DOI: 10.1002/cmdc.
201000334
Drugs for Hepatitis C: Unlocking a New Mechanism of
Action
Thomas W. Bell*[a]
The hepatitis C virus (HCV) infects approximately 200 million
people worldwide (~ 3 % of the population), including 4–5 mil-
lion in the United States. Most are unaware of HCV infections
that have not advanced enough to produce liver disease (cir-
rhosis or liver cancer). Approximately 12 000 Americans are es-
timated to die from this virus each year. Current treatment,
consisting of pegylated interferon-a (PEG-IFN-a) plus the nu-
cleoside antiviral drug ribavirin, is ineffective in more than
50 % of patients infected with genotype 1 HCV. This is the
most aggressive and common strain in the Americas, Europe,
Japan and China. Unfortunately, the conventional therapy is ac-
companied by poorly tolerated side effects. These facts have
led to aggressive competition between pharmaceutical compa-
nies striving to develop new, more effective anti-HCV drugs.[1–3]
Hepatitus C is an enveloped virus with a 9.6 kb RNA genome
that encodes a polyprotein consisting of about 3 000 amino
acids. Cellular and viral proteases process this polyprotein into
several structural proteins, plus several nonstructural proteins.
The latter are designated NS2, NS3, NS4A, NS4B, NS5A and
NS5B. NS2 and NS3 exhibit protease activity, and NS4A acts as
a cofactor for NS3. NS4B induces changes in cellular mem-
branes that are important for viral replication, while NS5B cat-
alyses RNA synthesis. NS5A has no known enzymatic function,
but it is essential for HCV replication and has been proposed
to function as a regulator for key replication events.[3] Over the
last several years, drug companies have mainly focused on in-
hibiting the protease function of NS3 or the RNA polymerase
activity of NS5B. Several such drugs are now in clinical trials,
with two (Vertex’s telaprevir and Merck’s boceprevir) in pha-
se III clinical trials at the time of this writing. According to a
May 2010 press release,[4] telaprevir produced a sustained viral
response in 75 % of patients, reducing the patient’s viral load
to below detectible levels. This is excellent news, but it does
not greatly affect the need for new anti-HCV drugs operating
by other mechanisms. Like many other viruses, HCV mutates
rapidly, so it is expected that combinations of drugs will be
needed to prevent and manage drug resistance, with the
eventual aim of eradicating the virus.
Traditionally, medicines were discovered by empirical test-
ing, first of natural products derived from plants, later of dyes
derived from coal tar or synthesized in the laboratory. Ehrlich’s
“magic bullet” hypothesis, stating that every drug has a specif-
[a] Prof. T. W. Bell
Department of Chemistry, University of Nevada, Reno, NV 89557-0216 (USA)
Fax: (+ 1) 775-784-6804
E-mail: twb@unr.edu
ChemMedChem 2010, 5, 1663 – 1665  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ic “receptor” (e.g., enzyme or receptor protein) that is responsi-
ble for its effect, eventually changed the focus of drug re-
search. Investigators began to look for small organic molecules
that could selectively interact with specific proteins that are
important in disease processes. By the late twentieth century,
genetic screens were used to identify protein function in many
organisms. This involves randomly producing mutations in
cells, screening the resulting mutants to generate a characteris-
tic of interest, and identifying the corresponding mutations in
specific genes. By the end of the twentieth century, an alter-
nate approach called “chemical genetics” was developed and
applied to the discovery of new drugs.[5] Chemical genetics, in
the same “forward” sense as the classical genetic screen just
described, involves screening libraries of small organic mole-
cules in cells, selecting a compound that produces the pheno-
type of interest, and identifying the corresponding protein
target.
The process of discovery and development of antiviral drugs
faces unique challenges.[6] Viruses do not replicate outside the
host cell, so drugs that inhibit viral replication must exploit
subtle biological differences between virus-infected cells and
normal, uninfected cells. Certain proteins and processes native
to the host cell can enable virion entry, replication of viral con-
stituents, or budding of nascent virus particles; hence, they are
included as potential targets for antiviral drugs. In order to
avoid toxicity and various side effects, the drug industry has
traditionally tried to achieve high potency and selectivity
against proteins that are characteristic of the virus, not of the
host cell. New drug leads are typically discovered by screening
compounds for inhibition of specific viral proteins in vitro, or
by screening compounds for inhibition of viral replication in
cells. The latter method, which resembles the chemical genet-
ics approach, is broader than specifically targeted screens be-
cause compounds that inhibit replication by any mechanism
can be discovered, including inhibitors of viral entry or bud-
ding of nascent virions (for screens conducted in cell cultures).
The dream of in silico drug design has only been realized for
one class of drugs: HIV protease inhibitors.[7, 8]
Historically, many drug companies have followed a conserva-
tive approach, developing new drugs that operate by the
same mechanism as previously developed drugs. A drug target
is fully validated by the existence of profitable drugs operating
by this mechanism of action; such drugs are often potent,
nontoxic, and produce minimal side effects. While more inter-
esting scientifically, validation of novel drug targets is risky and
very expensive, though also potentially more lucrative in the
long run. Validation of new targets for antiviral drugs is partic-
1663
 MED
ularly important because drugs operating by different mecha-
nisms are usually synergistic and combination therapy delays
the onset of resistant strains, which must simultaneously devel-
op multisite mutations conferring resistance to more than one
drug. A recent report of the first clinical validation of a novel
NS5A inhibitor can accordingly be considered a breakthrough
in anti-HCV drug research.[9]
Early in 2010, a team from Bristol–Myers Squibb reported
the novel HCV NS5A inhibitors shown in Figure 1.[10] These
compounds were identified by means of a high-throughput
screen that simultaneously measures inhibition of HCV replica-
tion, selectivity relative to a related virus (bovine viral diarrhea,
BVDV), and cellular toxicity in a 96-well format.[11] The HCV rep-
lication assay utilized human liver cells (Huh-7) transfected
with HCV RNA comprising the portion of the viral genome en-
coding proteins including NS3 through NS5B.[12] Intracellular
replication of this HCV “replicon” was detected by measuring
NS3 protease activity with a FRET assay employing a peptide
substrate bearing two fluorescent labels.[13] BVDV replication
was also detected by fluorescence, measuring luciferase pro-
duced in Huh-7 cells transfected with RNA to produce a BVDV
replicon containing the luciferase gene. Cytotoxicity was mea-
sured by a conventional method involving transformation of
Alamar blue dye by cellular enzymes. By means of this screen,
BMS-858 was identified as a novel anti-HCV lead. Initially, the
concentration of BMS-858 producing 50 % inhibition of wild-
type HCV replicon replication (EC50) was measured as 0.6–
1.0 mm, with low cytotoxicity (CC50 > 50 mm) and no detectible
activity toward the BVDV replicon. A series of related com-
pounds were then synthesized and screened, revealing BMS-
824 with an EC50 value of 5 nm against the wild-type HCV repli-
con. Remarkably, removal of just one atom from the structure
of BMS-858 results in a more than 100-fold increase in potency.
Figure 1. Structures of previously identified anti-HCV NS5A inhibitors.[10]
1664 www.chemmedchem.org  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
T. W. Bell
The HCV replicon system[12] provided the first reliable, cell-
based model for HCV replication and enabled the develop-
ment of high-throughput screens for anti-HCV drugs. Detect-
ing intracellular replication of a portion of the HCV genome
has powerful advantages for drug discovery, relative to assays
involving specific HCV proteins. By this means, drugs operating
by a broader range of mechanisms can be found, including
drugs that interact with viral proteins that do not have (or
have unknown) enzymatic activity. While the method has been
described as “mechanistically unbiased”,[9] in actuality, it is re-
stricted to processes related to intracellular replication and
cannot detect potential drugs that might be able to inhibit
binding/entry of the virus to host cells, or formation/budding
of infectious virus particles. In the case at hand, the target of
the BMS-858/824 series was identified as the HCV protein
NS5A by characterizing mutations in drug-resistant replicons.[10]
The replicon cell system then became a useful tool for investi-
gating interactions between NS5A and the drugs, enabling
testing with single-site mutants and various HCV genotypes.
Because of differences in the amino acid composition of the
NS5A protein, BMS-824 was found to be far less potent against
the genotype 1a HCV replicon than against genotype 1b, as
shown in Table 1. Further analogue studies unveiled the sym-
metrical compound BMS-665, which has similar potency
against genotype 1b as BMS-824, but also has significant abili-
ty to inhibit genotype 1a replication (Table 1).
Table 1. Half-maximum effective concentrations (EC50) for inhibition of
replication of HCV replicons by BMS drugs.
EC50 [nm]
genotype 1b genotype 1a
(strain=Con1) (strain=H77)
BMS-824[a] 18 > 10 000
BMS-665[a] 11 393
BMS-790052[b] 0.009 0.050
[a] Reference [10]. [b] Reference [9].
In May 2010, a more potent inhibitor of HCV NS5A was re-
ported, namely BMS-790052.[9] The structure of this drug is
shown in Figure 2 along with those of two analogues (1 and
2), which were used to confirm the mechanism of action. As
can be seen in Table 1, BMS-790052 inhibits replication of both
HCV replicon genotypes with EC50 values in the range of 9–
50 pm. This drug is also active against several other HCV repli-
con genotypes and has a therapeutic index (CC50/EC50) of at
least 100 000 in vitro. Despite having a molecular weight of
more than 700 g mol 1, BMS-790052 is orally bioavailable and
distributes effectively into the liver. It displays additive-to-syn-
ergistic effects with other anti-HCV drugs, and it has advanced
into clinical trials. Biotin-conjugated drugs 1 and 2, which bear
a much closer structural relationship to BMS-665, were report-
ed in the same publication as BMS-790052.[9] Compound 1,
with the same S,S-proline configuration as BMS-665, inhibited
replication of the genotype 1b HCV replicon with an EC50 value
ChemMedChem 2010, 5, 1663 – 1665
Treating Hepatitis C
Figure 2. Structures of an anti-HCV NS5A inhibitor in clinical trials (BMS-790052) and
biotin conjugates 1 and 2.[9]
of 33 nm, while the R,R-enantiomer (2) was inactive. These two
biotinylated compounds were added to separate flasks of HCV
1b replicon cells and their lysates were treated with streptavi-
din-agarose beads to isolate bound proteins, which were ana-
lyzed by gel electophoresis and immunoblotting. Compound 1
was found to bind HCV protein NS5A in this experiment, while
inactive control compound 2 did not.
NS5A is a 447 amino acid protein that apparently dimerizes
by self-association of domain I at the N terminus. The series of
drugs described here clearly bind to domain I of NS5A, accord-
ing to observed mutations in drug-resistant HCV replicon
strains. The symmetrical (pallindromic) structures of many of
these drugs suggest that they bind across the dimer interface
at the site of symmetry of the protein aggregate. This is remi-
niscent of the interaction of inhibitors with HIV protease,
which is smaller (only 99 amino acids) but also dimerizes, with
its active site pierced by the axis of symmetry of the assem-
bly.[7, 8] Chemists designed a number of C2-symmetric molecules
as HIV protease inhibitors, reasoning that affinity could be
maximized by matching the symmetry of a drug with that of
its receptor. Early observations of rapid onset of drug resist-
ance led to the concern that this approach had a serious weak-
ness: single-site mutation in a homodimer would weaken
drug–receptor interaction at two sites, rather than one. Only
non-C2-symmetric compounds eventually became viable anti-
HIV protease inhibitors, but it is not clear whether rapid onset
of resistance was really a practical limitation of the symmetrical
candidates. On the other hand, low aqueous solubility and
poor bioavailability have been associated with symmetry in
some HIV protease inhibitors.[14] It would be very interesting to
know in the case of the BMS NS5A inhibitors what is the influ-
ence of molecular symmetry on solubility, bioavailability, and
onset of drug resistance.
This drug discovery program aimed at inhibitors of HCV
NS5A is certainly an excellent example of increased willingness
of drug companies to invest in higher risk efforts to validate
new targets. Other HCV NS5A inhibitors have been report-
ChemMedChem 2010, 5, 1663 – 1665  2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ed,[15, 16] but BMS-790052 is apparently the most ad-
vanced in the clinic. The method of its develop-
ment—employing high-throughput screening of
drug libraries in cell-based replicon assays—is also
significant as a model for others to follow in the dis-
covery of drugs operating by unconventional mech-
anisms. This is a powerful demonstration of the utili-
ty of chemical genetics in drug discovery, though
some important questions remain unanswered. For
example, the exact manner in which the BMS com-
pounds interact with HCV NS5A has not been de-
scribed. One can hope that this question will be an-
swered soon, perhaps through the X-ray structure of
NS5A domain I bound to one of these drugs. Also,
NS5A is known to be essential in HCV replication,
but its exact role remains undiscovered. Again, a
potent NS5A inhibitor, such as BMS-790052, may be
the key to unlocking this secret.
Keywords: antiviral drugs · chemical genetics · drug
symmetry · hepatitis C · NS5A inhibitors
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Received: August 8, 2010
Published online on September 6, 2010
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