goebel et al.,1982;Huynh-Dinh et al.,1977;Kini et al., viral immediate-early protein,namely ORF45,that inter-
1989;Narang & Vince,1977;Revankar & Robins,1975; acts with cellular interferon-regulatory factor 7(IRF-7).
Sanghvi et al., 1988; Sidwell et al., 1979; Smejkal et al., IRF-7 phosphorylation therefore is inhibited and the accu-
1984; Srivastava et al., 1975, 1977; 1984; Storer et al., mulation of IRF-7 in the nucleus in response to viral infec-
1999; Tsilevich et al., 1987; Vijayaiakshmi & Yathindra, tion is blocked (Zhu et al., 2002). Since IRF-7 is a tran-
1980;Wood et al.,1985) Such research has demonstrated scription regulator responsible for virus-mediated activa-
that the structural requirements for antiviral activity are tion of type I interferon genes,the activation of interferon-
quite stringent.These include an absolute requirement for alpha and -beta genes during viral infection is shut down.
the 4-carboxamido group and the probable need for an In addition, a variety of other viruses (Grandvaux, 2002),
electron-rich hydrogen bond acceptor corresponding to such as paramyxoviruses (Gotoh,2001),herpes simplex-1,
330 ©2002 International Medical Press
RSV antivirals
Figure 6.The 2-5A system
vaccinia and encephalomyocarditis virus have evolved anti-
interferon defences. In at least some cases, these defences
Viral infection
are associated with the virulence and pathogenicity of
infection (Shors,1998;Xiang et al.,2002). 2-5A Synthetase
(active)
RSV seems no exception. Numerous observations dsRNA AMP
(Aberle et al., 1999; Chipps et al., 1993; Higgins et al., ppp5'(A2'p)nA
1990; Isaacs, 1989; Merolla et al., 1995; Nakayama et al., 2',5'-phosphodietsterase
1993;Pitkaranta & Hovi,1993;Roberts et al.,1992;Sung 2-5A Synthetase ATP
(inactive)
et al.,1993;Taylor et al.,1989;Young et al.,2000) exist to
2-5A-dependent RNase
show that although interferon is generated by RSV-infect- (inactive)
ed cells in culture and in humans, and while interferon
inhibits RSV replication in vitro, RSV is less sensitive to
2-5A-dependent RNase
interferon inhibition than other viruses such as influenza. (active)
Interferon is not induced effectively in RSV-infected
RNA
infants;moreover,exogenous interferon administration has
no effect on the course of RSV bronchiolitis.While neither RNA degraded
human or bovine RSV block the antiviral effect of interfer-
on by inhibiting interferon signalling or interferon induc-
tion, the bovine RSV NS1 and NS2 proteins antagonize mammalian cells through its activation of the latent 2-5A-
the antiviral effect of interferon through an unknown dependent RNase L which degrades mRNA, thereby
mechanism (Bossert & Conzelmann,2002). inhibiting translation (Player & Torrence, 1998). The
Important caveats exist to be added to the mix, unique feature of RNase L is that it is converted from a
however.Both γ-interferon(-/-) mice and mice treated with silent to an active form in response to 2-5A.Finally,2-5A
anti-γ-interferon developed more extensive inflammation is destroyed by and its cellular toxicity is limited by the
of the airways than control mice (van Schaik et al.,2000a). 2′,5′-phosphodiesterase (Player & Torrence, 1998). 2-5A
However, mice lacking γ-interferon showed less severe plays a key role in the anti-RSV effect of γ-interferon in
signs of airway obstruction, thereby suggesting a positive human epithelial cells in vitro(Behera et al.,2002).
role of γ-interferon in RSV infection in blocking virus pro- Covalent linkage of a 3′,5′-antisense oligodeoxyribonu-
duction and inflammatory responses. On the downside, cleotide and a 2′,5′-oligoadenylate activator of the 2-5A-
however,γ-interferon possessed a pathogenic role as a cause dependent RNase L provides a 2-5A-antisense composite
of airway obstruction.Thus, RSV-induced wheezing may nucleic acid that can,through its antisense domain,target a
be caused by γ-interferon, possibly through induction of
leukotriene release (van Schaik et al.,2000b).
Figure 7. Structures of key 2-5A congeners
Interferon, 2-5A, RNase L: clues to a chemotherapy
based on a natural defence? Interferon stimulated genes NH2
N
encode proteins that mediate all of the biological effects of N
interferons (De Clercq et al., 1978; Player & Torrence,
N N NH2
1998;Silverman,1994;Stark et al.,1998).There are three
essential components of the 2-5A system (Figure 6).First RO O N N
is the multienzyme family of 2-5A synthetases, which, H H
upon activation by double-stranded RNA,synthesize 2-5A H OH O H N N NH2
from ATP.Second is the unique latent and constitutive 2- O P O O N N
5A-dependent ribonuclease (RNase L),which,upon acti- O– H H
N
vation by 2-5A, degrades RNA. Third is the 2′,5′-phos- H OH O H N
phodiesterase that degrades 2-5A (Figure 7) to AMP and O P O O
ATP.Treatment with interferon protects cells through the R=H, ‘core’ O– H H
n H H
harmonious action of these three enzymes of the 2-5A sys- O O– OH OH
tem acting in the following way.Exposure of a cell to inter- R= P 2-5A 5’-monophosphate
O
feron induces enhanced levels of 2-5A synthetase. Then –O
O P
dsRNA,formed as an intermediate in viral replication,acti- O R=
vates the synthetase to generate 2-5A from ATP.2-5A pro- O P O O P O O–
vides an unambiguous signal to initiate RNA decay in R= –O P O O– P O– O–
–O
O– 2-5A 5’-diphosphate 2-5A 5’-triphosphate
Antiviral Chemistry & Chemotherapy 13:6 331
PF Torrence & LD Powell
Figure 8.Mechanism of selective destruction of RNA
A1 receptor and the subsequent relief of airway constric-
by 2-5A-antisense
tion caused by histamine,adenosine of dust-mite allergen
(Nyce,1997).
2p-55A'A-a2n'tpise[5n'seA2'p.]..2rN5p'ArN2p'rpNBpruNppBrNupprN[5p'rRdNNpNAr3N T'paprrN]g5.e.'.tN eca1Inomn9 mo92pat9-dplh;o5dleeyCArimet i-idtroaeh innntnao,ttot ia sbsrteeelyatovn recasgsrketle.ar ,talRos 1t ldeSi9RggiV9foiSf7e enVrs;reu:e Npcnogllynteiec cnoeace toht,itimdheo1eman9isct 9 i tch7thRaaa;rlrNvo PgefuAelo agtbr syhme (e RBertun haSle aertVsnt uuia aoclsmr.nced,e sR o1s esNfo9t ff 9tuA aw8l2lls).oy-.,;
p5'A2'p[5'A2'p]25'A2'pBupBup[5'dN3'p]5'N 2-5A-antisense:RNA hybrid 5A-antisense have been employed in these studies (see
| | | | | | | | | | | |
...rNprNprNprNprNprNprNprNprN... Table 1 and Figure 9).
The first set of RSV inhibition experiments were carried
RNase L p5'A2'p[5'A2'p]25'A2'pBup...BrNupprN[|5 p| 'r| dN| |Np | r3|N |' p|p |r ]N| 5|p'rNNprNprNprN... oanudt wpihtohs 2p-h5aAta-saen atcisteivnistiee cs hbiym tehrea sp srteasbeniliczee odf t ao 3e′x-o3n′-ulcinlekaesde
inactive monomer RNase L RNase L terminal nucleotide (Li et al.,1997) and 5′-thiophosphate
activated dimer (Xiao W, 1994); for instance, chimera Ia. This first
approach was premised on the fact that as RSV genomic
RNA transcription proceeds from the 3′→5′direction,the
p5'A2'p[5'A2R'Npa]se2 L5'A2'pBuRpNBasue pL[5'dN3'p]5'N...rrNNpprrNNpprrNNpprNprNRpNrNA. .s.cission products RAS gViv menR lNevAels oarfe R gNenAer adteesdtr iunc tdioecnr emasaiyn gh caovep ya ambuonrde apnrcoe-.
found effect upon the mRNAs of least abundance;namely,
RSV M2 and L mRNAs. Cirino and collaborators found
The antisense domain of a 2-5A-antisense binds to its targeted that replication of RSV strain A2 in human tracheal
RNA. RNase L monomer then binds to the 2-5A domain, effecting
endothelial cells was inhibited 50% by twice-daily admin-
RNase L dimerization (Dong & Silverman, 1995; 1997) and activa-
tion and subsequent RNA cleavage. The 2-5A-antisense chimera is istration of chimera Ia at 1 µM concentration.
still bonded to dimeric RNase L, ready to accept another substrate
Correspondingly, an 80% reduction on RSV M2 mRNA
molecule. Variations of this hypothetical sequence can be
envisaged. was effected by a single addition of chimera Ia at 3.3 µM
(Cirino et al.,1997).
particular chosen mRNA sequence which is then destroyed In a second approach, stability of 2-5A-antisense to
through localized activation of the latent 2-5A-dependent degradation by exonucleases was improved with antisense
RNase L (Adah et al.,2001;Lesiak et al.,1993;Silverman phosphorothioate internucleotide modifications (Player et
et al., 2000; Torrence, 1999; Torrence et al., 1993; 1997) al.,1998).Highly phosphorothioated oligonucleotides can
(Figure 8).The exact formulation of the linkage modality block activation of RNase L at sub-micromolar concentra-
between the 2′,5′-oligoadenylate moiety and the antisense tions (Player & Torrence,1999) (which could incidentally
domain was dictated by knowledge of 2-5A SAR (Imai et be problematical for clinical applications of fully substitut-
al.,1982a,1982b,1984,1985;Imai & Torrence,1981;Imai ed phosphorothioates). Therefore, a 2-5A-antisense for-
& Torrence,1983;Imai & Torrence,1984;Jamoulle et al., mulation was arrived at empirically by reduction of phos-
1984; Jamoulle et al., 1987; Kitade et al., 1989; 1991; phorothioate content of the oligonucleotide (Player et al.,
Krause et al.,1986;Lesiak et al.,1983;Lesiak & Torrence, 1998).A ‘gapmer’was generated,with only three internu-
1983,1985,1986,1987;Sawai et al.,1983,1985;Torrence cleotide thiophosphorylated linkages at the 5′- and 3′-ter-
et al.,1985,1984,1988,1992). mini of the antisense domain. In this second approach to
Based on the foregoing, we have speculated (Torrence, the inhibition of RSV replication (Player et al.,1998),the
1999) that the 2-5A-antisense strategy would be able to antisense nucleotide sequence for 2-5A-antisense
compensate, in part at least, for the deficient interferon (Chimera IIa) was targeted to the virus genomic(-) RNA.
responses characteristic of RSV infections. In addition, The conserved RNase L-sensitive uridylate-rich sequences
RSV replicates in the cytoplasm where the 2-5A-depend- that occur in gene-start,intergenic and gene-end signals of
ent RNase L is found (as well as in the nucleus) (Player & the genomic strand of RSV,were chosen as targets for 2-
Torrence, 1998). Aerosol administration of 2-5A-anti- 5A-antisense. The 17-mer antisense sequence 5′AAA
sense, akin to ribavirin therapy, could minimize problems AAT GGG GCA AAT AA3′ targeted sequences within
of oligonucleotide delivery and degradation.Further incen- the critical gene-end-intergenic-gene-start signals of RSV
tives for the application of 2-5A-antisense to RSV infec- genomic RNA.This 17-mer antisense cassette is a perfect
tion came from the success of DNA antisense oligonu- hybridization match for three vital RSV genomic RNA sig-
cleotides in the ablation of the mRNA for the adenosine nal sequences. This consensus oligonucleotide antisense
332 ©2002 International Medical Press
RSV antivirals
Table 1.2-5A-antisense chimeras
Target Chimera 2-5A-antisense domain Antisense sequence and structure
RSV mRNA Ia R=POX, X=S, n=2 5′ATG GTT ATT TGG GTT GTT3′p3′dT
2
Ia R=POX, X=S, n=0 5′ATG GTT ATT TGG GTT GTT3′p3′dT
2
Ic R=POX, X=S, n=2 5′TTG TTA TGT TGG GAT TTG3′p3′dT
2
RSV Genomic RNA IIa R=H, n=1 5′AsAsAs AAT GGG GCA A AsTs AsA3′
IIb R=POX, X=S, n=2 5′AsAsAs AAT GGG GCA A AsTs AsA3′
2
IIc R=POX, X=S, n=2 5′GsAsTs AGA AAT AGA AAsGs CsA3′
2
RSV Genomic RNA IIIa R=POX, X=S, n=0 [5′AsAsAs AAU GGG GCA A AsUs AsA3′]
2 m
IIIb R=POX, X=S, n=2 [5′AsAsAs AAU GGG GCA A AsUs AsA3′]
2 m
IIIc R=POX, X=S, n=2 [5′GsAsTs AGA AAT AGA AAsGs CsA3′]
2 m
IIId R=POX, X=O, n=2 [5′AsAsAs AAU GGG GCAs AsAs3′]
2 m
HIV mRNA IVa R=H, n=2 5′TTT TTT TTT TTT TTT TTT3′
IVb R=POX, X=O, n=2 5′TTT TTT TTT TTT TTT TTT3′
2
IVc R=POX, X=O, n=2 5′TTT T3′
2
Va R=POX, X=S, n=2 5′GTA CTA CTC CCT GCT TCT G3′
2
PKR mRNA Vb R=H, n=2 5′GTA CTA CTC CCT GCT TCT G3′
Vc R=POX, X=O, n=2 5′CAG AAG CAG GGA GTA GTA C3′
2
sequence could target other critical regions with significant applications and other targets,support a role for the 2-5A-
stringency. A single 2-5A-antisense chimera formulation dependent RNase L and the postulated mechanism of
could target several nucleotide sequences critical for tran- Figure 8 in the biological activities of 2-5A-antisense and
scription of the RSV genome. particularly in the RSV antiviral effects. (1) Anti-RSV
This approach combining enhanced degradation resist- properties and other biological activities of 2-5A-antisense
ance and strategic genomic sequences produced Chimera depend upon an intact functional 2-5A domain.For exam-
IIa with an EC of 0.3 µM compared with an EC of 30 ple (Torrence et al., 1993), in a Daudi cell-free extract,
50 50
µM for ribavirin in a virus yield reduction assay in human chimera Iva (Table 1) brought about cleavage of RNA con-
HEp-2 cells.Chimera IIa was a potent inhibitor of repre- struct target TAR:A25:vif;however,chimera IVb,bearing
sentative members of both A and B strains of RSV but did the inefficient 5′-non-phosphorylated RNase L activator,
not block replication of two closely related viruses of the A2′p5′A′2p5′A′2p5′A, was unable to produce
Paramyxoviridae family, measles and parainfluenza. TAR:A25:vif cleavage. Similarly, chimera Vb, directed
Chimera IIa was not virucidal nor did it induce interferon against the PKR mRNA and bearing the same 5′-non-
(Player et al.,1998). phosphorylated tetrameric 2′,5′-oligoadenylate, could not
The anti-RSV properties of a modified 2′-O-methylat- effect either cell-free or intact cell PKR RNA cleavage
ed RNA version of chimera IIa has been documented in an under conditions where chimera Va was highly effective at
African green monkey model of RSV infection (Leaman et cutting PKR mRNA (Maitra et al., 1995; Maran et al.,
al.,2002).The 2′-O-methylated RNA backbone analogue, 1994).A similar result was obtained with chimera Ia,one
chimera IIIa,was delivered by intranasal administration to of the first 2-5A-antisense formulations that exhibited
RSV-infected monkeys. Chimera doses of 10 and 50 anti-RSV activity (Cirino et al.,1997).This chimera,tar-
mg/kg provided significant reductions in nasal viral titres. geted to the RSV M2 mRNA,caused a substantial reduc-
Lower doses only delayed the onset of virus detection in tion in levels of M2 mRNA.The chimera Ib,with a dimer-
nasal swabs without any effect on virus shedding.At the 50 ic 2′,5′-oligoadenylate that cannot activate RNase L, was
mg/kg dosed,a 4 log reduction in virus titre resulted.Upon without effect on M2 mRNA levels and possessed only
termination of chimera IIIa administration, virus titres weak anti-RSV activity.Similar outcomes resulted from the
increased in the infected animals; however, these titres chimera series IIA and IIb as well as IIIa and IIIb targeted
never reached levels observed in untreated infected animals to the RSV genomic RNA (Cirino et al.,1997;Leaman et
(Leaman et al.,2002).No oligonucleotide controls,similar al., 2002; Player et al., 1998). (2) Anti-RSV properties as
to that executed in the above cell-free or tissue culture well as other bioactivites of 2-5A-antisense demand an
experiments,were carried out in these primate evaluations antisense domain that can complex with the targeted RNA.
of 2-5A-antisense (Leaman et al.,2002). Thus, the shortened version of chimera IVa, namely IVc,
was devoid of RNA cleavage ability in the Daudi/HIV
2-5A-antisense: an accumulation of supporting RNA cell-free system referred to above (Torrence et al.,
experiments
1993). In this case, the tetrameric deoxythymidine anti-
Several independent lines of evidence, from both RSV sense domain of chimera 1c could not form a complex of
Antiviral Chemistry & Chemotherapy 13:6 333
PF Torrence & LD Powell
Figure 9.2-5A-Antisense generic structure
NH2
2-5A domain N
N
N N NH2
RO O N N
H H
H H N N NH2
OH O
O P O O N N
O– H H Antisense tract
N
H H N Linker
OH O
O P O O
O– H H R’
n H H O O–
OH O O
P O O P CH2 O
O– O R’ H H
O
O
H H
2 OH Z
R=H or
OR
X O Z
O– OH
P X, W=O or S P O
–O ZR=’=OaMdee, thy, cyt, gua or ura W O– H H
n=0–2 m O
m=length of antisense chain
H H
H R’
sufficient Tm to survive under physiological conditions.For Player et al.,1998).Similar results were reported when the
PKR mRNA ablation in either intact HeLa cells or in a 2-5A-antisense targeted was the RNA template of human
cell-free system with pure human RNase L,chimera Vc,a telomerase (Kondo et al.,1998).(3) 2-5A-Antisense brings
‘sense’version of chimera Va,was without capacity to ablate about degradation of targeted RNA, but does not cause
PKR mRNA in HeLa cells (Maran et al.,1994);in addi- detectable degradation of non-targeted RNAs,even when
tion, it was more than an order of magnitude less potent they are of target virus origin.For instance,when the RSV
than chimera Va as an inducer of PKR mRNA destruction M2 mRNA was the target of the 2-5A-antisense,chimera
by pure RNase L .Introduction of mismatched bases into Ia produced an 80% reduction in M2 RNA in RSV infect-
the antisense chain of Va affected biological activity in ed HTE cells,but caused little,if any,reduction of levels of
accordance with the extent of mismatching (Xiao et al., RSV N or P mRNAs, nor did chimera Ia bring about a
1997).Thus,a single mismatch led to full retention of abil- reduction in cellular glyceraldehydes 3-phosphate dehydro-
ity to effect target PKR mRNA destruction in a cell-free genase mRNA (Cirino et al.,1997).In the case of chimeras
purified RNase L assay but not in intact cells. However, IIa and IIIa,potent inhibitors of RSV replication,RT-PCR
introduction of four mismatches or more into the antisense from chimera-treated RSV-infected cells demonstrated
region of chimera Va resulted in a total lack of RNA abla- that targeted RSV genomic RNA was destroyed with no
tion activity in both assays (Xiao et al.,1997).Length of the effect on cellular GAPDH or dsRNA-dependent protein
antisense chain can affect mRNA hybridization affinity. kinase (PKR) mRNAs (Leaman et al.,2002).In a separate
Thus,the antisense chain of Va could be shortened to a 15- cellular but non-viral system, 2-5A-antisense against
mer with no loss of PKR mRNA ablation activity in cells telomerase RNA (not telomerase mRNA) effected the
or with pure RNAse L.However,a dodecameric or short- complete disappearance of the RNA (template) component
er antisense region in chimera Va caused complete loss of of human telomerase,as well as the eventual death of the
activity (Xiao et al.,1997).For the chimeras Ia,IIa and IIIa, neuroblastoma cells employed (Kondo et al.,1998).(4) In
when the antisense nucleotide sequence was scrambled to demonstrable instances,2-5A-antisense targeted RNAs are
give chimera Ic, IIc and IIIc, respectively, there resulted cleaved at sites consistent with the binding sites of the 2-
substantial diminution in antiviral activity compared with 5A-antisense molecule. For example, in Daudi cell-free
parent chimeras (Cirino et al.,1997;Leaman et al.,2002; extracts, chimera IVa brought about multiple cleavages
334 ©2002 International Medical Press