Discovery of Potent Hepatitis C Virus NS5A Inhibitors

transcript

Discovery of Potent Hepatitis C Virus NS5A Inhibitorswith Dimeric Structures�

Julie A. Lemm,1 John E. Leet,2 Donald R. O’Boyle II,1 Jeffrey L. Romine,3 Xiaohua Stella Huang,4Daniel R. Schroeder,4 Jeffrey Alberts,5† Joseph L. Cantone,4 Jin-Hua Sun,1 Peter T. Nower,1

Scott W. Martin,3 Michael H. Serrano-Wu,3‡ Nicholas A. Meanwell,3Lawrence B. Snyder,3 and Min Gao1*

Department of Virology,1 Synthesis & Analysis Technology Team,2 Medicinal Chemistry,3 Discovery Analytical Sciences,4 andDiscovery Biotransformation,5 Bristol-Myers Squibb Research, 5 Research Parkway, Wallingford, Connecticut 06492

Received 3 February 2011/Returned for modification 10 March 2011/Accepted 6 May 2011

The exceptional in vitro potency of the hepatitis C virus (HCV) NS5A inhibitor BMS-790052 has translatedinto an in vivo effect in proof-of-concept clinical trials. Although the 50% effective concentration (EC50) of theinitial lead, the thiazolidinone BMS-824, was �10 nM in the replicon assay, it underwent transformation toother inhibitory species after incubation in cell culture medium. The biological profile of BMS-824, includingthe EC50, the drug concentration required to reduce cell growth by 50% (CC50), and the resistance profile,however, remained unchanged, triggering an investigation to identify the biologically active species. High-performance liquid chromatography (HPLC) biogram fractionation of a sample of BMS-824 incubated inmedium revealed that the most active fractions could readily be separated from the parental compound andretained the biological profile of BMS-824. From mass spectral and nuclear magnetic resonance data, theactive species was determined to be a dimer of BMS-824 derived from an intermolecular radical-mediatedreaction of the parent compound. Based upon an analysis of the structural elements of the dimer deemednecessary for anti-HCV activity, the stilbene derivative BMS-346 was synthesized. This compound exhibitedexcellent anti-HCV activity and showed a resistance profile similar to that of BMS-824, with changes incompound sensitivity mapped to the N terminus of NS5A. The N terminus of NS5A has been crystallized as adimer, complementing the symmetry of BMS-346 and allowing a potential mode of inhibition of NS5A to bediscussed. Identification of the stable, active pharmacophore associated with these NS5A inhibitors providedthe foundation for the design of more potent inhibitors with broad genotype inhibition. This culminated in theidentification of BMS-790052, a compound that preserves the symmetry discovered with BMS-346.

Hepatitis C virus (HCV) is the major causative agent ofnon-A, non-B hepatitis worldwide, which affects more than 3%of the world’s population. Of those infected with HCV, �70%proceed to a chronic state which can lead to severe liver dis-eases, including fibrosis, cirrhosis, or hepatocellular carcinoma(1, 7). There is currently no vaccine against HCV and nogenerally effective therapy for all HCV genotypes. The currentoptimal therapy is pegylated alpha interferon in combinationwith ribavirin, a regimen associated with significant side effectsand limited efficacy in the most prevalent patient population,consisting of genotype 1 (4). Therefore, there is an urgent needfor the development of more effective, HCV-specific antiviraltherapies with fewer side effects.

In the search for more efficacious, safer HCV therapies, themost actively pursued antiviral targets have been the NS3 pro-tease and NS5B RNA-dependent RNA polymerase, both es-sential enzymes for the replication of HCV (2, 11, 12). Excitingprogress has been demonstrated in clinical trials with multipleHCV NS3 serine protease inhibitors, as well as with both

nucleoside and nonnucleoside polymerase inhibitors. How-ever, due to the error-prone nature of the HCV polymerase,HCV is a highly heterogeneous virus and resistance variantsexist as part of the viral quasispecies. It is widely recognizedthat combinations of drugs with different resistance profiles arelikely to be required to effectively suppress the emergence ofresistant virus and achieve a sustained viral response. Thus,agents that inhibit HCV replication via novel targets are ofconsiderable interest.

With the development of HCV replicon and virus systems, itis now possible to identify inhibitors targeting nonenzymaticproteins via cell-based screens. The use of a cell-based repli-cation assay includes essential functions that previously couldnot be evaluated with in vitro enzyme assays. Inhibitors thattarget HCV NS5A, a protein with no known enzymatic func-tion, provide an interesting example of this approach. NS5A isa multifunctional protein required for several stages of theviral life cycle. It is a membrane-associated phosphoprotein (9,18) thought to be involved in interferon resistance that also hasbeen shown to interact with a number of host proteins, al-though its precise role in HCV replication is unknown (14).NS5A has recently been validated as a clinically relevant target(6), and inhibitors targeting this protein are actively beingpursued in clinical trials.

We recently reported the identification of compounds thatinhibit HCV replication in cell-based assays and target NS5A

* Corresponding author. Mailing address: 5 Research Parkway,Wallingford, CT 06492. Phone: (203) 677-6692. Fax: (203) 677-6088.E-mail: min.gao@bms.com.

† Present address: Eli Lilly, Indianapolis, IN.‡ Present address: Novartis Institute for BioMedical Research,

Cambridge, MA.� Published ahead of print on 16 May 2011.

(10). One such compound, BMS-824, is a potent and specificinhibitor of HCV RNA replication with a 50% effective con-centration (EC50) of �10 nM. Studies to further characterizethis compound revealed that BMS-824 was not stable in me-dium yet anti-HCV activity was maintained. In this report, wedescribe the use of an HCV bioactivity chromatogram assay(referred to here as a “biogram” [5]) to isolate and identify twotrace constituents from incubations of BMS-824 in assay me-dium that demonstrate exceptionally potent HCV inhibition inreplicons.

MATERIALS AND METHODS

Cell culture and compound. Both bovine viral diarrhea virus (BVDV) andHCV replicon cell lines were isolated as previously described (10, 15) and weremaintained in Dulbecco’s modified Eagle medium (DMEM) with 100 U/mlpenicillin-streptomycin, 10% fetal bovine serum (FBS), and 0.3 to 0.5 mg/mlGeneticin (G418). Huh-7 cells cured of a Con1 replicon were generated aspreviously described (10) and were propagated in DMEM with penicillin-strep-tomycin and 10% FBS. The compounds used in this study were synthesized atBristol-Myers Squibb.

Cell culture cytotoxicity and HCV inhibition assays. To assess HCV-inhibitoryactivity, HCV replicon cells were plated at a density of 104 per well in 96-wellplates 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 at37°C for 3 days prior to assaying for cytotoxicity and HCV inhibition. Cellviability was measured using an alamarBlue assay, and the CC50, the concentra-tion of compound which caused a 50% reduction in cell viability, was calculatedusing the median-effect equation.

HCV inhibition was measured using a fluorescence resonance energy transfer(FRET) assay which was performed as previously described (15). Briefly, afterstaining 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 theFRET peptide assay reagent per well. The assay reagent consisted of 1� lucif-erase cell culture lysis buffer, 150 mM NaCl, and 20 �M FRET peptide. Theplate containing the assay reagent was then read in kinetic mode in a Cytofluor4000 instrument which had been set to 340-nm excitation and 490-nm emissionwavelengths in automatic mode for 20 cycles. EC50s were calculated as thecompound 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 �25% confluence in the presence of0.5 mg/ml G418. Replicon cells maintained in the presence of DMSO were usedas a control. After 5 to 6 weeks, control DMSO-selected replicon cells andcompound-selected cells were tested for compound sensitivity using the HCVreplicon FRET assay.

cDNA cloning. Total RNA was isolated from both DMSO- and compound-selected cell lines using Trizol (Gibco-BRL) according to the manufacturer’sprotocol. To generate HCV cDNAs, the NS5A gene was amplified using theSuperScript One-Step reverse transcription-PCR kit (Gibco-BRL) and primerstargeting the NS4B and NS5B genes. Reaction products were cloned directly intopCR2.1-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 aluciferase reporter gene were synthesized in vitro using ScaI-digested DNAs andthe T7 MegaScript transcription kit (Ambion) according to the manufacturer’sdirections. For transient-replication assays, subconfluent cured Huh-7 cells in a35-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 withoutcompound 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).

Extraction and HPLC biogram. To begin isolation of active components de-rived from BMS-824, BMS-824 (5 �M final concentration) was incubated inassay medium at 37°C for 48 h in an initial 6-ml pilot experiment. After 48 h, 6ml acetonitrile was added to the incubation mixture and the resulting suspensionwas centrifuged. A 100-�l aliquot of the supernatant was subjected to HPLCfractionation. The HPLC (C18) conditions used included an Agilent HP-1100, a

Waters X-Terra 5-�m (C18) column (4.6 by 150 mm), a mobile phase consistingof 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 215liquid handler and dried under vacuum using a Savant SpeedVac. The driedmaterial was resuspended in medium, and a portion of it was tested for inhibitionin the replicon assay. The procedure was repeated using an enriched acetonitrileextract, which was prepared by freezing the aqueous medium-acetonitrile super-natant at �20°C, followed by recovery of the upper, acetonitrile phase, evapo-ration to dryness, and reconstitution in 200 �l methanol. In this manner, bioassayof all fractions revealed activity that correlated with a distinct yet minor late-eluting UV-detectable peak. The incubation procedure was scaled up (2 liters of5 �M BMS-824 in assay medium at 37°C, 5% CO2, and 95% humidity for 48 hwith the bottle cap closed). The incubation mixture was extracted with 2 liters ofacetonitrile, followed by centrifugation (Beckman GS-6R, 5,000 rpm, 20 min).The centrifuged aqueous medium-acetonitrile solutions were frozen at �20°C,and the resulting upper acetonitrile extract was recovered. The crude acetonitrileextract was dissolved in methanol-water at 65:35 (20 ml) and extracted twice withequal volumes of chloroform that had been presaturated with methanol-water at65:35. 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 lineargradient of 60:40 to 10:90 (vol/vol) over 20 min, holding at 0.01% trifluoroaceticacid–acetonitrile at 10:90 for 5 min, a 1.2-ml/min flow rate, and UV detection at254 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 subjectedto 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/minflow rate, and UV detection at 254 nm. Replicon active peaks 4 and 6 weremanually collected and submitted for biological evaluation.

A second scale-up incubation was conducted at a higher concentration ofBMS-824 (100 �M) in 1 liter of assay medium. A solution of BMS-824 (60 mg/60ml DMSO) was added to the medium, and the medium was divided into four500-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 repliconactive 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 MicromassQTOF-2 mass spectrometer in positive-ion mode with a full-width half-maximalresolution of 9,500, a LEU-enkephalin m/z 556 (M � H)� lock mass, and tandemMS (MS/MS) with a collision energy of 22 eV. NMR spectra were obtained ona 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 ammoniumacetate-acetonitrile at 95:5 (solvent A)–10 mM ammonium acetate-acetonitrileat 5:95 (solvent B) at an 85:15 to a 0:100 ratio of solvent A to solvent B over 25min, holding at 100% B solvent for 5 min, a flow rate of 1.2 ml/min, and UVdetection 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 ammoniumacetate-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 solventA to solvent B over 4 min, holding at 100% B solvent for 1 min, a flow rate of 0.5ml/min (methanol method) or 0.83 ml/min (acetonitrile method), and UV de-tection at 220 nm.

RESULTS

Characterization of HCV NS5A inhibitors. As part of thecharacterization 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 assaymedium for 72 h under the conditions used for the repliconassay. After incubation, medium containing the compound was

3796 LEMM ET AL. ANTIMICROB. AGENTS CHEMOTHER.

extracted with acetonitrile and examined by HPLC fraction-ation. A control sample of BMS-824 which was not incubatedin medium gave a single UV-detectable peak in the HPLCtrace. In contrast, the parent compound could not be detectedby HPLC in the sample of BMS-824 incubated in medium,providing evidence of parent compound reactivity under assayconditions (data not shown). The major products formed fromBMS-824 (compound 1) were identified as an oxidation prod-uct (compound 2) and a thiourea derivative (compound 3)derived from compound 2 by hydrolysis (Fig. 1). When testedin the HCV replicon assay, these products exhibited poor ac-tivity (EC50, �10 �M; data not shown), raising the possibilitythat undetected amounts of another highly potent compo-nent(s) were present. To assess this possibility, BMS-824 wasincubated with medium for various times (0 to 120 h) and themedium was subjected to the 3-day HCV replicon assay todetermine if the HCV-inhibitory activity remained. The EC50

remained constant at all time points, indicating that after 5days of incubation, potency was maintained even though theparent compound was no longer detectable (data not shown).This suggested that an active, stable component(s) was gener-ated from the parent compound during incubation in mediumand that the resulting new species was a potent HCV inhibitorthat could potentially be isolated and characterized.

Identification of fractions with HCV activity. To identify thesource of the potent inhibition, we first used a replicon HPLCbiogram assay to determine which fractions contained anti-HCV activity. The fraction collection utilized a time-basedprotocol, resulting in a direct relationship between an anti-HCV active well’s position on the plate and a correspondingarea on the HPLC chromatogram. Following incubation ofBMS-824 and extraction, the sample was subjected to HPLCfractionation (Fig. 2) and 80 fractions were collected and eval-uated in the HCV replicon assay. To ensure that any activitywe observed was specific for HCV, the fractions were tested

against the Y93H BMS-824-resistant replicon cell line andevaluated for cytotoxicity. BMS-824 and an HCV NS3 proteaseinhibitor were used as controls for the assay and yielded theexpected inhibitory profiles. Incubation of cells with 50 nMBMS-824 gave 72 and 0% inhibition on the wild-type andY93H replicon cells, respectively, while the protease inhibitorgave similar levels of inhibition on both cell lines. When all 80fractions were tested, HCV inhibition (�35% inhibition) wasreproducibly detected in fractions 56, 61, and 62 (Fig. 2). Thesefractions showed no significant activity toward the Y93H rep-licon, suggesting cross-resistance to BMS-824 with no overtcytotoxicity. Under the fractionation conditions used, a distinctUV peak is not detectable in the region of interest (fractions 61and 62); however it is clear from the biogram results that theactive component(s) eluted later than the parent compound,BMS-824 (compound 1) (fraction 56).

Isolation of active components. Following the incubation ofBMS-824 in medium, material was extracted using specificmodifications to facilitate separation (see Materials and Meth-ods) and the enriched sample was subjected to HPLC fraction-ation. A critical enrichment step prior to HPLC involved ex-traction of the complex medium matrix with acetonitrile,followed by centrifugation and freezing at �20°C to facilitatephase separation. The unfrozen acetonitrile extract was recov-ered and further refined using a chloroform-methanol-waterpartitioning step (i.e., modified Folch method [3]). TheHPLC-UV peaks in the enriched material consisted of startingmaterial (peak 3), plus four major peaks and one minor peakbetween peak 4 and peak 6 (Fig. 3). Each fraction was testedfor anti-HCV activity on both the wild-type and Y93H resistantcells, which show reduced susceptibility to BMS-824 (Table 1).Peak fractions were compared to those of control BMS-824,which yielded an EC50 �10-fold higher in this experiment thanthat normally observed (10). This was interpreted as a reducedability to convert to an active species in the 3-day replicon

FIG. 1. Structures of BMS-824 (compound 1) and medium-induced products (compounds 2, 3, and 4 to 6).

VOL. 55, 2011 NS5A INHIBITORS DERIVED FROM AN INTERMOLECULAR REACTION 3797

assay. As a control, a medium-only extraction-separation wasperformed as described above and tested in parallel. The con-trol extraction did not yield peaks of similar intensity, nor didthe gradient contain any significant inhibitory activity (data notshown). As shown in Table 1, peaks 3, 4, and 6 from the samplecontaining the incubated compound exhibited significant anti-HCV activity, with no detectable cytotoxicity. The EC50s ofthese peaks ranged from 43 nM to 600 pM. Importantly, thesethree peaks showed 30- to �3,800-fold resistance on the Y93Hcell line, suggesting that they contained components related tothe parent compound, BMS-824. Additional fractions repre-

senting the entire gradient were tested and did not contain anysignificant antiviral activity (data not shown). The parent com-pound, BMS-824 (compound 1), was identified in UV peak 3,the same peak in which control nonincubated BMS-824 alsoeluted. An oxidized form (compound 2) of BMS-824 was iden-tified in UV peak 2, and a thiourea component (compound 3)was identified in UV peak 1. These peaks were of less interestdue to their reduced HCV-inhibitory activity compared to thatof the other peaks and were not pursued further. Peaks 4 and6 did not correspond to parent compound BMS-824 yet con-tained very potent anti-HCV activity; in fact, peak 6 had morepotent HCV activity than the parent compound, which is in-dicative of conversion to a more active species.

Structure elucidation. A second scale-up was performed inorder to generate a sufficient quantity of the BMS-824-derivedactive species to allow structure determination. From ESI-HR-

FIG. 2. Biogram analysis to identify regions containing active components. BMS-824 was incubated in medium for 48 h and then subjected toextraction and HPLC fractionation. Eighty fractions were collected and tested for anti-HCV activity; the regions containing the peak inhibitoryactivity are indicated by arrows. Solid line, HPLC trace; dotted line, percent inhibition of HCV replicon activity; mAU, milliabsorbance units; Fr.,fraction.

FIG. 3. Fractionation of BMS-824 incubated in medium. BMS-824was incubated in medium for 48 h and then subjected to extractionwith acetonitrile and chloroform, followed by preparative HPLC. Thesix key peaks identified are labeled P1 to P6. Assignment of peakcomponents indicated: P1, thiourea; P2, oxidized BMS-824; P3, BMS-824. Other peaks did not contain enough material for structure deter-mination. Abs., absorbance.

TABLE 1. Anti-HCV activities of HPLC fractions

SampleEC50 (�M)a

Foldresistanceb

WT CC50(�M)c

WT Y93H

P1 �5 �5 1 �5P2 4.8 �5 1 �5P3 0.005 5 1,000 �5P4 0.043 1.3 30 �5P5 2.8 �5 �1.8 �5P6 0.0006 2.3 3,833 �5BMS-824 0.061 6.1 100 �50PI 0.200 0.209 1 �5

a Major peak fractions identified by HPLC fractionation were titrated onwild-type (WT) and resistant (Y93H) replicon cells and tested for antiviralactivity and toxicity. Nonfractionated BMS-824 and an HCV protease inhibitor(PI) were included as controls.

b Fold resistance � Y93H EC50/WT EC50.c Due to the amount of P1 to P6, the highest concentration used for CC50

determination was 5 �M.

MS, the molecular formula of compounds 4 and 6 was deter-mined to be C62H52N8O8F2S2 [observed mass of compounds 4and 6, m/z 1,139.3379 (M � H)�; calculated mass, m/z1,139.3396]. The NMR spectrum (500 MHz, acetone-d6) ofcompound 4 revealed that all of the substituents; phenacetyl,alanine, fluorobenzene, furan, and the thiazolidinone ring, thatwere in the starting material, BMS-824 (compound 1), werepresent. Key differences, however, included the loss of the

benzylic methine proton (� 5.33, BMS-824 [compound 1]) anda 13C resonance shift from � 51.4 (compound 1) to � 70.2(compound 4) (Fig. 4). From these data, it was surmised thatcompound 4 is a symmetrical (homo)dimer of BMS-824, withdimerization occurring at the benzylic carbon, and this wasconfirmed by mass spectral data (Fig. 5). In the course ofoptimizing NMR experimental conditions (i.e., solvent, tem-perature), we observed that upon prolonged NMR data acqui-

FIG. 4. 1H and 13C NMR chemical shifts for BMS-824 (compound 1) and dimer (compound 4). NMR solvents: a, acetonitrile-d3; b, ace-tone-d6.

FIG. 5. MS analysis of dimers 4 and 6 (positive-ion electrospray LC-MS and LC-MS/MS).

sition with compound 6 in acetonitrile-d3 at 55°C, this com-pound, similarly shown to be a homodimer of BMS-824(compound 1), converted to compound 4. This was concludedbased on coinjections on HPLC with orthogonal methods, byproton NMR, and mass spectral data. We therefore proposethat compounds 4 and 6 are atropisomers rather than diaste-reomers. The gross structure of compound 4 is depicted in Fig.6A, and further studies regarding its formation and the stere-ochemistry at the dimer linkage are currently under way.

Biological profile of a dimeric inhibitor. Due to the dimericstructure of compound 4, it appeared that the symmetry of themolecule was important for achieving potent HCV-inhibitoryactivity. To test this hypothesis, a simplified dimeric stilbenederivative, BMS-346 (Fig. 6B), was synthesized based on theprecise structure-activity relationship (SAR) associated withthe amino acid moiety (17). When tested in the HCV repliconassay, BMS-346 had an EC50 of 86 pM (Table 2), an approx-imately 70-fold enhancement of potency compared to that ofBMS-824. In contrast, the EC50 of BMS-346 on the BVDV

replicon was �10 �M, demonstrating that the dimeric in-hibitor has excellent potency against, as well as selectivityfor, HCV. Resistance of the Y93H replicon to BMS-346indicated the mechanistic relatedness of this compound toBMS-824 and suggested that NS5A is also the target of thiscompound (Table 2).

To further explore the antiviral activity of BMS-346, we usedthe compound to select for resistance on genotype 1b HCVreplicon cells. Mapping of the BMS-346-resistant cell line re-vealed an L31V NS5A substitution in 6 out of 6 clones, withtwo of the clones having L31V linked with a Q54L substitutionin NS5A. These are the same substitutions previously identi-fied from selection with BMS-824 (10), suggesting that BMS-346 binds in a manner similar to that of the active componentof BMS-824. When tested in a transient-replication assay, thesingle Q54L and L31V mutants conferred 30- to 80-fold resis-tance to BMS-346, respectively (Fig. 7). However, when theL31V and Q54L mutations were present together, resistance toBMS-346 increased significantly to �400-fold, suggesting thatboth changes are required to maximally affect compound po-tency. As expected, none of the NS5A mutants conferred re-sistance to a control HCV protease inhibitor (data not shown).

The chemical stability of BMS-346 was also examined byincubating the compound in cell medium for 72 h at 37°C,followed by HPLC fractionation. Under these conditions, lessthan 3% degradation of the parent structure was observed,indicating that BMS-346 is stable in medium and demonstrat-ing that this novel dimeric species has potent and selectiveanti-HCV activity.

In summary, the active components in replicon mediumwere identified whose symmetry afforded the necessary insightto prepare inhibitor BMS-346, a compound chemically inertunder assay conditions, and the NS5A-inhibitory activity ofBMS-346 was clearly established. Subsequently, this compoundprovided the basis for the design of BMS-790052, the firstNS5A inhibitor to show clinical efficacy.

DISCUSSION

A multiplexed HCV replicon screen was used to identifyspecific, nontoxic, low-molecular-weight inhibitors such as

FIG. 6. Dimeric structures. (A) Proposed structure of the activecomponent in peaks 4 and 6 based on NMR and MS. (B) Structure ofdimeric compound BMS-346.

TABLE 2. Biological activity of BMS-346a

Replicon EC50 (�M) CC50 (�M)

WT HCV 0.000086 �10Y93H 4 �10BVDV �10 �10

a EC50s were derived from this single experiment. On multiple test occasions,the average EC50s and standard deviations of BMS-346 were as follows: wild type(WT), 0.000052 0.0000076 �M; Y93H, 3.5 3.1 �M.

FIG. 7. Resistance analysis of BMS-346 selected substitutions.Huh-7 cells were transfected with wild-type (WT) or mutant repliconRNAs and incubated in the presence or absence of various concentra-tions of BMS-346. Luciferase activities were determined in lysates ofcells harvested at 72 h after transfection, and EC50s were determined.The n-fold resistance of the mutant RNAs relative to that of wild-typeRNA (mutant EC50/wild-type EC50) is depicted.

BMS-824 that target the NS5A protein. Careful evaluation ofpotency and specificity in a number of additional assays re-vealed that BMS-824 was chemically reactive under the assayconditions used and transformed into other species in tissueculture medium that were responsible for inhibitory activity. Itwas the surprising finding that anti-HCV activity was main-tained in the absence of detectable parent compound that ledus to pursue the identification of the exceptionally active com-ponents. To do so, we used a sensitive replicon HPLC biogramassay which allowed us to monitor fractions that containedanti-HCV activity. Inclusion of an NS5A-resistant cell line inthe experiments to demonstrate cross-resistance was key toconfirming relatedness to BMS-824 and allowing us to identifythe peak fractions of interest.

A powerful aspect of the HPLC biogram methodology lies inthe ability to use a functional assay to detect biologically activesubstances in crude matrices that are not readily detectable byphysical methods such as, for example, HPLC-UV (5, 8). Webegan the present study by incubating BMS-824 in HCV rep-licon assay medium prior to isolation experiments. Two essen-tial sample enrichment steps enabled the HPLC-UV detectionand isolation of two minor peaks that correlated with HCVreplicon inhibitory activity and allowed us to conduct structuredetermination using conventional spectroscopic techniques. Inthis manner, the active species was determined to be a larger,dimeric form of the parent molecule derived from a presumedintermolecular reaction rather than a smaller degraded deriv-ative. The dimerization of BMS-824 is currently believed tooccur through a radical mechanism, and this process is thesubject of continuing examination in order to determine theprecise reaction pathway and define the stereochemical rela-tionships.

The synthesis of BMS-346, a compound that symmetrizeselements of BMS-824 thought to be critical for HCV-inhibitoryactivity, yielded a compound with excellent activity againstHCV (EC50, �86 pM), confirming the hypothesis that potentantiviral activity could be derived from a symmetrical dimericmolecule. This was further demonstrated by extensive SARstudies (17). In addition, resistance generation and mappingyielded NS5A resistance substitutions similar to those identi-fied by selection with BMS-824, implying a common bindingsite for the BMS-824-derived active species and BMS-346.Based on resistance mapping, these NS5A inhibitors appear tointeract, either directly or indirectly, with the N terminus ofNS5A. The NS5A protein consists of three putative domains(I, II, and III), with the resistance mutations residing in do-main I. Domain I consists of the first 213 amino acids of theprotein and contains a membrane-anchoring -helix in theN-terminal 30 amino acids (16). The solid-state structure ofdomain I was recently determined, and it was shown to form adimeric complex via contacts near the N-terminal ends of themolecules, which can adopt different conformations (13, 19).The dimeric structure of BMS-346 complements the symmetryobserved in domain I, suggesting a functional role that allowsassociation with the NS5A protein across the dimer interface.The mechanism of action of NS5A inhibitors, like the role ofthe NS5A protein during the life cycle of HCV, is poorlyunderstood. The coincidence of dimeric structures for both theinhibitor and the NS5A protein, as well as the exceptionalpotency of the inhibitors, provides an opportunity to speculate

on and test different working models. Given that the resistancesubstitutions for BMS-346 lie in the vicinity of the dimer in-terface, it is possible that these inhibitors interfere with NS5Adimer formation. The exceptional potency of NS5A inhibitorssuggests that the anti-HCV effect may be amplified. It is con-ceivable that NS5A proteins form multimers during the for-mation of the replication complex and active viral RNA rep-lication. A single inhibitor may not only disrupt the formationof a single NS5A dimer but also affect adjacent NS5A dimers,thereby inactivating the function of an entire replication com-plex. Alternatively, the compounds may disrupt the proximal-helix that promotes essential membrane association or inter-action of NS5A with an unknown host or viral factor(s) re-quired for HCV RNA replication. Studies are in progress togain a better understanding of the multiple functions of NS5Aand the mode of inhibition of these NS5A inhibitors.

Efforts to identify the active component of compound BMS-824 revealed dimeric molecules generated from an intermolec-ular dimerization reaction, leading to the synthesis of a novelclass of symmetrical molecules that demonstrate excellent po-tency against HCV and which target the NS5A protein. Thesediscoveries provided the catalyst for an extensive investigationof further structural modifications of BMS-346 in which opti-mization efforts focused on broadening its genotype coverageand incorporating pharmacokinetic properties suitable for oraladministration. BMS-346 formed the foundation for the dis-covery of BMS-790052, an HCV NS5A inhibitor that exhibitspicomolar EC50s toward a broad range of HCV genotypes invitro and has shown excellent clinical efficacy following oraladministration to subjects chronically infected with HCV ge-notype 1 (6).

ACKNOWLEDGMENTS

We thank our Bristol-Myers Squibb colleagues for helpful discus-sions during the course of this work and Mark Cockett for his contin-ued support.

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Discovery of Potent Hepatitis C Virus NS5A Inhibitors

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