A COMPOSITION METHOD AND FORMULATION FOR PEPTIDE BASED INHIBITOR OF HEPATITIS C VIRUS

REPLICATION

FIELD OF THE INVENTION

The present invention generally relates to the field of molecular biology and more specifically embodiments of the invention relates to peptide based inhibitors of Hepatitis C Virus (HCV) replication.

BACKGROUND

Hepatitis C infection is a major health concern across diverse human population and is primarily caused by HCV. Since the discovery of the virus, considerable progress has been made in the understanding of the virus life cycle. HCV is a positive sense single stranded RNA virus belonging to the family, Flaviviridae. The genome of HCV comprises 9600 nucleotides and has a single open reading frame (ORF) that encodes structural proteins and non structural proteins. Both the structural and non structural proteins have been the target for development of HCV therapies. Of these, the proteases namely, NS2 cysteine auto-protease and NS3-4A serine protease, encoded by the viral genome have been targeted for curbing the HCV infection. HCV non-structural protein 3 (NS3) plays a critical role in polyprotein processing and viral RNA replication. This multifunctional enzyme has two major domains: the serine protease domain and helicase domain. Saumitra Das group have demonstrated that the NS3 protease domain alone can specifically bind to HCV-IRES (Internal Ribosomal Entry Site) near initiator AUG and dislodge the binding of a host factor, the human La protein. The interplay leads to inhibition of translation in favor of replication.

One of the methods of curbing HCV infection has been through inhibition of activity of NS3-4A. Various inhibitors have been designed and targeted at the NS3-4A. The inhibitors include but are not limited to small molecules, derived synthetically and obtained from natural sources, oligonucleotides and peptides. The introduction of treatment with pegylated interferon (IFN) and ribavirin increased the proportion of patients who achieved a sustained antiviral response. Despite this, the toll of patients suffering from HCV infection continues to increase and a proportion fail to respond to the therapy (Bartenschlager, 1997; Pawlotsky, 1999; Mannus et al, 2001). The response of the patients also depends on the viral genotype and non responders to previous IFN-based therapies often fail after re-treatment (Yoshida et al, 2009). Moreover, side effects due to the current therapies add to the complexity.

To date most NS3/4A inhibitors in clinical trials are peptide-based derived from the cleavage products and target the protease cleavage site. In spite all efforts, the virus has been able to successfully overcome these compounds due to the genetic heterogeneity that results in selection of genomes which already contain a resistant imprint (Pawlotsky, 2003; Mirani et al, 2011).

In addition, Valopicitabine was developed against the HCV RNA dependent RNA polymerase (Toniutto et al, 2007) and there are reports of attempts to inhibit HCV replication by several different agents including peptides, small RNA decoy, DNA or RNA aptamers, si RNA, shRNA, Ribozymes, DNAzymes etc. (Dasgupta et al, 2004; Pawlotsky et al, 2007; Roy et al, 2008; Subramanian et al, 2009).

As the genetic variability of the virus enables it to develop antiviral resistance, it becomes a challenge to develop specific HCV inhibitors. Additionally toxicity is another aspect to be taken care of. So, keeping all these limitations in mind it is a need of the hour to come forward with naive anti-HCV agents. Therefore, it is essential to identify newer drug targets and develop more efficacious anti-HCV drugs that are at the same time less toxic.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to various embodiments, some of which are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

FIG. 1 shows the RNA binding residues in the C-terminal end of the NS3-4A as predicted by various prediction tools.

FIG. 2A shows the binding of the C-terminal construct with the RNA-IRES site as determined by the dose dependent shift observed through gel retardation assay.

FIG. 2B shows a graph indicating % translation inhibition obtained with the C-terminal construct of the NS3-4A, N-terminal construct of NS3-4A and the full construct including the C-
terminal construct and the N-terminal construct as determined through translation assay.

FIG.3A shows a schematic representation of a C-terminal peptide obtained from the C-terminal construct obtained from the predictions.

Fig.3B shows a considerable binding of NS3pr0C-C15 to HCV- IRES through UV-crosslinking in tris-tricine gel.

FIG.3C shows the binding of C-terminal peptide to the HCV- IRES site as determined through filter binding assay.

FIG.3D shows a graph indicating % translation inhibition obtained with the C-terminal peptide as determined through translation assay.

FIG.4A shows inhibition of IRES mediated translation by the Hexa-arginine tagged NS3proC-C15 peptide.

FIG.4B shows concentration dependent inhibition of IRES mediated translation by the Hexa-arginine tagged NS3proC-C15 peptide.

FIG.4C shows inhibition of replication by the hexa-arginine tagged NS3proC-C15 peptide as compared to NS3pr0C-N15 peptide, and non specific peptide showed no inhibition.

SUMMARY OF THE INVENTION

One aspect of the invention provides a peptide, derived from the RNA binding region of the NS3-4A, as an inhibitor for targeting the HCV replication.

Yet another aspect of the invention is to provide a delivery mechanism for targeting the peptide derived from the RNA binding region of the NS3-4A for inhibiting the HCV replication.

BRIEF DESCRIPTION OF INVENTION

The definitions, terms and terminology adopted in the disclosure have their usual meaning and interpretations, unless otherwise specified. Various embodiments of the invention provide a peptide derived from the RNA binding region of the NS3 protease domain of the HCV. In an embodiment of the invention the HCV is a wild type virus. The RNA protein interaction between NS3 protein and the highly conserved viral RNA is targeted, the chance of generation of viral escape mutant is very low. The sequence of a stem loop IV (SLIV) located within the IRES element (320-359) near iAUG is highly conserved. One embodiment of the invention includes peptide derived from the RNA binding region of the NS3 protease domain. Thus if the RNA protein interaction between NS3 protein and the highly conserved viral RNA is targeted, the chance of generation of viral escape mutant is very low. The various embodiments and their functioning shall be described herein below. FIG. 1 shows the RNA binding residues in the C-terminal end of the NS3-4A as predicted by various prediction tools. Prediction of probable RNA binding residues is carried out using software prediction tools.

The software tools employed include but are not limited to RNABindR, BindN, and Pprint. The prediction result yields a consensus sequence. The consensus sequence obtained reveals that the RNA binding residues are concentrated in the C terminal half of the NS3-4A and is identified as NS3pr0. NS3pro is expressed as two regions, AN-NS3pro and AC-NS3pro. In an embodiment of the invention, mRNAs are transcribed from different plasmid (what are the plasmids used, what different type of plasmids can be used) constructs. The transcription includes the steps of Labeling the mRNAs with a radioisotope a [32P] UTP; Transcribing the radiolabeled mRNAs in presence of a RNA polymerase to obtain a capped bi-cistronic RNA; and Translating the capped bi-cistronic RNA in presence of a translation mixture to obtain a polypeptide; In an embodiment of the invention the RNA polymerase is a T7 RNA polymerase. The T7 RNA polymerase is obtained from Promega. The radiolabeled mRNAs are labeled with a [32P] UTP and transcribed in vitro using T7 RNA polymerase. In vitro translation of capped bi-cistronic RNA containing the HCV IRES is carried out in the presence of a translation mixture. In an embodiment of the invention, the translation mixture includes nuclease treated rabbit reticulocyte lysate (RRL) (Promega), amino acid mix and RNase inhibitor.

Translation was measured by renilla and firefly luciferase activities according to the dual luciferase assay system as defined by Promega protocol. The obtained polypeptide is tested for Renilla and firefly luciferase activity was measured according to the Promega protocol using the dual luciferase assay system as defined by Promega protocol. The binding of the C-terminal construct with the RNA-IRES is estimated through gel retardation assay. FIG. 2A shows the binding of the C-terminal construct with the RNA-IRES site as determined by the dose dependent shift observed through gel retardation assay. In an embodiment of the invention, the gel retardation assay includes the steps of.

Incubating the a[32P]UTP labeled HCV SLIV RNA with the protein of interest at 30°C for 20 minutes in RNA binding buffer to allow reaction of the components; and loading the reacted components on a gel for electrophoresis.

In an embodiment of the invention, the RNA binding buffer comprises of 5mM HEPES pH 7.6, 25mM KCI, 2mM MgCI2, 3.8% Glycerol, 2mM DTT and 0.1 mM EDTA.. The reactions were loaded on a 4% polyacrylamide gel and separated by electrophoresis at 12mA in 4°C until the dye front reached the lower half of the gel. The gel was then fixed and dried and the results collected using a phosphor imager.

Increasing concentrations of AN-NS3pro showed a dose dependent shift indicating that the C terminal region interacted with HCV IRES RNA whereas the binding was insignificant with AC-NS3pro. We also showed that NS3pro inhibits IRES-mediated translation upon RNA binding (Ray and Das, 2011). As a result, the effect of AN-NS3pro and AC-NS3pro on IRES-mediated translation was determined in vitro using a dicistronic construct in which firefly luciferase translation is controlled by the HCV IRES and renilla luciferase is controlled by cap-dependent translation. An in vitro translation assay showed that AN-NS3pro, by virtue of its binding to the HCV IRES, also showed considerable inhibition of IRES-mediated translation (Fig. 2B). FIG.3A shows a schematic representation of a C-terminal peptide obtained from the C-terminal construct obtained from the predictions. Based upon the predictions as described herein above, a 30 residue peptide (IPVRRRGDSR GSLLSPRPISYLKGSSGGPL) of NS3 (114-143) is designed from the C terminal half of the NS3 protease. The peptide shows significant binding to the HCV IRES in an UV crosslinking assay and the affinity is determined using filter binding assay (Fig 3B, FIG 3C).

In an embodiment of the invention, the a[32P]UTP labeled HCV RNA is incubated with the peptide of interest at 30°C for 15 minutes in RNA binding buffer. The RNA binding buffer includes 5 millimolar (mM) of HEPES at pH 7.6, 25mM of KCI, 2mM of MgCI2, 3.8% Glycerol, 2mM DTT and 0.1 mM EDTA). Subsequent to incubating with the RNA binding buffer, the a[32P]UTP labeled HCV RNA is subjected to loading reactions on a nitrocellulose filter previously equilibrated with RNA binding buffer. In an embodiment of the invention, the loading reaction includes the steps of placing the nitrocellulose filters on the plurality of sockets provided on a filter binding apparatus;preequilibriating each of the nitrocellulose filter with the RNA binding buffer; loading the RNA protein mixture onto the pre- equilibrated nitrocellulose filter; incubating the RNA protein mixture loaded nitrocellulose filter at 25°C - 30°C for at least 4minutes; washing the nitrocellulose filter at least once with the RNA binding buffer and dried. The dried nitrocellulose filter is then measured for retained counts in a scintillation counter. The protein concentration was plotted against the percentage RNA retained on Y axis. Each experiment is repeated at least three times for recording statistically acceptable values.

The effect of the peptide is determined in an in vitro translation assay. The results of the assay show a concentration-dependent inhibition of the translation. In order to study the specificity of
binding and the inhibition, the 30-mer peptide is further split into two peptides of equal length, namely the NS3pr0C-N15 (IPVRRRGDSRGSLLS) and the NS3pr0C-C15 (PRPISYLKGSSGGPL). FIG.3B shows the binding of C-terminal peptide to the HCV-IRES site as determined through filter binding assay.

The NS3proC-C15 peptide shows higher affinity as compared to the NS3proC-N15 peptide structure. The difference in binding affinity is attributed to the rigid structure of NS3proC-N15, which contains a beta hairpin turn. The presence of the beta hairpin turn on the NS3proC-N15 peptide results in decreased interaction of the NS3proC-N15 peptide with RNA, as demonstrated by the reduced binding affinity. The higher affinity of the NS3proC-C15 peptide is attributed to an alpha helical structure formed by the region 131-137 (PISYLKG) which facilitates RNA interaction. The in vitro translation assay shows significant inhibition of IRES mediated translation by the NS3proC-C15 peptide compared to the NS3proC-N15 peptide (Fig.3D).

In another embodiment of the invention, the effect of the NS3proC-C15 peptide and the NS3proC-N15 peptide on HCV IRES translation in mammalian cells is studied. The mammalian cells chosen for the study is a liver cell. In an example of the invention the liver cell is a Huh7 cell. Each of the NS3proC-C15 peptide and the NS3pr0C-N15 peptide is tagged with a hexa-arginine tag. The hexa-arginine tag facilitates peptide transduction. In order to determine specificity of the peptide binding, a randomly generated peptide of the NS3proC-C15 peptide is also tagged with a hexa-arginine tag. In an embodiment of the invention, the process of studying the effect of the NS3proC-C15 peptide on mammalian cells include Tranfecting the RNA into the mammalian cell;
Inducing transduction by adding hexa arginine tagged peptides to the transfected mammalian cell; Harvesting the cells for estimating levels of protein expression; and

Determining enzyme activity for estimating effect of peptides on the mammalian cells; RNA obtained from the bicistronic construct of HCV is transfected into a monolayer of Huh7 cells in a plurality of petri dishes of at least 35mm dia. The medium for transfection included Lipofectamine 2000 reagent (Invitrogen) in antibiotic free medium. The Huh 7 cells had a confluence of at least eighty percent (80%). Alternatively, the RNA can also be transfected into a monolayer of Huh7.5 cells. Subsequent to initiation of transfection into Huh7 cells, the hexa arginine tagged peptides are added to the Huh7 cells. In an embodiment of the invention, the hexa-arginine tagged peptides are introduced into the transfected Huh7 cells after a time interval of four hours from the time of transfection. The medium of introduction of the hexa-arginine tagged peptide into the transfected Huh7 cells includes DMEM supplemented with 10% fetal bovine serum. The cells are harvested after 24 hours later using Tri reagent (Sigma) for RNA preparation and RT-PCR. To investigate levels of protein expression, the cell lysates are prepared using passive lysis buffer (Promega) and luciferase activity measured using the Luciferase assay reagent (Promega).

A toe printing assay is performed to locate putative RNA binding residues of a protein. Unlabeled HCV SLIV RNA is incubated in the presence or absence of increasing concentrations of protein/peptide at 30 C for 20 minutes and then annealed to an end labeled primer (75 fmoles) with sequence complementary to the 3' end of the RNA. The RNA-protein complex was reverse-
transcribed with three units of AMV-RT (Promega) at 45°C for 30 min, followed by denaturation of the AMV-RT at 95 °C for 3 min. The cDNAs were then subjected to phenol chloroform extraction, then precipitated with 0.3 M sodium acetate and 2.5 volumes of absolute alcohol. The pellets were redissolved in nuclease free water and resolved on an 7 M urea-8% acrylamide gel. In parallel, a sequencing reaction using the same end labeled primer was examined by electrophoresis to indicate the exact position of the RT stops.

RNA was isolated from Huh 7 or Huh7.5 cells post transfection and transduction. The RNAs were reverse transcribed with HCV 5' primer and GAPDH 3' primer using MMLV RT (Promega) for amplification of negative strand of HCV RNA. The resulting ; cDNA was used for PCR amplification corresponding to the HCV IRES. The PCR products were examined by gel electrophoresis. GAPDH was used as an internal control. FIG.4A shows inhibition of IRES mediated translation by the Hexa-arginine tagged NS3proC-C15 peptide. The inhibition of the
) IRES mediated translation by NS3proC-C15 peptide is 60% inhibition of IRES mediated translation (Fig. 4A). Increasing concentrations of the NS3proC-C15 peptide showed 80% inhibition of translation (Fig. 4B). Furthermore, the effect of the peptides on HCV replication was confirmed by transient transfection of a HCV monocistonic replicon RNA followed by peptide transduction. Twenty four hours after peptide transduction, total RNA was isolated and the level of the HCV negative strand was measured using semi-quantitative RT-PCR.

C15 peptide showed greater inhibition of IRES-directed translation as compared to N15, and the scrambled C15 peptide showed no inhibition (Fig. 4C).

In an embodiment of the invention, a method for delivery of the peptide into an animal model is also provided. The animal model chosen is mice. In an example of the invention, female BALB/C mice are chosen for delivery of peptide. The above results showed that the C15 peptide was able to bind to the HCV SLIV in the HCV IRES and was able to inhibit HCV IRES-mediated translation. Consequently, the peptide was able to inhibit HCV RNA replication in cell culture system. To determine if the peptide is able to inhibit HCV IRES mediated translation in an animal model, the HCV bicistronic construct and the FITC conjugated C15 peptide were encapsulated in F-virosome of Sendai virus origin. The F protein on the virosome specifically interacts with the asialoglycoprotein receptor on the surface of the mouse hepatocyte thereby leading to membrane fusion and release of the contents in the cytoplasm. The encapsulation of the peptide was confirmed by western blot analysis using anti-NS3 antibody (Fig. 5A). Fusion-mediated delivery of the virosome was confirmed by incubating Huh7 cells with the virosomes for 2 hours at 37°C and subjecting the cells to inverted fluorescence microscopy 24 hours later (Fig. 5C and D). The strong signal obtained as shown by green fluorescence (Fig. 5C and D) indicates that the virosomes could efficiently deliver the peptides. Using the same experimental conditions, luciferase expression was also measured to examine the effect of the peptide on HCV IRES mediated translation. These results suggested -60% reduction in translation (Fig. 5A). The loaded virosomes were tested ex vivo in Huh7 cells before injecting mice. It showed significant inhibition of HCV IRES mediated translation in Huh 7 cells (Fig 5B). F-virosomes loaded with the HCV bicistronic construct and the NS3 peptide were then injected into BALB/c mice via the tail vein. Two days post-injection, the animals were sacrificed, the hepatocytes were isolated and luciferase activity was measured. The C 15 peptide showed approximately 52% inhibition of HCV IRES-mediated translation, but the non specific peptide showed no inhibition (Fig. 5E). Thus the C15 peptide when delivered to the hepatocytes, in which protein expression is controlled by the HCV IRES, is able to inhibit IRES-directed translation and thus reduce the level of expression of a reporter molecule. Monolayers of Huh7 cells were incubated with loaded F-virosomes (0.3 mg F-protein) containing 4 ug of the bicistronic HCV plasmid in the presence or absence of the NS3 peptide or a nonspecific peptide in serum free medium. Two hours post fusion, 10% serum containing DMEM was added and the cells were then incubated for 24 hrs prior to analysis of luciferase expression.

Alternatively, the vehicle for delivery of the C15 peptide can be a chitosan based nanocapsule. Further, the vehicle for delivery can also be dendrimers. Each of the above mentioned vehicle for delivery is explained herein below as alternate embodiments of the invention.

Delivery of C15 peptide preferentially into liver cells using Chitosan based nanocapsules:

In one embodiment of the invention, a biocompatible and biodegradable polyelectrolyte nano-container is obtained. The nano-container is provided with targeting ligands for the efficient internalization and transfection of the C15 peptide in Human hepatocellular carcinoma (Huh7) cells. The method of targeting C15 peptide comprises of:

• Preparing a nano-sized polyelectrolyte capsule by the Layer-by-Layer (LbL) technique.

• Optimizing encapsulation of C15 peptide.

• Surface functionalization of the capsules with a directing ligand, for example, protamine-asialoorosomucoid (PRO-ASOR) for site-specific delivery through receptor mediated endocytosis preferentially to liver cells.

Delivery of NS3 peptide using functionalized dendrimers: Use of liposomes and polymer as non-viral gene delivery vectors have progressed in the past decade. The hyperbranched macromolecule, namely, dendrimers, has evolved as potential high-performance gene delivery vectors in recent years (Paleos 2007). Dendrimers offer the benefits of a macromolecule, yet being mono-dispersed with maximum branching throughout the structure. Cationic poly(amido amine) (PAMAM) dendrimers presenting primary amines at their peripheries were demonstrated early-on for their potential as gene delivery systems, occurring due to facile electrostatic interactions with nucleic acids (Eichman 2000, Maiti 2006, Nandy 2012). Issues related to the toxicity and biocompatibility (Patil 2011, Chen 2010) were also addressed and attempts were made to develop novel dendrimer-based (Patil 2011, Agrawal 2009) delivery systems. For example, surface modification of PAMAM dendrimers with poly(ethylene glycol) not only reduced the toxicity significantly, but also increased transfection efficiency by several folds (Luo 2002, Kim 2004). Dendritic structures have also been used for peptide delivery and the mechanism for such delivery is believed to be very similar to the siRNA delivery (Hamilton 2009).

PETIM dendrimer series represent a class of dendrimers possessing tertiary amine as the branch points, ether as the linkers that connect the branch points through n-propyl spacer. A previous study on this dendrimer having oxygen as the core showed gene transfection abilities and their toxicity profiles surpassed those reported known for other dendrimers (Thankappan2011).

Encouraged by the lower toxicities of this series of dendrimers in comparison to other similar dendrimer types, we undertook further studies, pertaining specifically to queries relating to the complexation with Peptides. We plan to conjugate the galactose attached PETIM and/or PANAM dendrimer with the NS3 peptide to preferentially deliver the antiHCV peptides to liver cells. An embodiment of the invention also provides a method for detecting the HCV infection. "Detecting" or "identifying" as used herein refers to determining the nature or the identity of a condition (say of a disease like HCV infrection). Detection may be accompanied by a determination as to the severity of the . condition. "Quantitating" as used herein refers to determining the number of organisms in the given test sample that is attributing to the condition, for example HCV infection. The detection or quantitating the organisms in the sample can be done by any of the techniques known in the art, for example ELISA, immunofluorescence but not limited to these techniques. As used herein "detecting the presence of virus" refers to a measurable parameter of the presence of binding event between the antibody raised against the peptide and the epitope displayed by the host cell. For example, presence of binding can be detected using several well-recognized binding assays, for example the ELISA described in the further sections. Cell free assays can be used to measure the binding of the peptides of the current invention by various means. Various "means", for example, fluorometric, flow cytometric means may be used. The assays and means mentioned herein are examples, and by no way limiting.

The invention provides a composition method and formulation for a peptide based inhibitor of hepatitis c virus replication. The peptide synthesized is specific to the C-terminal of the HCV NS3 protease. The peptide significantly inhibits the HCV replication through inhibition of IRES mediated translation. The invention also provides a method for delivery of the peptide into a mammalian cell for inhibition of HCV replication. The aforesaid description is enabled to capture the nature of the invention. It is to be noted, however, that the aforesaid description and appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.

References:

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We Claim:

1. A polypeptide for inhibiting HCV replication, wherein the polypeptide is specific to the C-terminal end of the protease.

2. The polypeptide according to Claim 1, wherein the polypeptide is a 15mer.

3. The polypeptide according to Claim 1, wherein the amino acid sequence of the polypeptide is PRPISYLKGSSGGPL.

4. The polypeptide according to Claim 1, wherein the inhibiton of the HCV replication is achieved through inhibition of IRES-mediated translation of the virus.

5. A method for delivery of the polypeptide into a mammalian cell for targeting HCV replication, wherein the method comprises of conjugating the polypeptide with a fluorescent tag;

selecting a bicistronic construct of HCV; and selecting a vehicle for encapsulating the conjugated polypeptide;

6. The method of delivery according to claim 5, wherein the vehicle for encapsulating the polypeptide is selected from the group comprising of virosomes, dendrimers and nanocapsules.

7. The method of delivery according to claim 5, wherein the delivery is a fusion mediated delivery.

8. The method of delivery according to Claim 5, wherein the targeting of HCV replication includes inhibition of IRES-mediated translation of the virus.

9. A polypeptide as described in the specification and as illustrated in the accompany drawings.