WO2013178782A1

WO2013178782A1 - Allosteric inhibitors of ns3 protease from hepatitis c virus

Info

Publication number: WO2013178782A1
Application number: PCT/EP2013/061256
Authority: WO
Inventors: Adrian Velazquez-Campoy; Javier Sancho Sanz; Olga Abian Franco; Sonia VEGA SANCHEZ
Original assignee: Universidad De Zaragoza; Fundación Agencia Aragones Para La Investigación Y El Desarollo; Instituto Aragonés De Ciencias De La Salud; Universiteit Gent
Priority date: 2012-05-30
Filing date: 2013-05-31
Publication date: 2013-12-05

Abstract

The present invention provides compounds for use in a method for treatment of hepatitis C, wherein the compound binds to the inactive conformational state of the Zn+2-free NS3 protease of the hepatitis C virus (HCV), stabilizing said conformation, and therefore preventing the activating effect of the interaction with the two cofactors, Zn+2 and NS4A, and, thus, effectively inhibiting allosterically the catalytic activity of the protease.

Description

ALLOSTERIC INHIBITORS OF NS3 PROTEASE FROM HEPATITIS C VIRUS

Field of the invention

The present invention provides compounds capable of binding the NS3 protease of the hepatitis C virus (HCV) which are useful for the treatment of hepatitis C.

Background art

The hepatitis C virus (HCV) infection is a worldwide health problem, HCV infected people amount to more than 200 million, with 80% of them becoming chronic patients, and many of them

progressing to cirrhosis and hepatocellular carcinoma. The viral RNA is directly translated in infected cells into a precursor polyprotein which must be processed for successful viral

maturation. Host cellular proteases are implicated in the

processing of the structural viral proteins, whereas two different proteolytic activities encoded in the viral polyprotein, NS2 (or HS2/3) and NS3 , are involved in the processing of the nonstructural viral proteins. Most of the non-structural cleavage sites (NS3 -NS4A to NS5A-NS5B) are processed by the NS3 protease, which is a Zn+2 -dependent serine protease comprising the NS3 N- terminal domain.

Since its identification., the NS3 protease active site has been considered a pharmacological target for drug discovery. Although very few protease competitive inhibitors have entered clinical trials, a broad variety of competitive inhibitors has been developed. Very recently, two protease inhibitors have been approved by the FDA for therapeutic treatment, namely Telaprevir and Boceprevir . However, new antiviral agents are urgently needed because resistance mutations causing an efficacy reduction for these two drugs have already been identified.

Brief Description of the Figures Fig. 1: Biophysical characterization of the Zn+2-free NS3 protease from the hepatitis C virus. Fig la: Fluorescence emission spectra

(/.ex = 280 nm) ; pH 5, 25°C, [Protein] = 4 μ. ; 2n+2 2 mM (continuous line) ; Zn+2 0 mM (dotted line) . This figure illustrates how the removal of zinc causes a red-shift in the spectrum (towards higher wavelengths) indicating exposure of tryptophans to the solvent. Fig lb; Far-UV circular dichroism spectra; pH 5, 25°C , [Protein] =

4 μΜ; Zn+2 2 mM (continuous line) ; Zn+2 0 mM (dotted line) . This figure illustrates how the removal of zinc causes a slight change in. the spectrum, indicating that most of the secondary structure is maintained. Fig Ic: ANS binding; pH 5, 25°C, [Protein] = 2 μΜ; Zn+2 2 mM (continuous line) ; Zn+2 0 mM (dotted line) , This figure illustrates how the removal of zinc causes an increase in ANS fluorescence , indicating exposure of hydrophobic surf ce in the protein. Fig Id: Thermal stability: Differential scanning

calorimetry ; pH 5, [Protein] = 40 μΜ; Zn+2 40 μΜ ( continuous 1 ine ) ; Zn+2 0 μΜ ( dotted line) . This figure illustrates how the removal of zinc causes a marked destabilization, lowering the structural stability of the protein. Fig le : Chemical stability: Fluorescence; pH 5, 25°C, [Protein] = 4 μΜ; Zn+2 40 μΜ (closed squares) ,- Zn+2 0 μΜ (open squares) . This figure illustrates how the removal of zinc causes a marked destabilization, lowering the structural stability of the protein.

Fig. 2: This figure shows thermal denaturation curves for NS3 protease in the presence of different compounds followed by ANS fluorescence in sodium acetate 100 mM, at pH5 in the absence of Zn+2.

Fig. 3: Evaluation of potency and cytotoxicity of the selected compounds capable of binding the Zn÷2-free NS3 protease of hepatitis C virus in cell assays .

Fig . 4 : Evaluation of potency and cytotoxicity of the selected compounds capable of binding the active conformation of NS3 protease of hepatitis C virus in cell assays . Detailed description

It is the obj ect underlying the present invention to provide compounds, compositions and formulations that are useful for the treatment of hepatitis C,

The present invention thus provides compounds for use in a method for treatment of hepatitis C, wherein the compound binds to the inactive conformational state of the Zn+2 -free NS3 protease of the hepatitis C virus (HCV) , stabilizing said conformation, and therefore preventing the activating effect of the interaction with the two cofactors, Zn+2 and NS4A, and, thus , effectively

inhibiting allosterically the catalytic activity of the protease .

NS3 protease is a 20 KDa serine protease structurally homologous to other extracellular serine proteases , like trypsin and

chymotrypsin . The catalytic active site of this protease is located in a groove between the N-terminal and C-terminal domains . The secondary structure of this protease is dominated by beta strands and turns , this is confirmed by its circular dichroism spectrum. Extracellular proteases homologous to HS3 protease from hepatitis C virus, show disulfide bridges which role is to stabilize the molecular structure of the protease . However, NS3 protease from hepatitis C virus fails to contain said disulfide bridges and instead comprises a Zn+2 ion tetra-coordinated by three cysteine residues and a histidine residue in its C-terminal domain. The Zn+2 ion is required by the NS3 protease for its hydrolytic activity, since its removal leads to the inactivation of the protease . Consequently, the Zn+2 ion is considered to have a structural role by stabilizing the active conformation of the NS3 protease of the hepa i is C virus .

Calorimetric and. spectroscopic data suggest a considerable global conformational change upon Zn+2 binding which mainly involves the tertiary structure of the protein . Thus , the disruption of the NS3 -Zn+2 interaction leads to significant unfolding of the molecule .. This conformational change leads to enzyme inactivation.

Therefore, in the absence of Zn+2 , NS3 protease is mostly

unstructured and can be considered an intrinsically (partially) disordered protein that folds upon binding to its Zn+2 cofactor.

The Zn.+2-free NS3 protease shows very little stabilization energy, 0.4 kcal/mol at 20°C, which leads to 35% of unfolded protein and 65% of un- structured-native inactive protein at that temperature . Because unfolding thermal and chemical transitions are observed for Zn+2 -free NS3 protease , the Zn+2 -free conformational state is not equivalent to the fully unfolded protein and has a residual structure. Other homologous Zn+2 -dependent proteases do not show an unfolding transition in the absence of Zn+2 , an indication of complete loss of structure upon Zn+2 removal . A complete biophysical characterization of the Zn+2 -free NS3 protease from the hepatitis C virus is provided herein in Figures 1 (a) to 1 (e) .

As stated before , the inventors have found compounds capable of binding to the inactive conformational state of the Zn+2 -free NS3 protease , stabilizing and trapping said conformation, preventing the activating effect of the interaction with the two cofactors , Zn+2 and NS4A, and, thus, effectively inhibiting aliosterically the catalytic activity of the protease .

In order to identify said compounds , a ligand. screening procedure was performed for NS3 protease under conditions where the inactive partially- folded Zn+2 -free NS3 protease conformation is

significantly populated, (basically, at pH 5 in the presence of a saturated concentration of EOTA) . This procedure is fully detailed in examples 1 and 2 below. A set of compounds were selected based on the stabilizing effect induced on HS3 protease against thermal denaturation ( see examples 1 and 2 and figure 2 ) . These inhibitors showed a non-competitive mechanism of action and provide several improvement s over known NS3 clinical inhibitors showing a competitive mechanism of action, these improvements are at least the following: 1) inhibition by a different and new mechanism of action, trapping the enzyme into a non-productive partially-folded conformation; 2} reduced impact of known resistance-associated mutations; 3} inhibition of NS4A binding (NS A not only enhances NS3 protease catalytic activity, but also localize NS3 pxOtease on appropriate membrane regions within the replication complex) ; and 4} possibility of combination therapy together with the current approved clinical inhibitors. Once the compounds were selected, the potency as HCV-replication inhibitors and the cytotoxicity of these compounds was evaluated in cell assays ( see example 3) based on theses assays eight compounds were finally selected (see table I below)

Table i , Se ecte compoun s

The results of the evaluation of the potency and cytotoxicity of the selected compounds in cells assays are provided in figure 3 and table 2 below.

Table II. Evaluation of potency and cytotoxicity of the selected compounds in cells assays. ECS0, effective concentration 50%; LC5G, lethal concentration 50%; EC90, effective concentration 90%; LC90, lethal concentration 90%.

These results illustrate the viability of these compounds as HCV- inhibitors . Thus, suitable compounds which can be used according to the present invention include compounds of the following formulae

Compound 1 (Nocodazole) Compound 2 (Dipyridamole)

Compound 5 {Tacrine hydrochloride hydrate)

CIH

From these compounds three are particularly preferred; these are Nocodazole, Camptothecine, dydrogesterone as well as analogues or derivatives of any of these compounds thereof. Thus, a first group of preferred compounds which can be used according to the present invention includes compounds of the following formula:

Formula (I.)

wherein l is S or 0;

R2 , R3 , R5, R6 , R7 , R8 and R9 are each independently of each other hydrogen or a Cl-C4-alkyl , a C2-C4-alkenyl or a C2-C4-alkynyl group ; and

R4 is hydrogen or a C1-C4 -alkyl , a C2 -C4 -alkenyl , a C2 -C4-alkynyl group or a -CH2-NH2, -CH2-OH or CH3-CN.

In particular, compounds of the following formula: Formula (11)

A second group of preferred compounds which can be used according to the present invention includes compounds of the following formula : Formula (III)

wherein l, R2 and R3 are each independently of each other hydrogen or a Cl-C4-alkyl, a C2-C4-alkenyl or a C2 -C4 -alkynyl group ; and R4 is an -0R5 or an OCOR5 wherein R5 is a hydrogen , CI -C4 -alkyi , CI -C4-alkenyl or CI -C4 -alkynyl ;

More particularly, compounds of the following formula: Formula (IV)

A third group of preferred compounds which, can be used according to the present invention includes compounds of t e following formula :

Formula (V)

wherein

Rl and R4 are independently of each other a hydrogen , an oxo group or an -OR5 or an -OCOR5 wherein R5 is a hydrogen, Cl-C4-alkyl, Cl- C4-alkenyl or C1-C4 -alkynyi ;

R5 and R6 are each independently of each other hydrogen or a Ci- C4-alkyl, a C2-C4-alkenyl or a C2 -C4-alkynyi group;

R2 is a hydrogen, or a Cl-C4-alk l, a =CH2 , a C2 -C4 -alkenyl or a

C2 ~C4 -a1kyny1 group R3 is an oxo group or a -COR5 wherein R5 is a C1-C4 -alkyl , C1-C4 - alkenyl or CI -C4 -alkynyi ;

In particular, compounds of the following formula: Formula (VI)

In a further aspect, the present invention also provides a method for identifying compounds that can be used according to the present invention. Hence, the present invention further provides a method for screening a compound capable of binding and stabilizing the Zn+2 -free NS3 protease conformation of the hepatitis C virus in a non-native inactive partially- folded conformation, wherein a compound library is subjected to a binding assay as illustrated herein in example 1 and 2 ,

Further-more, the present invention provides for a method of production of a compound suitable for use in a method for

treatment of hepatitis C, which comprises the following steps : a. Subjecting a compound library to a binding assay to the Zn+2 -free NS3 protease conformation of the hepatitis C virus ; and b. Selecting a compound, from the compounds of step a) , capable of binding and stabilizing the Zn+2 -free NS3 protease conformation of the hepatitis C virus in a non- native inactive partially- folded conformation.

In the context of the present invention, it is understood that a compound capable of binding and stabilizing the Zn+2 -free NS3 protease conformation of the hepatitis C virus in a non-native inactive partially- folded conformation, is a compound capable of establishing favourable interactions and forming a complex with the Zn+2 -free HS3 protease and capable of enhancing the stability of said non-native inactive partially- folded conformation against thermal denaturation .

Additionally, the present invention further provides compounds for use in a method for treatment of hepatitis C, wherein the compound binds to the active conformational state of the NS3 protease of the hepatitis C virus (HCV) , and, thus , effectively inhibiting the catalytic activity of the protease through a competitive mechanism of action.

In order to identify said compounds , a ligand screening procedure was performed for 1TS3 protease under conditions where the active conformation is significantly populated. This procedure is fully detailed in example 5. A set of compounds were selected as potential ligands due to their capacity to inhibit the NS3 protease of the hepatitis C virus.

The selected compounds were betulinic acid (from hereina ter compound 9); ursolic acid (from hereinafter compound 10);

mometasone furoate ( from hereinafter compound 14) ; tenoxicam (from hereinafter compound 15); Butoconazole nitrate (from hereinafter confound 11} ; Amphotericin B (from hereinafter compound 12) and '4 -benzyl- 5- ({ [1- { -methylphenyl ! -4- {2-thienyl) -lH-imidazol-2- yl] sulfanyl }methyl ) - 2 , 4 -dihydro-3H- 1 , 2 , - triazole-3 -thione ( from hereinafter compound 13 ) .

The potency as HCV-replication inhibitors and the cytotoxicity of the set of compounds selected was evaluated in cell assays; based on theses assays five compounds were finally selected ( see table 3 below) . The results of the evaluation of the potency and cytotoxicity of the selected compounds in cells assays are provided in figure 4 and table 3 below .

Table III, Evaluation of potency ana c otoxi city of the selecte compounds in cells assays.

These results illustrate the viability of these compounds as HCV- inhibitors .

Thus , suitable compounds which can be used according to the present invention include compounds of the following formulae : Compound 9 (betulinic acid} Compound 10 (ursolic acid)

Compound 11 {Butoconazole nitrate]

Dound 12 {Amphotericin B)

OH

OH OH

Compound 13 { ' 4 -benzyl -5- { { [1- (4 -methyl henyl ) - - ( 2 -t ienyl ) - imidazol-2 -yl] sulfanyl }methyl) -2 , 4 -dihydro- 3H- 1,2, 4 -triazole-3 - tiiione)

From these compounds, compound 13, as well as analogues or derivatives thereof, is particularly preferred.

It is important to note that all of the compounds of the invention include pharmaceutically acceptable salts, esters, amides, and prodrugs therof , including but not limited to carboxylase salts, amino acid addition salts, esters, amides , and prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, irritation allergic response, and the like, commensurate with a reasonable

benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the invention. The term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Representative salts include the hydrobromide ,

hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate , stearate , laurate, borate, benzoate, lactate, phosphate, tosylate , citrate, maleate , fumarate ,

succinate , tartrate, naphthylate mesylate, glucoheptonate , laccobsonate , and laurylsulphonate salts, and the like . These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium , and amine cations including, but not limited to amnionium,

tetramethylammonium, tetraethylammonium, methylamine ,

dimethylamine , trimethylamine , triethylamine , ethylamine, and the like.

Accordingly, a further aspect: of the present invention includes pharmaceutical compositions comprising as one or more compounds of the invention disclosed above, associated with a pharmaceutically acceptable carrier. For administration, the compounds are

ordinarily combined, with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide , sodium and calcium salts of phosphoric and sulfuric acids , acacia, gelatin, sodium alginate , polyvinylpyrrolidine , and/or polyvinyl alcohol , and tableted or encapsulated for conventional

administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol , carboxymethy1 cellulose colloidal, solutions , ethanol , corn oil , peanut oil , cottonseed oil , sesame oil , tragacanth gum , and/or various buffers. Other adjuvants and modes of

administration are well known in the pharmaceu ical art. The carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the ar .

Examples of pharmaceutically acceptable, non-toxic esters of the compounds of this invention include C1-C6 alkyl esters, wherein the alkyl group is a straight or branched substituted or

unsubstituted, C5-C7 cycloalkyl esters , as well as aryialkyl esters such as benzyl and triphenylmethyl . C1-C4 esters are preferred, such as methyl, ethyl, 2,2, 2 -trichloroethyl , and tert- butyl . Esters of the compounds of the present, invention may be prepared according to conventional methods. Examples of pharmaceutically acceptable, non- to ic amides of the compounds of this invention include amides derived from ammonia, primary C1-C6 alkyl amines and secondary C1-C6 dialkyl amines , wherein the alkyl groups are straight or branched. In the case of secondary amines, the amine may also be in the form, of a 5- or 6-membered

heterocycle containing one nitrogen atom. Amides derived from ammonia, C1-C3 alkyl primary amines and C, -C2 dialkyl secondary amines are preferred. Amides of the compounds of the invention may be prepared according to conventional methods.

The term "prodrug" refers to compounds that are rapidly

transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. A thorough

discussion of prodrugs is provided in . Higuchi and V . Stella, "Pro-drugs as Novel Delivery Systems , " Vol . 14 of the A. C . S .

Symposium Series , and in Bioreversible Carriers in Drug Design, ed. Edward B . Roche , American Pharmaceutical Association and Pergamon Press, 1987 , both of which are hereby incorporated, by reference .

These compounds can be administered individual ly or in

combination, usually in the form of a pharmaceutical composition. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound .

Accordingly, a further aspect of the present invention includes pharmaceutical compositions comprising as one or more compounds of the invention disclosed above , associated with a pharmaceutically acceptable carrier . For administration, the compounds are

ordinarily combined with one or more adjuvants appropriate for the indicated route of administration. The compounds may be admixed with lactose , sucrose , starch powder, cellulose esters of alkanoic acids , stearic acid, talc , magnesium stearate , magnesium oxide , sodium alginate , polyvinylpyrrolidine , and/or polyvinyl alcohol , and tableted or encapsulated for conventional administration. Alternatively, the compounds of this invention may be dissolved in saline, water, polyethylene glycol, propylene glycol,

carboxymethyl cellulose colloidal solutions, ethanol, corn oil, peanut: oil, cottonseed oil, sesame oil , tragaeanth gum, and/or various buffers. Other adjuvants and modes of administration are well known in the pharmaceutical art. The carrier or diluent ma- include ime delay material, such as glyceryl monoscearate or glyceryl distearate alone or with a wax, or other materials well known in the arc .

The following examples are intended for the sole purpose of

illustrating the present invention.

Examples

Example 1; MATERIALS AND METHODS 1.1 Inh.I > i t s

Hepatitis C virus (HCV) inhibitors were identified and evaluated for their anti-HCV activities using replicon systems. The

compounds were supplied by Prestwick chemical company. 1.2 NS3 protease purification

In vitro NS3 protease assays iHTS and 1TC) have been performed with the isolated N -terminal domain from full-length NS3 protein, which exhibits similar properties (enzymatic activity, inhibition constants , and allosteric activation mechanism) zc those of the full-length protein.

1,3 Cells and rep 1_ pons

The highly permissive cell clone Huh ?-Lunet (V. Lohmann and R. Bartenschlager) as well as Huh 7 cells containing subgenortiic HCV replicons I389luc-ubi-neo/NS3 -3 ^"/5.1 (Huh 5-2) , I377NS3-3^'/ t (Huh 9-13) or I389/hygro-ubi-NS3-3/5.1 have been described recently 22- 26. Cells were grown in Dulbecco's modified Eagle's Medium (DMEM; Gibco , Merelbeke , Belgium) supplemented with 10% heat- inactivated fetal bovine serum ( PAN-BIOTECH GmbH, Germany) , IX non-essen ial amino acids (Gibco , 100 IU/mL penicillin (Gibco) , 100 ^.g mL streptomycin (Gibco), and 250 jttg/mL Geneticin (G418, Gibco) for Huh 5-2 cells

1_._4 Anti-HCV Assay in Huh 5-2 Cells Huh 5-2 cells were seeded at a density of 5X 103 per well in a tissue culture-treated white 96 -well view plate [Techno Plastic Products AG, Switzerland) in complete DMEM supplemented with 250 /ig/iTiL G418. After incubation for 24 hours at 37°C (5% COG } medium was removed and 2-fold serial dilutions in complete DMEM (without G418) of the test compounds were added in a total volume of 100 μΐι. After 3 days of incubation at 37°C, cell culture medium was removed arid luciferase activity was determined using the Bright- GioTM Luciferase Assay System (Promega Corporation, Leiden, The Netherlands) the luciferase signal was measured using a Synergy HT Multimode Reader (BioTek Instruments, Inc, Vermont, USA) . The 50% effective concentration (EC50) was defined as the

concentration of compound that reduced the luciferase signal by 50%,

1.5 C tostatic Assay Huh 5-2 or Huh mono cells were seeded at a density of SX 103 cells per well of a 96 -well plate in complete DMEM with the appropriate concentrations of G418. Serial dilutions of the test compounds in complete DMEM without or G418 were added 24 hours after seeding. Cells were allowed to proliferate for 3 days at 37°C, af er which the cell number was determined by CeliTiter^® 96 AQueous One

Solution Cell Proliferation Assay (Promega Corporation) . The 50% cytostatus concentration (CC50 ) was defined as the concentration that inhibited the proliferation of exponentially growing cells by 50% . 1.6 High-Throughput: Screening for compounds capable of binding and stabilizing the Zn+2 - free NS3 protease of the hep. . . ^" . ims

The NS3 protease exhibits a complex conformational landscape where a non-native inactive partially- folded conformation is populated in the absence of Zn+2. Ligands targeting the inactive partially- folded NS3 protease state have been identified as those compounds inducing a stabilizing effect against thermal denaturation in the absence of Zn+2. The protein unfolding transition can be monitored by following ANS (8-anilino 1- naphthalene sulphonic acid) emission fluorescence, which greatly increases when the protein exposes its internal hydrophobic residues during the thermal unfolding process. ANS is a fluorescent probe that binds to hydrophobic regions in proteins with an increase in its fluorescence quantum yield.

HTS for ligand- induced NS3 stabilization was performed by

monitoring the thermal denaturation of recombinant pure NS3 protease in the presence of the extrinsic fluorescent probe ANS ( Sigma -Aldrich) . HTS experiments were performed in a FluoDia T7Q High Temperature Fluorescence Microplate Reader (Photon Technology International) . Protein- ligand solutions (100 p.l) were dispensed into 96 -well microplates (ThermoFast 96 skirted plates, from Thermo Scientific) and overlaid with 20 μΐ of mineral oil to prevent evaporation. Protein solutions contained 2 μΜ NS3 protease in 100 mM Sodium Acetate, 2 mM EDTA, pK 5.0, and 100 μΜ ANS,

Ligands dissolved in DMSO were added at 100 μΜ (with a final DMSO 2.5% residual concentration) to microplates containing the protein solutions and incubated at 25°C for 30 minutes before loading into the microplate reader . Control experiments with NS3 protease samples with/without DMSO and/or Zn+2 were routinely performed in each microplate . Thermal denaturation was monitored by following the increase in ANS fluorescence intensity associated with protein unfolding {kexc = 395 and Kern - 500 nm, where Aexc is the

excitation wavelength and he is the emission wavelength) ,

Unfolding curves were registered from 25 °C to 75 °C in PC steps. The system was allowed to equilibrate at each temperature for 1 minute before each fluorescence acquisition. In practice, this represents an operational heating rate of 0.25 °C/min

approximately . The specific steps taken for the HTS of ligand- induced NS3 stabilization were as follows :

First, preparation of the following materials:

Buffer A: Sodium acetate 100 mMpH5 - Buffer B : Sodium acetate 100 mMpHS supplemented with

ΙΟΟ Μοί Zn

Buffer C: Sodium acetate 100 mM pHS supplemented with 2mM of EDTA

Psol : Purified NS3 protease 20 μΜ in Buffer B, - Fsol: ANS 20 mM in Buffer A

Lsol : Ligand 5mM in D SO

Dsol : DMSO

Msol : Mineral oil

Secondly, preparation of the following stocks solutions: 1) StockNS3 /EDTA . Preparation of a stock solution of NS3 protease in presence of EDTA: mix 1000 μΐΐ of Psol and 3750 μΐ of Buffer C.

2 ) StockNS3/Zn. Preparation of a stock solution of NS3 protease in presence of Zn: mix 120 μΐ» of Psol and 450 uL of Buffer B.

3) StockAKB/EDTA, Preparation of a stock solution of ANS in presence of EDTA: mix 60 μL of Psol and 5940 μΐ, of Buffer C.

4) ScockANS/Zn. Preparation of a stock solution of A S in presence of Zn: mix 1 μΒ of Fsol and 999 μη of Buffer B.

Thirdly, preparation of the 96 well plates :

1) Controls : Only in column 1, A to H wells: Addition of 47.5 μΐ, of StockNS3/Zn Addition of 50 μΐ, of StockANS/Zn Addition of 2.5 μL of Dsoi

2) Controls : Only in column 12, A to H wells : - Addition of 47.5 μΐ, of StockNS3/EDTA

Addition of 50 μη of StockANS/EDTA Addition of 2.5 μΐ, of Dsol

3) Samples: In columns 2 to 11, A to H wells :

Addition of 47.5 μη of StockNS3 /ED - Addition of 50 μL of StockANS/EDTA

Addition of 2,5 μL of Lsol

4) In controls and samples:

Addition of 20 Τ of Msol without mixing since the mineral oil must be placed on the surface of the mixture. Fourthly, reading the fluorescence in a FIuoDia T70 High

Temperature Fluorescence Microplate Reader (Photon. Technology International) using the following parameters: a) Aexc = 395 and hem = 500 n:m (Aexc - excitation 'wavelength and hem emission wavelength). b) 25°C to 75°C in PC steps. c } Operational heating rate of 0.25°C/min.

Lastly, analysis of the results by representing the curves Fluorescence signal {A. U) versus Temperature ( °C) (see fig. 2)

1.7 Isothermal Titration Calorimetry (ITC)

Ligand binding to NS3 protease was determined with a high- sensitivity isothermal titration VP -ITC microcalorimeter (MicroCal) . Protein samples and reference solutions were properly degassed and carefully loaded into the cells to avoid bubble formation during stirring. Experiments were performed with freshly prepared buffer-exchanged protein, solutions. Experiments were performed at 25 °C in 100 mM Sodium Acetate, 2 mM EDTA, pH 5.0. Experiments were carried out titrating 20 μΜ of NS3 protease solution in the calorimetric ceil with a 300 μΜ solution of compound. The heat evolved after each ligand injection was obtained from the integral of the calorimetric signal. The heat due to the binding reaction was obtained as the difference between the reaction heat and the corresponding heat of dilution, the latter estimated as a constant heat throughout the experiment, and included as an adjustable parameter in the analysis . The

association constant. (Ka) and the enthalpy change (ΔΗ) were obtained through non- linear regression of experimental data to a model for a protein with a single binding site. Data were analyzed using software developed in our laboratory implemented i Origin 7 (OriginLab) . The dissociation constant (Kd) , the free energy change (£s.G) and the entropic change (AS) were obtained from, basic thermodynamic relationships .

Example 2s Identification of HCV compounds capable of binding and stabilizing the Zn+2 - free NS3 protease of the hepatitis C virus by HTS Although in the absence of Zn+2 NS3 retains some structure , it- shows very low stability against thermal denaturation .

Approximately 40% of the molecules are unfolded at 25 °C,

Therefore, the native baseline in the pre-unfolding region is absent in the thermal denaturation assays (Figure 2) . Compounds binding to the NS3 protease are identified as those compounds stabilizing the protein against thermal unfolding, i.e. those compounds increasing the mid- transition temperature . The mid- transition temperature is defined as the temperature for maximal slope in the unfolding curve or the temperature at which half of the maximal change in the signal is achieved. The absence of the native pre-unfolding baseline makes somewhat difficult the evaluation of the mid- transition temperature following this second operational methodology. Therefore, hits were identified as those compounds shifting the unfolding transition to higher

temperatures, compared to the internal controls in each microplate (NS3 protease + A S + DMSO) .

Example 3. Antiviral activity of selected compounds capable of binding and stabii isittg the Ζπ,+2-free NS3 protease of the

hepatitis C virus in HCV subgenoitiic replicon cells

Compounds selected in the HTS step are ligands targeting the inactive partially-folded NS3 protease conformation. Therefore, those compounds will act as allosterlc inhibitors stabilizing and trapping the. IIS3 protease into an inactive conformation. The potency of these compounds was assessed by in vitro HCV

replication assays with genotype lb Conl HCV subgenomic replicons {Huh 5-2) . Some of the selected compounds by HTS inhibited HCV replicon replication (measured as luciferase signal) in a dose- dependent manner (Table 2, Figure 3) . The estimated EC50 values for these compounds ranged from nanomolar to micromolar . The anti- HCV activity was not the result of a cytostatic effect, since the LC50 for these compounds were significantly higher than the EC50.

Example 4, Characterization, of KS3 protease binding to selected compounds capable of binding and stabilizing the Zn+2-free NS3 protease of the hepatitis C virus by ITC

ITC allows the direct determination, of intermolecular

interactions, by determining the association, constant Ka (or the dissociation constant Kd = l/Ka) , the binding enthalpy I;H, and the binding stoichiometry .. The dissociation constants for the

interaction between NS3 protease WT and drug-resistance -associated variants with the selected compounds are summarized in. Tables 4 and 5 below. These results indicate that these compounds are able to bind to NS3 protease variants associated with drug i'esistance with the same strength,

Table IV. Dissociation constan.cs for selected compounds binding to S3 protea

Wild type at 25° in 100 mM Sodium Acetate, 2 mM EDTA, pii 5,

Compound 1 K_d (M) Compound 2 I Compound 5 Kd (M)

S139A 1.4 x ICT⁵ S139A 8.4 x lO^"6 S139A 5.5 xlO^"6

S139A/D168A 2.6 x 10^"6 S139A/D168A 1.1 x 10^"5 S139A/D168A 8.5 x 10^{' 6}

S139A/R155K 5.3 xlO^"6 S139A/R155K 2.3 xlO^-5 S139A/R155K 5.9 x 10-6

S139A/A156T S139A/A156T S139A/A156T 8.3 x 10^'5

S139A/R155Q S139A/R155Q 2.1 xlO^-5 S139A/R155Q 7.4 xlO^"5

Compound 6 K_d (M) Compound 7 K_d (M) Compound 8 _d CM)

S139A 2.4 xlO^"5 S139A l.B lO^'5 S139A 3.4 xlO^"5

S139A/D168A 3.6 x icr⁵ S139A/D168A 6,5 xlO^"6 S139A/D168A 1.6 xlO^'5

S139A/R155K 6.1 x 10^{" 5} S139A/R1S5 1.6 xlO^-5 S139A/R155K 9.2 xlO^"6

S139A/A156T 4.5 x 10^{' 5} S139A/A156T 5,8 x 10^'6 S139A/A156T 2.2 xlO^-5

S139A/R155Q 2.3 x 10^~5 S139A/R155Q 4.2 x 10^"6 S139A/R155Q x If)-⁵ Table v. Dissociation constant for selected compounds binding to drug-resistance- associated NS3 protease variants in at 25°C in 100 mM Sodium Acetate, 2 mM EDTA, pH 5.

Example 5. Identification of compounds capable of binding and stabilizing the active conformation of the NS3 protease of the hepatitis C virus by HTS

The NS3 protease catalyzes the hydrolytic cleavage of the

polypeptide translated from the viral RNA. Competitive inhibitors targeting the substrate binding site have been identified as those compounds blocking the enzymatic hydrolytic activity.

Measurement of protease activity is accomplished using a

Fluorescence Resonance Energy Transfer (FRET) peptide substrate. The substrate has two fluorophore groups , EDA S and DABCYL, separated by a few aminoacid residues . Quenching of the EDANS fluorophore by distance -dependent FRET to the DABCYL quencher is eliminated upon cleavage of the intervening peptide linker .

Therefore, the enzymatic activity can be determined by monitoring the fluorescence emission from the EDANS fluorophore ( exc = 390 nm, Kern = 500 nm) . HTS for ligand- induced NS3 inhibition was performed by monitoring the enzymatic activity of recombinant pure., active NS3 protease with a peptidic substrate Si (Ac-DED (EDAMS) EE-Abu -L-Lactoyl - SK (DABCYL) -NH₂ , BACHEM) . HTS experiments were performed in a

FluoDia T70 Fluorescence Microplate Reader (Photon Technology International) . Protein-ligand solutions (200 ul) were dispensed into 36 -well microplates (ThermoFast 96 skirted plates, from.

Thermo Scientific) . Protein solutions contained 15-30 nM NS3 protease in 50 mM Tris pH 7.0, 0.1% CHAPS , 50% glycerol, 5 μ zinc chloride and 30 mK DTT . Ligands dissolved i DMSO were added at 100 μΜ (with a final DMSO 2.5% residual concentration) to

microplates containing the protein solutions and incubated at 25 °C for 30 minutes . Enzymatic reaction, was initiated by addin

substrate SI at a final concentration of 4 μΚ, Control experiments with NS3 protease samples with/without DMSO and/or NS3 protease and/or substrate SI were routinely performed in each microplate . Enzyme activity was monitored at 25°C by following the increase in EDANS fluorescence intensity associated with substrate cleavage lAexc = 390 and Aero = 500 nm, where Aexc is trie excitation wavelength and ΛΘΗΙ is the emission wavelength} . Compounds inhibiting the NS3 protease are identified as those compounds blocking the hydrolytic activity (small and slow increase in substrate fluorescence, compared to the internal controls in each micropiate) of the enzyme .

Example 6. Antiviral activity of selected compounds capable of binding and stabilizing the active conformation, of the NS3 protease of the hepatitis C virus in HCV subgenomic rep1icon cells

Compounds selected in the HTS step of example 5 are ligands targeting the folded NS3 protease active conformation. The potency of these compounds was assessed by in vitro HCV replication assays with genotype lb Conl HCV subgenomic repiicons (Huh 5-2) . Some of the selected compounds by HTS inhibited HCV replicon replication (measured as luciferase signal) in a dose -dependent manner {Figure 4) ,

Claims

aims

A compound for use in a method for treatment of hepatitis C. wherein the compound binds to the inactive conformational state of the Zn+2-free NS3 protease of the hepatitis C virus (HOT) , stabilizing said conformation.

The compound for use in a method for treatment according to claim 1, wherein the compound has an 1C50 of 0,01 riM to 120 μΜ for stabilizing the inactive conformational state of the Zn+2- free NS3 protease of the hepatitis C virus,

A method for the production of a compound suitable for use in a method for treatment of hepatitis C, which comprises the following steps:

a. Subjecting a compound library to a binding assay to the Zn+2 -free NS3 protease conformation of the hepatitis C virus; and

b. Selecting a compound, from the compounds of step a) , capable of binding and stabilizing the Zn+2 -free NS3 protease conformation of the hepatitis C virus in a non-native inactive partially- folded conformation.

The compound for use in a method for treatment according to any one of claims i or 2, wherein the compound is a. compound of the following general formula:

Formula. (V)

wherein

Rl and R4 are independently of each, other a hydrogen, an oxo group or an -ORB or an -0C0R5 wherein R5 is a hydrogen, Cl-C4~alkyl, Cl- C4-alkenyl or CI -C4 -alkynyl ;

R5 and R6 are each independently of each other hydrogen or a Cl- C4-alkyi, a C2 -C4-alkenyl or a C2-C4- alkynyl group

R2 is a hydrogen or a Cl-C4-alkyl, a =CH2 , a C2-C4-alkenyl or a C2 -C4 - a1kynyl group ,- and R3 is an oxo group or a -COR5 wherein R5 is a CI -C4 -alkyl , C1~C4- alkenyl or CI -C -alkynyl .

5. The compound for use in a method for treatment according to claim 4, wherein the compound comprises the following formula ; Formula (VI)

or a pharmaceutically acceptable salt, ester, amide, and prodrug thereof ,

6. The compound for" use in a method for treatment according to any one of claims 1 or 2, wherein the compound is a compound of the following general formula :

Formula (III)

wherein

RI , R2 and R3 are each independently of each other hydrogen or a Cl-C4-alkyl, a C2 -C -alkenyl or a C2 -C4 -alkynyl group; and

R4 is an -ORB or an 0C0R5 wherein R5 is a hydrogen, C1-C4 -alkyl , CI - C -alkenyl or CI -C4~alkynyl .

The compound for use in a method for treatment according to claim 6, wherein the compound comprises the following formula :

Formula (IV)

or a pharmaceutically acceptable salt, ester, amide, and prodrug thereof .

8. The compound for use in a method for treatment according to any one of claims 1 or 2, wherein the compound is a compound of the following general formula:

Formula (I)

wherein

Rl is S or 0;

R2 , R3 , R5 , R6, R7 , R8 and R9 are each independently of each other hydrogen or a Cl-C4-alkyl, a C2-C4-alkenyl or a C2-C4 -alkynyl group ; and

R4 is hydrogen or a Cl-C4-alkyl , a C2 -C4 -alkenyl , a C2-C4-alkynyl group or a -CH2-NH2, -CH2-GH or CH3-CN,

9. The compound for use in a me od for treatment according to claim 8, wherein the compound comprises the following formula :

Formula (xl)

or a pharmaceutically acceptable salt, ester, amide , and prodrug thereof . The compound for use in a method for treatment according to any one of claims 1 or 2, wherein the compound comprises any one of the following formulae: a .

or a pharmaceutically acceptable salt, ester, amide, and prodrug thereof

11. Method for screening a compound for the treatment of

hepatitis C virus, wherein a compound library is subjected to a binding assay wherein a compound is selected if it is capable of binding and stabilizing the Zn+2 -free NS3 protease conformation of the hepatitis C virus in a non- native inactive partially-folded conformation.

12. A compound for use in a method for treatment of hepatitis C,, wherein the compound comprises the following formula:

or a pharmaceutically acceptable salt, ester, amide, and prodrug thereof .