3796
LEMM ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
Waters X-Terra 5-m (C18) column (4.6 by 150 mm), a mobile phase consisting
of a 0.01% trifluoroacetic acid–acetonitrile gradient (8), a flow rate of 1.2 ml/min,
and 254-nm UV detection. For the HPLC biogram (replicon assay) analyses,
fractions were collected in Beckman 96-deep-well plates using a Gilson 215
liquid handler and dried under vacuum using a Savant SpeedVac. The dried
material was resuspended in medium, and a portion of it was tested for inhibition
in the replicon assay. The procedure was repeated using an enriched acetonitrile
extract, which was prepared by freezing the aqueous medium-acetonitrile super-
natant at Ϫ20°C, followed by recovery of the upper, acetonitrile phase, evapo-
ration to dryness, and reconstitution in 200 l methanol. In this manner, bioassay
of all fractions revealed activity that correlated with a distinct yet minor late-
eluting UV-detectable peak. The incubation procedure was scaled up (2 liters of
5 M BMS-824 in assay medium at 37°C, 5% CO2, and 95% humidity for 48 h
with the bottle cap closed). The incubation mixture was extracted with 2 liters of
acetonitrile, followed by centrifugation (Beckman GS-6R, 5,000 rpm, 20 min).
The centrifuged aqueous medium-acetonitrile solutions were frozen at Ϫ20°C,
and the resulting upper acetonitrile extract was recovered. The crude acetonitrile
extract was dissolved in methanol-water at 65:35 (20 ml) and extracted twice with
equal volumes of chloroform that had been presaturated with methanol-water at
65:35. The biogram fractionation on the enriched chloroform extract was con-
ducted with an Agilent HP-1100, a YMC Pro-C18 5-m column (4.6 by 150 mm),
a mobile phase consisting of a 0.01% trifluoroacetic acid–acetonitrile linear
gradient of 60:40 to 10:90 (vol/vol) over 20 min, holding at 0.01% trifluoroacetic
acid–acetonitrile at 10:90 for 5 min, a 1.2-ml/min flow rate, and UV detection at
254 nm. In this manner, bioassay of all fractions revealed two active UV peaks
(17.8 min [peak 4] and 19.0 min [peak 6]). The chloroform extract was subjected
to preparative HPLC with a Beckman System Gold workstation, a YMC Pro-C18
5-m column (20 by 150 mm), a mobile phase consisting of a 0.01% trifluoro-
acetic acid–acetonitrile linear gradient of 60:40 to 10:90 (vol/vol) over 20 min,
holding at 0.01% trifluoroacetic acid–acetonitrile at 10:90 for 5 min, a 20-ml/min
flow rate, and UV detection at 254 nm. Replicon active peaks 4 and 6 were
manually collected and submitted for biological evaluation.
(10). One such compound, BMS-824, is a potent and specific
inhibitor of HCV RNA replication with a 50% effective con-
centration (EC50) of ϳ10 nM. Studies to further characterize
this compound revealed that BMS-824 was not stable in me-
dium yet anti-HCV activity was maintained. In this report, we
describe the use of an HCV bioactivity chromatogram assay
(referred to here as a “biogram” [5]) to isolate and identify two
trace constituents from incubations of BMS-824 in assay me-
dium that demonstrate exceptionally potent HCV inhibition in
replicons.
MATERIALS AND METHODS
Cell culture and compound. Both bovine viral diarrhea virus (BVDV) and
HCV replicon cell lines were isolated as previously described (10, 15) and were
maintained in Dulbecco’s modified Eagle medium (DMEM) with 100 U/ml
penicillin-streptomycin, 10% fetal bovine serum (FBS), and 0.3 to 0.5 mg/ml
Geneticin (G418). Huh-7 cells cured of a Con1 replicon were generated as
previously described (10) and were propagated in DMEM with penicillin-strep-
tomycin and 10% FBS. The compounds used in this study were synthesized at
Bristol-Myers Squibb.
Cell culture cytotoxicity and HCV inhibition assays. To assess HCV-inhibitory
activity, HCV replicon cells were plated at a density of 104 per well in 96-well
plates in DMEM medium containing 10% FBS (assay medium). Following in-
cubation overnight, compounds or high-performance liquid chromatography
(HPLC) fractions (detailed below) were added to cell plates and incubated at
37°C for 3 days prior to assaying for cytotoxicity and HCV inhibition. Cell
viability was measured using an alamarBlue assay, and the CC50, the concentra-
tion of compound which caused a 50% reduction in cell viability, was calculated
using the median-effect equation.
A second scale-up incubation was conducted at a higher concentration of
BMS-824 (100 M) in 1 liter of assay medium. A solution of BMS-824 (60 mg/60
ml DMSO) was added to the medium, and the medium was divided into four
500-ml Erlenmeyer shake flasks, sealed with a semipermeable membrane (Bio-
wrap), and placed in an incubator-orbital shaker at 37°C and 100 rpm for 67 h.
Workup and isolation as described above yielded sufficient amounts of replicon
active components for structure elucidation (peak 4, 1.1 mg; peak 6, 1.1 mg).
High-resolution MS, NMR and HPLC. Electrospray ionization–high-resolu-
tion mass spectrometry (ESI-HR-MS) data were obtained with a Micromass
QTOF-2 mass spectrometer in positive-ion mode with a full-width half-maximal
resolution of 9,500, a LEU-enkephalin m/z 556 (M ϩ H)ϩ lock mass, and tandem
MS (MS/MS) with a collision energy of 22 eV. NMR spectra were obtained on
a Bruker DRX 500-MHz spectrometer equipped with a 5-mm TXI CryoProbe.
Proton and carbon chemical shifts are reported in ppm relative to tetramethyl-
silane (compound 1, acetonitrile-d3; compound 4, acetone-d6 solvent) (see Fig.
4). HPLC coinjection analyses were conducted using a (i) Waters Sunfire C18
column (5 m, 4.6 by 150 mm), a mobile phase consisting of 10 mM ammonium
acetate-acetonitrile at 95:5 (solvent A)–10 mM ammonium acetate-acetonitrile
at 5:95 (solvent B) at an 85:15 to a 0:100 ratio of solvent A to solvent B over 25
min, holding at 100% B solvent for 5 min, a flow rate of 1.2 ml/min, and UV
detection at 254 nm or (ii) a Waters Acquity UPLC BEH C18 or a Shield RP18
(1.7 m, 2.1 by 50 mm) column, a mobile phase consisting of 10 mM ammonium
acetate-acetonitrile or methanol at 95:5 (solvent A)–10 mM ammonium acetate-
acetonitrile or methanol at 5:95 (solvent B) at a 100:0 to a 0:100 ratio of solvent
A to solvent B over 4 min, holding at 100% B solvent for 1 min, a flow rate of 0.5
ml/min (methanol method) or 0.83 ml/min (acetonitrile method), and UV de-
tection at 220 nm.
HCV inhibition was measured using a fluorescence resonance energy transfer
(FRET) assay which was performed as previously described (15). Briefly, after
staining with alamarBlue, replicon cell plates were washed with phosphate-
buffered saline and then used for the FRET assay by the addition of 30 l of the
FRET peptide assay reagent per well. The assay reagent consisted of 1ϫ lucif-
erase cell culture lysis buffer, 150 mM NaCl, and 20 M FRET peptide. The
plate containing the assay reagent was then read in kinetic mode in a Cytofluor
4000 instrument which had been set to 340-nm excitation and 490-nm emission
wavelengths in automatic mode for 20 cycles. EC50s were calculated as the
compound concentrations which caused a 50% reduction in HCV FRET activity.
Isolation of resistant replicons. Selection of resistant replicon cells was per-
formed by growing genotype 1b replicon cells in medium containing a concen-
tration of 5 M BMS-346. Medium containing compound was added to mono-
layers of HCV 1b-377-neo replicon cells at ϳ25% confluence in the presence of
0.5 mg/ml G418. Replicon cells maintained in the presence of DMSO were used
as a control. After 5 to 6 weeks, control DMSO-selected replicon cells and
compound-selected cells were tested for compound sensitivity using the HCV
replicon FRET assay.
cDNA cloning. Total RNA was isolated from both DMSO- and compound-
selected cell lines using Trizol (Gibco-BRL) according to the manufacturer’s
protocol. To generate HCV cDNAs, the NS5A gene was amplified using the
SuperScript One-Step reverse transcription-PCR kit (Gibco-BRL) and primers
targeting the NS4B and NS5B genes. Reaction products were cloned directly into
pCR2.1-TOPO using a TOPO TA cloning kit (Invitrogen), and the DNA se-
quence of the NS5A coding region was determined for multiple clones.
Transient-replication assays. RNA transcripts of HCV replicons containing a
luciferase reporter gene were synthesized in vitro using ScaI-digested DNAs and
the T7 MegaScript transcription kit (Ambion) according to the manufacturer’s
directions. For transient-replication assays, subconfluent cured Huh-7 cells in a
35-mm dish were transfected with 2.5 g of RNA transcript using DMRIE-C
(Invitrogen) according to the manufacturer’s directions. Four hours later, trans-
fectant was removed and replaced with DMEM–10% FBS with or without
compound and then incubated at 37°C. At various time points, cells were har-
vested and luciferase activity was determined using the Renilla luciferase assay kit
(Promega).
RESULTS
Characterization of HCV NS5A inhibitors. As part of the
characterization of the NS5A inhibitor BMS-824 (Fig. 1) (10),
it became apparent that under certain conditions this com-
pound exhibited some chemical reactivity, leading to experi-
ments designed to ascertain its chemical behavior in medium.
Toward this end, BMS-824 was incubated in cell-free assay
medium for 72 h under the conditions used for the replicon
assay. After incubation, medium containing the compound was
Extraction and HPLC biogram. To begin isolation of active components de-
rived from BMS-824, BMS-824 (5 M final concentration) was incubated in
assay medium at 37°C for 48 h in an initial 6-ml pilot experiment. After 48 h, 6
ml acetonitrile was added to the incubation mixture and the resulting suspension
was centrifuged. A 100-l aliquot of the supernatant was subjected to HPLC
fractionation. The HPLC (C18) conditions used included an Agilent HP-1100, a