Identification of a series of compounds with potent antiviral activity for the treatment of enterovirus infections

Article abstract of DOI:10.1021/ml400095m

Rhinovirus (genus enterovirus) infections are responsible for many of the severe exacerbations of asthma and chronic obstructive pulmonary disease. Other members of the genus can cause life-threatening acute neurological infections. There is currently no antiviral drug approved for the treatment of such infections. We have identified a series of potent, broad-spectrum antiviral compounds that inhibit the replication of the human rhinovirus, Coxsackie virus, poliovirus, and enterovirus-71. The mechanism of action of the compounds has been established as inhibition of a lipid kinase, PI4KIIIβ. Inhibition of hepatitis C replication in a replicon assay correlated with enterovirus inhibition.

Full text of DOI:10.1021/ml400095m

Letter
pubs.acs.org/acsmedchemlett
Identification of a Series of Compounds with Potent Antiviral Activity
for the Treatment of Enterovirus Infections
Angus M. MacLeod,*,†,∥ Dale R. Mitchell,†,∥ Nicholas J. Palmer,† Herve Van de Poel,† Katja Conrath,§
́
̈
Martin Andrews,§ Pieter Leyssen,‡ and Johan Neyts‡
†Medicinal Chemistry Department, BioFocus, Chesterford Research Park, Cambridge, CB10 1XL, U.K.
‡Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, University of Leuven, Leuven, Belgium
§Galapagos NV, Generaal de Wittelaan, 2800 Mechelen, Belgium
S
* Supporting Information
ABSTRACT: Rhinovirus (genus enterovirus) infections are responsible
for many of the severe exacerbations of asthma and chronic obstructive
pulmonary disease. Other members of the genus can cause life-threatening
acute neurological infections. There is currently no antiviral drug approved
for the treatment of such infections. We have identified a series of potent,
broad-spectrum antiviral compounds that inhibit the replication of the
human rhinovirus, Coxsackie virus, poliovirus, and enterovirus-71. The
mechanism of action of the compounds has been established as inhibition
of a lipid kinase, PI4KIIIβ. Inhibition of hepatitis C replication in a
replicon assay correlated with enterovirus inhibition.
KEYWORDS: antiviral, enterovirus, HCV, inhibitor, PI4KIIIβ
ver the last 15 years, evidence has gathered that viral
Oinfections of the respiratory tract are a major cause of
exacerbations in both chronic obstructive pulmonary disease
(COPD)1 and asthma.2 Of these infections, half to two-thirds
are caused by human rhinovirus (HRV), the most well-known
etiologic agent of the common cold.3 While these infections are
merely inconvenient in otherwise healthy individuals, exacer-
bation of the symptoms of COPD and asthma are serious,
leading to very large numbers of hospitalizations and significant
mortality. In fact, the prevalence of COPD is increasing rapidly,
and it is predicted to become the third leading cause of death
Figure 1. HCV 1b replicon screening hit.
globally by 2030.4 Hence, the identification of compounds that
can interfere with rhinovirus replication may lead to drugs that
have important clinical value in the management of a growing
medical need.
oviruses, a genus of the Picornaviridae family, including HRV,
polio virus (PV), and Coxsackie virus (CV). In most cases, the
compounds were 3−5-fold more active against enteroviruses,
which, in common with HCV, are positive-sense single-
stranded RNA (+ssRNA) viruses. The compounds proved
inactive against other RNA, DNA, and retroviruses and were
not cytotoxic at antiviral concentrations, indicating a specific
and selective antiviral effect.
Enteroviruses present a range of threats to human health,
from the common cold to life-threatening infections, including
encephalitis and viral meningitis.9 Polio is largely considered to
be a disease of the past because of the success of the World
Health Organization’s Global Polio Eradication Initiative in
In the past, several drug candidates have been progressed
into clinical trials for the treatment of HRV infections. These
include Rupintrivir,5 a viral 3C protease inhibitor, and a number
of compounds that prevent virus entry into cells by binding to
the viral capsid: Pirodavir,6 Pleconaril,7 and BTA798.8 Only the
latter of these is currently in clinical development, the others
having been terminated due to lack of clinical efficacy or
unacceptable side effects.
As part of an antiviral program directed toward hepatitis C
(HCV), we identified a series of imidazo-pyrazines with modest
(micromolar) activity in a genotype 1b replicon screen (Figure
1). These compounds were derived from a BioFocus SoftFocus
library (SFK28) initially designed as potential kinase inhibitors.
Additional screening of the imidazo-pyrazines against a panel
of other viruses revealed similar activity against the Enter-
Received: March 8, 2013
Accepted: May 8, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
reducing the incidence of polio over the past two decades.
However, as the initiative moves toward its final goal, it has
become apparent that polio antiviral drugs will be required for
emergency use in a postvaccination era to deal with the threat
of outbreaks caused by circulating vaccine-derived poliovirus
(VDPV), including treatment of infections transmitted by
immunocompromised patients who chronically shed vaccine-
derived poliovirus.10
Table 1. Antiviral Activities against HRV-14
To improve on the antiviral potency of hit compound 1 and
its congeners, we explored the effect of varying the substituents
on the core scaffold, the aryl ring at the 3-position, and on the
8-position benzylamine. The synthesis of analogues (Scheme 1)
a
Scheme 1. Synthesis of Imidazopyrazines
a
i
Reagents and conditions: (a) BrCH2CHO, PrOH reflux; (b) NBS,
DCM; (c) R1NH2.HCl, Pr2EtN, BuOH, 108 °C; (d) ArB(OH)2,
Pd(0), DMF, microwave.
i
i
followed the methodology developed for the original library
production and HRV14 in a Hela Rh cell line was used as the
primary target for tracking potency optimization. The synthetic
route was sufficiently flexible to readily allow variation of the
substituents at both the 3- and 8-position of the core scaffold.
Introduction of substituents at the 2-position of the core
scaffold was achieved by the use of the appropriate haloacetate
(replacing bromoacetaldehyde) under similar reaction con-
ditions. Enhancement in antiviral activity was achieved by
varying the position and nature of substituents on the terminal
aryl rings, and modification at C2 of the imidazo-pyrazine ring.
In particular, polar substituents at the meta position of the
benzylamine and H-bond donor/acceptor groups para
substituted on the C3 aryl ring were potency enhancing
(Table 1). Methylation of the C2 position of the imidazopyr-
azine generally had no effect on activity or gave a small increase
in potency. Methylation at the benzylic position of the
benzylamine was also well tolerated with the (racemic)
analogue 6 retaining good potency.
a
Activity in nM.
a
Table 2. Cross-Species Antienteroviral Activity
compd
HRV14
CVB3
PV1
EV71
1
2
3
4
5
6
3900
150
90
3000
550
490
68
3000
140
150
38
470
80
34
39
15
31
15
19
11
Cytotoxicity of the compounds in the Hela Rh cells was
assessed in parallel with antiviral efficacy. The compounds were
found to have a high selectivity index for antiviral activity
relative to cellular toxicity with CC50 values generally >1000-
fold higher than the EC50 values.
15
66
a
EC50 values in nM
Comparison of the HRV14 activity of 1−6 against other
members of the enteroviruses (Table 2) showed the rank order
of potencies is essentially the same regardless of the species.
This includes enterovirus-71, an emerging virus that has been
the cause of several outbreaks in Asia, resulting in a number of
deaths through neurological inflammation.11 The consistency in
activity across the enteroviruses suggested that the molecular
target of the compounds is either a viral target with very high
conservation of sequence within the family or a single common
target, most likely a host cell protein essential for the replication
of the viruses.
Compound 5 (BF738735) was selected for more extensive
antienterovirus profiling and revealed a remarkable consistency
in activity across a wide range of species (Table 3). Pleconaril
(Figure 2), an anti-HRV agent that acts by preventing viral
entry into cells by binding to the viral capsid, had a range of
B
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ACS Medicinal Chemistry Letters
Letter
a
Table 3. Comparison of the Antienteroviral Activity of
BF738735 with Pleconaril
virus
BF738735
pleconaril
HRV2
HRV9
HRV14
HRV15
HRV29
HRV39
HRV41
HRV42
HRV45
HRV59
HRV63
HRV70
HRV72
HRV85
HRV86
HRV89
CVA9
9
7
6
83
31
21
5
177
166
26
4
44
17
27
25
6
655
100000
8900
471
85
6
24
28
24
24
24
21
15
11
19
13
5
578
1800
883
89
75
33
CVB3
13000
13400
24200
1084
84
Figure 3. Correlation of activity against HRV and HCV. EC50 values
were determined for inhibition of HRV replication in Hela Rh cells
and against an HCV genotype 1b replicon in Huh 5.2. The correlation
coefficient for the line of best fit is 0.81. The straight line drawn is for y
= x.
EV71
PV1
PV2
PV3
a
EC50 values in nM
the antiviral effect of the compounds. Time of drug-addition
studies revealed that, whereas Pleconaril is only effective against
HRV14 when added together with, or prior to, the addition of
virus, BF738735 is active both when added before or after virus
infection, indicating the mechanism is not at an early stage of
the virus replication cycle. No significant activity of BF738735
was observed against isolated viral enzymes, which, taken
together with the very narrow range of potencies seen within
the genus enterovirus, pointed to a mechanism of action
involving a host cell factor. The high correlation in activity
against HCV suggested the mechanism is shared by this virus.
Representative compounds were selected for broad profiling
against a range of enzymes, receptors, and ion channels and
showed no significant activity at 1 μM. The highest activity
observed in screening against a panel of >400 protein kinases
was in the low micromolar range, considerably higher than the
nanomolar antiviral activity that was observed in the virus-cell-
based assays.
Concurrent with the work detailed here, several publications
appeared detailing the results of target identification genomic
screening to identify novel host targets involved in HCV
replication. From these studies, the lipid kinases phosphatidy-
linositol-4 kinase III alpha and beta (PI4KIIIα and PI4KIIIβ)
emerged as enzymes that appear to be important in the
replication of the HCV virus. An siRNA study by Borawski et
al.13 showed that knockdown of either PI4KIIIα or PI4KIIIβ
led to reduced production of viral RNA for HCV genotypes 1a
and 1b. Studies by Reiss et al.14 and Berger et al.15 concluded
that only enzymatic activity of PI4KIIIα is required for HCV
replication, whereas van Kuppeveld et al.16 demonstrated that
PI4KIIIβ mediated enrichment of phosphatidyl inositol-4-
phosphate in endoplasmic reticulum membranes is required
for HCV replication. Arita et al.17 recently disclosed the
discovery of T-00127-HEV1 (Figure 4), which was demon-
strated to be a selective PI4KIIIβ inhibitor (IC50 60nM) with
micromolar antiviral activity against PV1; T-00127-HEV1 was
Figure 2. Pleconaril.
more than 3 orders of magnitude in EC50 values and was
essentially inactive against some serotypes.
Pleconaril reached phase III clinical trials but did not
progress to registration due to drug−drug interactions with oral
contraceptives and insufficient efficacy in treating infections.12
The relatively weak activity of Pleconaril against many of the
HRV serotypes may have contributed to its poor clinical
efficacy in a community setting where patients presenting with
similar symptoms are infected with a spectrum of HRV
serotypes. The broad, uniformly potent activity of BF738735
observed in the test panel offers a clear advantage over
Pleconaril.
Although the potency of the compounds against HCV was
generally slightly lower than that against HRV14 (e.g.,
BF738735 HCV1b EC50 56 nM), a strong correlation in
activity (Figure 3) was observed during the optimization
process, again suggesting a common mechanism of action.
Because HCV belongs to a different virus family (Flaviviridae),
this observation reinforced the likelihood that the target may be
a host cell protein rather than a viral enzyme or structural
protein.
While medicinal chemistry efforts to optimize the potency of
this series of compounds were carried out, parallel efforts were
undertaken to determine the mechanism of action. Resistance
selection experiments showed that, in contrast to Pleconaril,
there is a high genetic barrier for HRV to become resistant to
C
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ACS Medicinal Chemistry Letters
Letter
REFERENCES
■
(1) Papi, A.; Bellettato, C. M.; Braccioni, F.; Romagnoli, M.; Casolari,
P.; Caramori, G.; Fabbri, L. M.; Johnston, S. L. Infections and airway
inflammation in chronic obstructive pulmonary disease severe
exacerbations. Am. J. Respir. Crit. Care Med. 2006, 173, 1114−1121.
(2) Corne, J. M.; Marshall, C.; Smith, S.; Schreiber, J.; Sanderson, G.;
Holgate, S. T.; Johnston, S. L. Frequency, severity, and duration of
rhinovirus infections in asthmatic and non-asthmatic individuals: a
longitudinal cohort study. Lancet 2002, 359, 831−834.
(3) Mallia, P.; Contoli, M.; Caramori, G.; Pandit, A.; Johnston, S. L.;
Papi, A. Exacerbations of asthma and chronic obstructive pulmonary
disease (COPD): focus on virus induced exacerbations. Curr. Pharm.
Des. 2007, 13 (1), 73−97.
(4) WHO. World health statistics 2008. http://www.who.int/
whosis/whostat/EN_WHS08_Full.pdf.
Figure 4.
(5) Hayden, F. G.; Turner, R. B.; Gwaltney, J. M.; Chi-Burris, K.;
Gersten, M.; Hsyu, P.; Patick, A. K.; Smith, G. J.; Zalman, L. S. Phase
II, randomized, double-blind, placebo-controlled studies of ruprintrivir
nasal spray 2-percent suspension for prevention and treatment of
experimentally induced rhinovirus colds in healthy volunteers.
Antimicrob. Agents Chemother. 2003, 47 (12), 3907−3916.
(6) Hayden, F. G.; Hipskind, G. J.; Woerner, D. H.; Eisen, G. F.;
Janssens, M.; Janssen, P. A. J.; Andries, K. Intranasal Pirodavir
(R77,975) treatment of rhinovirus colds. Antimicrob. Agents Chemother.
1995, 39 (2), 290−294.
(7) Hayden, F. G.; Herrington, D. T.; Coats, T. L.; Kim, K.; Cooper,
E. C.; Villano, S. A.; Liu, S.; Hudson, S.; Pevear, D. C.; Collett, M.;
McKinlay, M. Efficacy and safety of oral pleconaril for treatment of
colds due to picornaviruses in adults: results of 2 double-blind,
randomized, placebo-controlled trials. Clin. Infect. Dis. 2003, 36 (12),
1523−1532.
not active against HCV. In contrast, the dual PI4KIIIα
/PI4KIIIβ inhibitor AL-9,18 which has a 5-fold preference for
activity against PI4KIIIα (IC50 0.57 vs 3.08 μM), does inhibit
HCV replication (EC50 0.29 μM for the 1b genotype)
suggesting a greater dependence of this virus on the PI4KIIIα
isoform.
Screening BF738735 against a panel of lipid kinases revealed
potent and highly selective inhibitory activity on PI4KIIIβ, with
an IC50 of 5.7 nM. Inhibition of the related isoform PI4KIIIα
was substantially weaker with an IC50 of 1700 nM, and no
activity was found against a range of other lipid kinases (IC50
10 000 nM).
>
In summary, we have discovered a novel series of antiviral
compounds with broad applicability for the treatment of
enterovirus infections and with a potential for use against HCV
in combination with drugs working by complementary
mechanisms. The clinical application of PI4KIIIβ inhibitors
will depend on the therapeutic index that can be achieved,
particularly in nonlife-threatening infections. A recent pub-
lication19 has suggested that compounds acting by this
mechanism cause reductions in T-cell count as a side effect
that potentially will limit the use of this type of drug. Further
studies will define the scope under which this may be managed.
(8) Feil, S. C.; Hamilton, S.; Krippner, G. Y.; Lin, B.; Luttick, A.;
McConnell, D. B.; Nearn, R.; Parker, M. W.; Ryan, J.; Stanislawski, P.
C.; Tucker, S. P.; Watson, K. G.; Morton, C. J. An orally available 3-
ethoxybenzisoxazole capsid binder with clinical activity against human
rhinovirus. ACS Med. Chem. Lett. 2012, 3 (4), 303−307.
(9) De Palma, A. M.; Vliegen, I.; De Clercq, E.; Neyts, J. Selective
inhibitors of picornavirus replication. Med. Res. Rev. 2008, 28 (6),
823−884.
(10) Barrett, S. Polio eradication: strengthening the weakest links.
Health Aff. 2009, 28 (4), 1079−1090.
(11) McMinn, P. C. Recent advances in the molecular epidemiology
and control of human enterovirus 71 infection. Curr. Opin. Virol. 2012,
2 (2), 199−205.
ASSOCIATED CONTENT
* Supporting Information
■
(12) Fleischer, R.; Laessig, K. Safety and efficacy evaluation of
pleconaril for treatment of the common cold. Clin. Infect. Dis. 2003, 37
(12), 1722.
S
Detailed experimental procedures for the synthesis of
compounds and antiviral assays. This material is available free
of charge via the Internet at http://pubs.acs.org.
(13) Borawski, J.; Troke, P.; Puyang, X.; Gibaja, V.; Zhao, S.;
Mickanin, C.; Leighton-Davies, J.; Wilson, C. J.; Myer, V.;
Cornellataracido, I.; Baryza, J.; Tallarico, J.; Joberty, G.; Bantscheff,
M.; Schirle, M.; Bouwmeester, T.; Mathy, J. E.; Lin, K.; Compton, T.;
Labow, M.; Wiedmann, B.; Gaither, L. A. Class III phosphatidylino-
sitol 4-kinase alpha and beta are novel host factor regulators of
hepatitis C virus replication. J. Virol. 2009, 83 (19), 10058−10074.
(14) Reiss, S.; Rebhan, I.; Backes, P.; Romero-Brey, I.; Erfle, H.;
Matula, P.; Kaderali, L.; Poenisch, M.; Blankenburg, H.; Hiet, M. S.;
Longerich, T.; Diehl, S.; Ramirez, F.; Balla, T.; Rohr, K.; Kaul, A.;
AUTHOR INFORMATION
Corresponding Author
*(A.M.M.) E-mail: angus.macleod@glpg.com.
■
Author Contributions
∥These authors contributed equally to this work. The
manuscript was written through contributions of all authors.
Buhler, S.; Pepperkok, R.; Lengauer, T.; Albrecht, M.; Eils, R.;
̈
Notes
Schirmacher, P.; Lohmann, V.; Bartenschlager, R. Recruitment and
activation of a lipid kinase by hepatitis C virus NS5A is essential for
integrity of the membranous replication compartment. Cell Host
Microb. 2011, 9 (1), 32−45.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We would like to acknowledge Stijn Delmotte and Tom Bellon
for their excellent assistance in generating the antiviral data.
■
(15) Berger, K. L.; Cooper, J. D.; Heaton, N. S.; Yoon, R.; Oakland,
T. E.; Jordan, T. X.; Mateu, G.; Grakoui, A.; Randall, G. Roles for
endocytic trafficking and phosphatidylinositol 4-kinase III alpha in
hepatitis C virus replication. Proc. Natl. Acad. Sci. U.S.A. 2009, 106
(18), 7577−7582.
ABBREVIATIONS
COPD, chronic obstructive pulmonary disease; HRV, human
rhinovirus; PV, poliovirus; CV, Coxsackie virus
■
(16) Hsu, N. Y.; Ilnytska, O.; Belov, G.; Santiana, M.; Chen, Y. H.;
Takvorian, P. M.; Pau, C.; van der Schaar, H.; Kaushik-Basu, N.; Balla,
D
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ACS Medicinal Chemistry Letters
Letter
T.; Cameron, C. E.; Ehrenfeld, E.; van Kuppeveld, F. J.; Altan-Bonnet,
N. Viral reorganization of the secretory pathway generates distinct
organelles for RNA replication. Cell 2010, 141 (5), 799−811.
(17) Arita, M.; Kojima, H.; Nagano, T.; Okabe, T.; Wakita, T.;
Shimizu, H. Phosphatidylinositol 4-kinase III beta is a target of
enviroxime-like compounds for antipoliovirus activity. J. Virol. 2011,
85 (5), 2364−2372.
(18) Bianco, A.; Reghellin, V.; Donnici, L.; Fenu, S.; Alvarez, R.;
Baruffa, C.; Peri, F.; Pagani, M.; Abrignani, S.; Neddermann, P.; De
Francesco, R. PLoS Pathog. 2012, 8 (3), e1002576.
(19) Lamarche, M. J.; Borawski, J.; Bose, A.; Capacci-Daniel, C.;
Colvin, R.; Dennehy, M.; Ding, J.; Dobler, M.; Drumm, J.; Gaither, L.
A.; Gao, J.; Jiang, X.; Lin, K.; McKeever, U.; Puyang, X.; Raman, P.;
Thohan, S.; Tommasi, R.; Wagner, K.; Xiong, X.; Zabawa, T.; Zhu, S.;
Wiedmann, B. Anti-hepatitis C virus activity and toxicity of type III
phosphatidylinositol-4-kinase beta inhibitors. Antimicrob. Agents
Chemother. 2012, 56 (10), 5149−5156.
E
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