FR2815358A1 - Screening for polypeptide having mutant RNA recognition site, useful for controlling viral infections, especially hepatitis C, by binding to internal ribosome entry site - Google Patents

Abstract

Screening for recombinant polypeptide (I) that contains at least one mutant RNA recognition motif (mMRR), from a protein naturally able to bind RNA, that binds to an IRES (internal ribosome entry site), or one of its domains, in an RNA molecule with affinity at least equal to that of the native motif (nMRR). Screening for recombinant polypeptide (I) that contains at least one mutant RNA recognition motif (mMRR), from a protein naturally able to bind RNA, that binds to an IRES (internal ribosome entry site), or one of its domains, in an RNA molecule with affinity at least equal to that of the native motif (nMRR). At least one nMRR is cloned, subjected to mutagenesis, then (I) expressed and the affinity of (I) for IRES (or its domains) determined. (I) with the required affinity are selected. An Independent claim is also included for (I) produced by the new method.

Description

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The present invention aims to provide a method intended for screening for polypeptides capable of being used as a medicament for treating hepatitis C. More particularly, the present invention relates to a new method for screening for recombinant polypeptides comprising at least one recognition motif d Mutated RNA from a naturally occurring RNA-binding protein, such as eIF3, said motif being capable of binding to the IRES sequence of a viral RNA molecule with an affinity greater than or equal to that of the motif (MRR ) of said protein which naturally binds said RNA molecule. The invention also relates to said recombinant polypeptides capable of being obtained by the screening method which can be used as a medicament for the prevention and / or treatment of viral pathologies, in particular hepatitis C.

 In 1989, the hepatitis C virus (HCV) was identified as the causative agent of non-A, non-B hepatitis transmitted by blood transfusions. HCV belongs to the family of Flaviridae in which we can cite the Yellow Fever, Dengue and Japanese Encephalitis viruses. The genome of this enveloped virus is in the form of a single strand of RNA containing a reading frame and two 5 'and 3' untranslated regions.

 Like HIV or the influenza virus, HCV has great variability. The rapid mutation of some of its genes, notably the envelope gene, allows this virus to escape from the host's immune system and makes vaccine development problematic. The most preserved area is the

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région 5'non-codante (IRES-International Ribosome Entry Site) (92% d'homologie entre les souches de VHC), ce qui laisse suggérer un rôle important de cette région dans le cycle viral. En effet, cette région possède une structure secondaire qui permet au virus d'initier la traduction de son ARN d'une façon capindépendante. Les protéines virales sont synthétisées sous forme d'un seul polypeptide-précurseur qui est ensuite clivé par des protéases cellulaires et virales.

5 ′ non-coding region (IRES-International Ribosome Entry Site) (92% homology between the strains of HCV), which suggests an important role for this region in the viral cycle. Indeed, this region has a secondary structure which allows the virus to initiate the translation of its RNA in a capindependent manner. Viral proteins are synthesized as a single precursor polypeptide which is then cleaved by cellular and viral proteases.

 At present, the treatment of hepatitis C is based on the administration of interferon (inhibition of protein synthesis via PKR), combined with ribavirin (nucleoside analogue). Several undesirable effects of these two compounds arise from the non-specific nature of their action. In addition, certain virus genotypes are not very sensitive to these substances, which motivates research into new, more specific anti-viral agents. Among those which are developed, mention may be made of (i) inhibitors of viral enzymes, such as antiproteases, inhibitors of helicase and of polymerase; (ii) anti-sense oligonucleotides, capable of binding to the internal ribosome entry site (IRES, for Internal Ribosome Entry Site) and thus of inhibiting the synthesis of viral proteins, represent another class of potential therapeutics; this approach made it possible to clearly demonstrate that the IRES located in the non-coding region of HCV RNA can be considered as an attractive therapeutic target, since various previous genetic and biochemical studies have demonstrated that IRES is essential for viral translation.

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However, the use of oligonucleotides in vivo remains fairly random because of their rapid degradation by host nucleases and their low intracellular penetration. There is therefore a real need to develop a new class of anti-viral therapeutic molecules capable of binding to IRES and which is not sensitive to nucleases.

 The present invention therefore proposes to provide a method for screening for recombinant polypeptides which specifically bind the IRES of uncapped viral RNA, such as HCV viral RNA. Such polypeptides, capable of specifically recognizing IRES of viral RNA are therefore capable of being used as inhibitors of the synthesis of viral proteins in vitro and in vivo.

Such polypeptides do not have the drawbacks of therapeutic molecules of the prior art. The present invention further relates to the polypeptide capable of being selected by the screening method as a medicament.

moins un motif de reconnaissance d'ARN (MRR) muté d'une protéine liant naturellement l'ARN, ledit motif étant capable de se lier à la séquence IRES ou l'un de ces domaines d'une molécule d'ARN avec une affinité supérieure ou égale à celle du motif (MRR) de ladite protéine liant naturellement ladite molécule d'ARN, ledit procédé comportant les étapes de : a) clonage d'au moins le motif (MRR) de ladite protéine liant naturellement l'ARN.

b) mutagénèse d'au moins ledit motif (MRR), More specifically, the method of screening for recombinant polypeptides of the invention comprising at least

at least one mutated RNA recognition motif (MRR) of a naturally occurring RNA-binding protein, said motif being capable of binding to the IRES sequence or one of these domains of an RNA molecule with affinity greater than or equal to that of the motif (MRR) of said protein which naturally binds said RNA molecule, said method comprising the steps of: a) cloning at least the motif (MRR) of said protein which naturally binds RNA.

b) mutagenesis of at least said motif (MRR),

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c) expression dudit polypeptide recombinant comprenant au moins le motif (MRR) muté, d) détermination de l'affinité dudit motif (MRR) muté dudit polypeptide recombinant pour ladite séquence IRES ou l'un de ses domaines ; e) sélection des polypeptides recombinants dont le motif (MRR) muté a une affinité supérieure ou égale à celle du motif (MRR) de ladite protéine liant naturellement l'ARN.

c) expression of said recombinant polypeptide comprising at least the mutated motif (MRR), d) determination of the affinity of said mutated motif (MRR) of said recombinant polypeptide for said IRES sequence or one of its domains; e) selection of recombinant polypeptides whose mutated motif (MRR) has an affinity greater than or equal to that of the motif (MRR) of said protein which naturally binds RNA.

l'ARN telles le facteur initiateur de la traduction chez les eucaryotes eIF3 (Birney E, 1993) et UlsnRNP ainsi que les régulateurs de traduction tels que la Polyadenylate binding protein (PABP). By RNA recognition motif (MRR) is intended to denote a motif shared by a large number of proteins which naturally bind RNA. Among these proteins, mention may be made of the maturation proteins of

RNA such as the translation initiating factor in eukaryotes eIF3 (Birney E, 1993) and UlsnRNP as well as translation regulators such as Polyadenylate binding protein (PABP).

 The consensus sequence of the MRR, of approximately 80 amino acids, is characterized by the presence of two highly conserved sequences RNP1 (8AA) and RNP2 (6AA).

peptidique se fait dans l'ordre Pl--al-p2-03-a2-B4 La structure tri-dimensionnelle de plusieurs MRR a été résolue par radiocristallographie et RMN. Elle se présente généralement sous forme d'un feuillet ss de quatre brins, contenant les signatures RNP-1 et RNP-2 et de deux hélices a. Ces données structurales ont permis d'identifier des domaines de la protéine impliqués dans l'interaction avec l'ARN. Ainsi, les boucles ss2-ss3 et a2-ss4 se déplacent lors de la formation The arrangement of secondary structures on the sequence

peptide is done in the order P1 - al-p2-03-a2-B4 The three-dimensional structure of several MRRs was resolved by X-ray crystallography and NMR. It is generally in the form of a sheet of four strands ss, containing the signatures RNP-1 and RNP-2 and two propellers a. These structural data made it possible to identify domains of the protein involved in the interaction with RNA. Thus, the loops ss2-ss3 and a2-ss4 move during training

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du complexe polypeptide-ARN et par conséquent peuvent être impliquées dans l'interaction avec l'ARN. Les données obtenues grâce à la mutagenèse dirigée dans la boucle ss2-ss3 ont démontré que la taille de cette boucle est importante pour la stabilité du complexe MRR-ARN (Laird-Offringa et al., 1995). Un modèle mécanistique d'interaction a été suggéré et compare la boucle protéique P2-03avec un bouton dont la taille détermine la stabilité de la fermeture.

of the polypeptide-RNA complex and therefore may be involved in the interaction with RNA. The data obtained by site-directed mutagenesis in the ss2-ss3 loop have demonstrated that the size of this loop is important for the stability of the MRR-RNA complex (Laird-Offringa et al., 1995). A mechanistic interaction model has been suggested and compares the P2-03 protein loop with a button whose size determines the stability of the closure.

 According to a particular embodiment, said recombinant polypeptide according to the invention is isolated by the technique called phage display also called molecular evolution.

In this technique, the protein of interest is fused with one of the proteins of the phage capsid and thus exposed on the surface of the bacteriophage. Systematic variations in the protein gene sequence create a library of different phages exposing different molecular forms of the protein on its surface. The aim of the approach is to select and amplify phage particles capable of clinging to a selected ligand and makes it possible to choose the peptides capable of specifically recognizing the target.

 The most striking application of the phage display technique is the design of new proteins capable of interacting in a specific way with DNA. A protein domain called "zinc fingers" capable of binding to DNA can be exposed on the surface of the bacteriophage capsid and mutated (Klug, 1999). Selection-amplification cycles of phage particles capable of clinging to different

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séquences d'ADN ont permis d'exprimer des motifs "doigts de zinc"capables de reconnaître les séquences de l'ADN très spécifiquement. Plusieurs études effectuées dans ce domaine ont permis de déduire"le code de reconnaissance"reliant la séquence d'acides aminés caractéristiques des motifs"doigts de zinc" (environ 20 AA) et celle des bases d'acides nucléiques (triplets) (Wolfe et al., 1999). La combinaison de plusieurs"doigts de zinc"différents permet ainsi de concevoir rationnellement des polypeptides aptes à se lier à une séquence choisie de l'ADN.

DNA sequences have made it possible to express "zinc finger" motifs capable of recognizing DNA sequences very specifically. Several studies carried out in this field have made it possible to deduce "the recognition code" linking the amino acid sequence characteristic of the "zinc finger" motifs (approximately 20 AA) and that of the nucleic acid bases (triplets) (Wolfe and al., 1999). The combination of several different "zinc fingers" thus makes it possible to rationally design polypeptides capable of binding to a chosen sequence of DNA.

 The new proteins thus designed then have a considerable affinity and specificity vis-à-vis the ligand (Ko <2.1 x 10-15M) (Kim et al., 1998).

 The use of phage display technology allows rapid obtaining of many mutated polypeptides specific for the IRES sequence of interest, preferably the IRES sequence of the hepatitis C virus.

 The term IRES sequence is intended to denote the 5 'untranslated region of certain viral RNAs (Flaviviridae, Picornaviridae, Coronaviridae) which have a typical secondary structure which serves as a recruitment site for ribosomes. This region allows the viruses concerned to initiate the translation of their RNA in a CAP-independent manner. Preferably, said IRES sequence according to the invention, or one of its domains, is the IRES sequence of a viral RNA. The domain of the IRES sequence is preferably chosen from the nucleic sequences directly in contact with the MRR.

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Said IRES sequence or one of its domains comes from viral RNA of viruses which, during their viral replication cycle, use a cap-independent translation step of a viral RNA sequence. Preferably, said virus is an RNA virus; preferably, said virus belongs to the family of Flaviviridae, Picornaviridae, Coronaviridae.

 Among the Flaviviridae, mention should be made in particular of the human hepatitis C virus (HCV), the bovine diarrhea virus (BVDV), the swine fever virus. According to a preferred embodiment of the invention, it is the hepatitis C virus.

 Among the Picornaviridae, mention should be made of (i) Enteroviruses, in particular human polioviruses, Coxsackieviruses, human Echoviruses, pigs and cattle; (ii) Rhinoviruses, and in particular the cold virus; (iii) Aphtoviruses, in particular the foot-and-mouth disease virus; (iv) cardioviruses, in particular the encephalomyocarditis virus (EMC), the Mengo virus; (v) hepatitis A virus.

 Among the Coronaviridae, mention should be made of the transmissible piglet gastroenteritis virus, the avian bronchitis virus, the calf diarrhea virus, the human diarrhea virus, the hepatitis virus of the mouse.

 In the context of the present invention, the IRES sequence is preferably the IRES sequence of the hepatitis C virus RNA of sequence SEQ ID N 1, and the domains of the IRES sequence are preferably, the sequence IIIa loop. SEQ ID NO2, the IIIb loop of

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séquence SEQ ID N 3, la boucle IIIc de séquence SEQ ID ? 4.

sequence SEQ ID N 3, the loop IIIc of sequence SEQ ID? 4.

By IRES sequence within the meaning of the present invention is also intended to denote the natural variants of the IRES sequences and in particular the natural variants of the IRES sequence of the HCV. The term “natural variant” is intended to denote the IRES sequences originating from the genetic polymorphism of the population of viral RNAs; these natural IRES sequence variants do not have a substantially modified activity compared to a consensus wild IRES sequence.

 The term IRES sequence domain is intended to denote a sequence fragment which has the capacity to link the MRR motif of an RNA-binding protein.

la protéine p110 à se fixer sur la séquence IRES de l'ARN viral du virus de l'hépatite C. Cette protéine eIF3pno se fixe spécifiquement sur la partie apicale de la région III de l'IRES du VHC et notamment sur les boucles IIIb ou IIIc (Sizova et al., 1998). D'après According to a preferred embodiment, said protein which naturally binds RNA is selected from the group composed of RNA processing proteins and factors regulating translation; more particularly, they are the ribonucleoproteins snRNP and hnRNP and the subunits of the eukaryotic translation initiation factor 3 (eIF3). According to a particular embodiment, the protein which naturally binds RNA molecules is the multiprotein factor initiating human eukaryotic translation 3 (eIF3). More particularly, the protein is the P110 protein (eIF3pno) of the eIF3 complex. The inventors have demonstrated the ability to

the protein p110 to bind to the IRES sequence of the viral RNA of the hepatitis C virus. This protein eIF3pno binds specifically to the apical part of region III of the HCV IRES and in particular to the IIIb loops or IIIc (Sizova et al., 1998). According to

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l'analyse de la séquence en acides aminés, la protéine eIF3pllo (SEQ ID NO 5 ; NO d'accession AAB42010 dans la banque NCBI) possède un"Motif de Reconnaissance"d'ARN dans sa partie centrale (MRR). Ce domaine (SEQ ID NO 6, correspondant aux acides aminés 175 à 283 de la séquence SEQ ID NO 5) est donc responsable de la fixation de la protéine eIF3pllO sur la boucle III de l'IRES du VHC.

analysis of the amino acid sequence, the protein eIF3pllo (SEQ ID NO 5; accession NO AAB42010 in the NCBI library) has a "Reason for Recognition" of RNA in its central part (MRR). This domain (SEQ ID NO 6, corresponding to amino acids 175 to 283 of the sequence SEQ ID NO 5) is therefore responsible for the binding of the protein eIF3pl10 on loop III of the HCV IRES.

d'acides aminés 175 à 283 de la séquence SEQ ID NO 5 ou l'un de ses fragments ou l'un de leurs variants naturels. Par fragment du MRR, on entend désigner un fragment de la séquence d'acides aminés du MRR qui conserve l'aptitude à se lier à une séquence IRES. De préférence, le fragment du MRR selon l'invention correspond à la séquence d'acides aminés 185 à 270 de la séquence SEQ ID NO 5. Par variants naturels, on entend désigner les MRR présentant des variations dans la séquence du MRR par rapport à la séquence consensus sauvage, de telles variations représentant les polymorphismes rencontrés dans la population et qui ne changent pas de manière substantielle l'affinité dudit MRR pour sa séquence cible. According to a preferred embodiment, said RNA recognition motif (MRR) is the MRR of eIF3plo of sequence SEQ ID? 6 corresponding to the sequence

amino acids 175 to 283 of the sequence SEQ ID NO 5 or one of its fragments or one of their natural variants. By fragment of the MRR is meant a fragment of the amino acid sequence of the MRR which retains the ability to bind to an IRES sequence. Preferably, the fragment of the MRR according to the invention corresponds to the amino acid sequence 185 to 270 of the sequence SEQ ID NO 5. By natural variants is meant the MRR having variations in the sequence of the MRR with respect to the wild consensus sequence, such variations representing the polymorphisms encountered in the population and which do not substantially change the affinity of said MRR for its target sequence.

 Mutations are introduced into the MRR of the polypeptide of the invention in order to increase the affinity of the MRR for the target IRES sequence. Such mutations are introduced by random mutagenesis, or by targeted mutagenesis according to techniques known to those skilled in the art. The use of the phage display technique allows the introduction of a large number of

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mutation de manière alétoire dans ledit MRR et ainsi permettre d'obtenir rapidement un grand nombre de mutants à cribler.

mutation in a random manner in said MRR and thus make it possible to rapidly obtain a large number of mutants to be screened.

 The term “mutation” is intended to denote any changes which have occurred in the sequence of the MRR of the polypeptide according to the invention and in particular of the MRR of eIF3pllo, other than the changes corresponding to those of the natural variants, and which substantially modify the affinity of the polypeptide according to the invention to said IRES sequence. Among the mutations capable of being introduced into the MRR of the polypeptide according to the invention, mention should be made of point mutations, deletions, insertions, substitutions. However, it is advisable to select only the mutations introduced into the MRR of a protein which binds naturally the RNA and which confer on this mutated MRR an affinity greater than or equal to the affinity of the non mutated MRR of the same naturally binding protein. RNA.

est le MRR de eIF3plo de séquence SEQ ID NO 6 (séquence d'acides aminés 175 à 283 de la séquence SEQ ID NO 5) ou l'un de ses fragments ou de leurs variants naturels. De préférence, il s'agit du fragment d'acides aminés

185 à 270 de la séquence SEQ ID NO 5 du MRR de eIF3pno ; ledit MRR de eIF3pno, ou l'un de ses fragments, ou leurs variants est (sont) muté (s) à au Preferably, the mutations introduced into the MRR are located in the motifs directly in contact with the RNA, that is to say preferably in the very conserved sequences RNP1 and RNP2, and more particularly in the ss2-ss3 and a2-ss4 of MRR. According to a preferred mode, the MRR according to the invention

is the MRR of eIF3plo of sequence SEQ ID NO 6 (amino acid sequence 175 to 283 of the sequence SEQ ID NO 5) or one of its fragments or their natural variants. Preferably, it is the amino acid fragment

185 to 270 of the sequence SEQ ID NO 5 of the MRR of eIF3pno; said MRR of eIF3pno, or one of its fragments, or their variants is (are) mutated to at

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moins une des positions suivantes de la séquence SEQ ID No 5. la valine en position 188, le glutamate en position 224 et 225, l'aspartat en position 226 et 251, la glycine en position 227 et 252, la lysine en position 228 et 248, la tyrosine en position 232 et 253, la phénylalanine en position 234, - l'asparagine en position 249, - l'alanine en position 250.

minus one of the following positions of the sequence SEQ ID No 5. valine in position 188, glutamate in position 224 and 225, aspartate in position 226 and 251, glycine in position 227 and 252, lysine in position 228 and 248, tyrosine in position 232 and 253, phenylalanine in position 234, - asparagine in position 249, - alanine in position 250.

 The invention also relates to the recombinant polypeptide capable of being obtained by the method according to the invention. Said polypeptide comprises at least one MRR motif. According to a preferred embodiment, the polypeptide according to the invention comprises at least the minimal motif of functional RNA recognition (MRR).

 It is within the scope of the invention to produce fusion proteins with said polypeptide of the invention to improve, for example, the purification, the solubility, the bioavailability, the lifetime, the targeting of the polypeptide according to the invention.

 According to a preferred embodiment, the polypeptide of the invention corresponds to at least the mutated MRR of eIF3pnoOul'one of its fragments or one of their natural variants. More particularly, the polypeptide according to the invention comprises the amino acid sequence 185 to 270 of the sequence SEQ ID NO 5 in which mutations have been introduced at the positions mentioned above.

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The polypeptide of the invention is a recombinant protein which is capable of interacting with the IRES sequence of RNA viruses with an affinity greater than or equal to that of cellular or viral proteins which bind naturally to this sequence during viral infection . The IRES sequence indeed has the ability to form a secondary loop structure with which the polypeptide of the invention is capable of interacting. Such interactions are easily demonstrated by a person skilled in the art by RNA gel delay experiments and / or by UV coupling experiments. The polypeptide of the invention therefore constitutes a competitive inhibitor of naturally binding proteins. on the viral IRES sequence, among which eIF3 should be mentioned. The polypeptide of the invention decreases or inhibits the replication of viral RNA.

 The polypeptide according to the invention is therefore an active principle intended to be used as a medicament and more particularly as a medicament for the treatment of viral pathologies involving a virus, which during its cycle of viral replication, implements a CAP-independent translation step of an RNA sequence. Among these viruses, mention should be made of the previously mentioned viruses. In general, the viral pathology which the polypeptide according to the invention proposes to treat corresponds to the pathology involving the virus of which the IRES sequence of the RNA, or one of its domains, was used in the screening process according to the invention. Preferably, said virus is the hepatitis C virus.

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C'est donc un objet de la présente invention de fournir un polypeptide selon l'invention à titre de médicament destiné à inhiber la traduction du génome viral, et ainsi empêcher sa réplication. Le polypeptide selon l'invention à titre de médicament est préférentiellement sous forme soluble, associés à un véhicule pharmaceutiquement acceptable. Par véhicule pharmaceutiquement acceptable, on entend désigner tout type de véhicule employé habituellement dans la préparation de compositions injectables, c'est-à-dire un diluant, un agent de suspension tel une solution saline isotonique ou tamponnée. De préférence, ces composés seront administrés par voie systémique, en particulier par voie intraveineuse, par voie intramusculaire, intradermique ou par voie orale. Leurs modes d'administration, posologies et formes galéniques optimaux peuvent être déterminés selon les critères généralement pris en compte dans l'établissement d'un traitement adapté à un patient comme par exemple l'âge ou le poids corporel du patient, la gravité de son état général, la tolérance au traitement et les effets secondaires constatés, etc. Quand l'agent est un polypeptide, on peut l'introduire dans des tissus ou des cellules hôtes par un certain nombre de façons, incluant la micro-injection ou la fusion de vésicules.

It is therefore an object of the present invention to provide a polypeptide according to the invention as a medicament intended to inhibit the translation of the viral genome, and thus prevent its replication. The polypeptide according to the invention as a medicament is preferably in soluble form, associated with a pharmaceutically acceptable vehicle. The term “pharmaceutically acceptable vehicle” is intended to denote any type of vehicle usually used in the preparation of injectable compositions, that is to say a diluent, a suspending agent such as an isotonic or buffered saline solution. Preferably, these compounds will be administered by the systemic route, in particular by the intravenous route, by the intramuscular, intradermal route or by the oral route. Their optimal modes of administration, dosages and dosage forms can be determined according to the criteria generally taken into account in establishing a treatment adapted to a patient such as for example the patient's age or body weight, the severity of his general condition, tolerance to treatment and side effects observed, etc. When the agent is a polypeptide, it can be introduced into host tissues or cells in a number of ways, including microinjection or fusion of vesicles.

 It is therefore one of the objects of the present invention to use a polypeptide comprising at least one RNA recognition motif (MRR) of a protein which naturally binds RNA to inhibit CAP-independent translation of a coding region of an IRES sequence of said RNA sequence. More particularly, the present invention proposes to use the polypeptide according to the invention for the preparation of a medicament

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destiné au traitement de pathologies virales impliquant des virus qui au cours de leur cycle de réplication virale mettent en oeuvre une étape de traduction CAPindépendante d'une séquence d'ARN. Parmi ces virus, il convient de citer les virus précédemment évoqués et sélectionnés parmi les Flaviviridae, les Picornaviridae, les Coronaviridae. Ledit Flaviriridae étant de préférence choisi parmi le virus de l'hépatite C, le virus de la peste porcine, le virus de la diarrhée bovine. De préférence, ledit Flaviviridae est le virus de l'hépatite C.

intended for the treatment of viral pathologies implying of the viruses which during their cycle of viral replication implement a step of CAPindependent translation of a sequence of RNA. Among these viruses, mention should be made of the viruses previously mentioned and selected from Flaviviridae, Picornaviridae, Coronaviridae. Said Flaviriridae being preferably chosen from the hepatitis C virus, the swine fever virus, the bovine diarrhea virus. Preferably, said Flaviviridae is the hepatitis C virus.

 The polypeptide according to the invention is used for the preparation of a medicament intended for the treatment of hepatitis C, bovine diarrhea, calf diarrhea, human diarrhea, swine fever, gastroenteritis transmissible from piglets, foot and mouth disease, encephalo-myocarditis (CME), hepatitis A, mouse hepatitis, colds, avian bronchitis.

 More particularly, the polypeptide according to the invention is used for the preparation of a medicament intended for the treatment of hepatitis C.

 Finally, the invention aims to provide a pharmaceutical composition for the preventive and curative treatment of a pathology chosen from hepatitis C, bovine diarrhea, calf diarrhea, human diarrhea, swine fever, transmissible gastroenteritis of piglets, foot and mouth disease, encephalo-myocarditis (CME), hepatitis A, mouse hepatitis, colds, avian bronchitis, characterized in that it contains a therapeutically effective amount of a polypeptide

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 according to the invention and a pharmaceutically acceptable vehicle.

 Other characteristics and advantages of the present invention will be better demonstrated on reading the following examples.

Figure 1: The amino acid sequence of the RNA recognition motif (MRR) of the protein eIF3pllo.

 The areas of the protein involved in the interaction with RNA that are likely to be mutated are in larger letters.

Figure 2: Structure of the HCV IRES
Enlarged, the structure of the interaction site with eIF3pllo- is shown. Figure 3: Diagram of the IIIb loop synthesis of the HCV IRES in vitro (Kruger et al.,
1999)

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EXEMPLES

EXEMPLE 1 : Clonage et expression du fragment polypeptidique correspondant au motif MMR du eIF3plo.

EXAMPLES

EXAMPLE 1 Cloning and expression of the polypeptide fragment corresponding to the MMR motif of eIF3plo.

5'-TCAGTGGTGGTGGTGCTCGAGTCGACCTCGTCACTGATCGTCAT (SEQ ID NO 8). Le fragment amplifié est coupé et cloné dans le plasmide pET-30B (NOVAGEN) en fusion avec His-tag sur C-terminus coupé au préalable avec NdeI et Xhol. La protéine est produite dans E. coli (souche BL21 LysS) et purifiée sur la colonne de Ni2±NTA agarose. 1.1 The DNA fragment coding for amino acids 185-270 of eIF3pllo (SEQ ID NO 5) and corresponding to a fragment of the RNA recognition motif, is amplified by PCR using human DNA extracted from cells of the peripheral blood as matrix and primers 5'-GGAGATATACATATGAGCCAGGAAGCAGATGGA (SEQ ID NO 7) and

5'-TCAGTGGTGGTGGTGCTCGAGTCGACCTCGTCACTGATCGTCAT (SEQ ID NO 8). The amplified fragment is cut and cloned into the plasmid pET-30B (NOVAGEN) in fusion with His-tag on C-terminus cut beforehand with NdeI and Xhol. The protein is produced in E. coli (strain BL21 LysS) and purified on the column of Ni2 ± NTA agarose.

 1.2. Cytotoxicity of the eIF3pno fragment.

 Before studying the antiviral activity of the polypeptide, its possible toxic effect is evaluated by staining with Trypan blue. Possible undesirable effects (non-specific effects on protein synthesis) are estimated by measuring the incorporation of methionine [S35] into the proteins of HeLa cells.

EXAMPLE 2:
2.1 Cloning of HCV IRES (40-372 nt)

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L'ADNc correspondant à l'IRES du VHC minimal requis pour la traduction cap-indépendante comprenant les nucléotides 40-372 de l'ARN viral du VHC, est amplifié par RT-PCR à partir de sérum provenant de patients VHC-positifs et cloné dans un vecteur pGEM-luc (Promega) sous le contrôle du promoteur T7. La transcription est effectuée in vitro en utilisant T7polymérase et le plasmide linéarisé par NdeI.

The cDNA corresponding to the minimum HCV IRES required for cap-independent translation comprising nucleotides 40-372 of the viral HCV RNA, is amplified by RT-PCR from serum from HCV-positive and cloned patients. in a vector pGEM-luc (Promega) under the control of the T7 promoter. Transcription is carried out in vitro using T7polymerase and the plasmid linearized by NdeI.

2.2. Summary of the different parts of IRES
Previous work shows that eIF3pllo is attached to part III of the IRES (Sizova et al., 1998).

Knowing that the MRR domains recognize RNA sequences up to 20 bases in length, the inventors have located the interaction site more precisely by using oligoribonucleotides corresponding to loops IIIa, IIIb and IIIc. Instead of cloning, the inventors use a faster method previously developed for ribozymes. Two overlapping oligonucleotides, one of which contains the T7 polymerase promoter and the other an IRES sequence, are used to synthesize a double stranded fragment using the Klenow fragment.

The linear double-stranded DNA fragment is used as a template for the synthesis of RNA in vitro. The same procedure will be used for the synthesis of loops IIIa and IIIc (using the corresponding oligonucleotides) (Figure 3).

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EXEMPLE 3 : Etude de l'affinité du polypeptide pour l'ARN viral
L'affinité du polypeptide pour l'IRES est mesurée in vitro par retard de l'ARN sur gel d'acrylamide (gel

shift). L'ARN marqué par [32p] lors de la transcription est incubé avec le polypeptide en présence de Mg2+ (2, 5 mM) nécessaire pour maintenir sa structure tertiaire stable (Kieft et al., 1999) et déposé sur gel d'acrylamide (8%). Les gels sont analysés par Phosphorimager STORM (Amersham).

EXAMPLE 3 Study of the Affinity of the Polypeptide for Viral RNA
The affinity of the polypeptide for IRES is measured in vitro by delay of the RNA on acrylamide gel (gel

shift). The RNA labeled with [32p] during transcription is incubated with the polypeptide in the presence of Mg2 + (2.5 mM) necessary to maintain its tertiary structure stable (Kieft et al., 1999) and deposited on acrylamide gel ( 8%). The gels are analyzed by Phosphorimager STORM (Amersham).

EXAMPLE 4 The anti-viral activity of the polypeptide
The ability of the polypeptide to inhibit IRES-dependent translation is estimated in vitro. The fragment of the HCV cDNA comprising the nucleotides 40-372 is recloned in fusion with the luciferase gene under the T7 promoter. The effect of the polypeptide is evaluated in vitro by following the inhibition of the synthesis of luciferase in the lysate of reticulocytes.

EXAMPLE 5 Structural of the polypeptide-RNA complex
NMR studies of the polypeptide-RNA complex make it possible to precisely identify the sites of these interactions and thus locate sites of mutations capable of increasing the affinity of the two partners. The structure of the complex between the protein

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et l'ARN est étudiée par RMM. Ces analyses structurales sont effectuées en parallèle avec la recherche par "phage display"qui débute à partir d'un modèle théorique. Compte tenu de la taille du domaine (environ 90 acides aminés), un marquage isotopique à l'azote-15 permet de cartographier ("mapper") le site d'interaction de la protéine avec la boucle d'ARN de façon simple par l'analyse des variations des déplacements chimiques des fonctions amides du squelette peptidique.

and the RNA is studied by RMM. These structural analyzes are carried out in parallel with research by "phage display" which starts from a theoretical model. Given the size of the domain (approximately 90 amino acids), isotopic labeling with nitrogen-15 makes it possible to map ("map") the site of interaction of the protein with the RNA loop in a simple manner by l analysis of variations in the chemical shifts of the amide functions of the peptide skeleton.

EXAMPLE 6 Site-directed mutagenesis of the polypeptide and selection of mutants with high affinity for viral RNA (phage display)
In order to obtain a library of the polypeptide fragment corresponding to the MRR motif of eIF3pllo, the inventors use the RPAS system (Amersham) conventionally intended for the construction of banks of variable fragments of antibodies. The DNA fragment coding for the polypeptide corresponding to amino acids of sequence 185-270 of the sequence SEQ ID? 5 is recloned in fusion with the gene for protein III of the capsid of phage M13 in a phagemid (pCANTAB 5E, Amersham). The cells transfected with phagemid and infected with phage-helper will be selected thanks to their resistance to ampicillin (phagemid) and kanamycin (phage helper). The phage particles produced by the selected cells will express MRR on their surface. The extracellular localization of the

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MRR sera confirmée par la capacité des particules phagiques de fixer l'ARN radiomarqué.

MRR will be confirmed by the ability of phage particles to bind radiolabelled RNA.

 To prepare the library of MRR mutants, each amino acid in the ss2-ss3 loop (5 amino acids) involved in the interaction with RNA, will be "randomized" by PCR amplification (using an oligonucleotide-primer in which each codon to be mutated will be exchanged for a degenerate codon). This PCR amplified fragment will be inserted into a phagemid instead of the "wild" fragment. Thus prepared, the phagemid library will be introduced into E. coli and amplified. Infection of cells with phage-helper will allow production of the phage library of fusion.

 The selected loop RNA is immobilized on streptavidin beads using complementary biotinylated oligodeoxynucleotides. These beads are used to capture phages expressing mutants of MRR motifs having a strong affinity for RNA (the selection is carried out in the presence of an excess of tRNA).

Captured phage clones are amplified and reselected. The clones selected after 3-4 cycles are sequenced. Sequence comparison makes it possible to identify amino acids promoting interaction with the target RNA.

 The corresponding polypeptides are expressed and purified. Their affinities for viral RNA, their specificity as well as the structure of the RNApolypepdid complexes are studied as described above. The same procedure is used to build other combinatorial libraries by introducing a

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mutation au niveau de la boucle a2-ss4 (6 acides aminés) ou de certaines positions dans RNP1 et RNP2.

mutation at the level of the a2-ss4 loop (6 amino acids) or of certain positions in RNP1 and RNP2.

EXAMPLE 7 Study of the affinity for the viral RNA of the designed neo-polypeptides
The genes of the polypeptides having a strong affinity for viral RNA are cloned in fusion with His-tag, expressed in E. coli and purified. Their affinity and specificity for the target RNA, as well as their capacity to inhibit protein synthesis are studied in vitro as described above.

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 REFERENCES Kieft et al., J. Mol. Biol. 292 (3): 513-529 Kim et al., 1998, Proc. Natl. Acad. Sci. 95: 2812-2817 Klug, 1999, J. Mol. Biol. 293: 215-218 Krüger et al., 1999, Methods in Enzymology pp207-225 Laird-Offringa et al., 1995, Proc. Natl. Acad. Sci.

 92 (25): 11859-11863 Sizova et al., 1998, J. Virol. 72: 4775-4782 Wolfe et al., 1999, J. Mol. Biol. 285: 1917-1934 Xu et al., 1997, Structure 5: 559-570

Claims (25)

 1. A method of screening for recombinant polypeptides comprising at least one RNA recognition motif (MRR) mutated from a protein which naturally binds RNA, said motif being capable of binding to the sequence IRES or one of these domains of an RNA molecule with an affinity greater than or equal to that of the motif (MRR) of said protein naturally binding said RNA molecule, said method comprising the steps of: a) cloning at least the motif (MRR) of said protein which naturally binds RNA. b) mutagenesis of at least said motif (MRR), c) expression of said recombinant polypeptide comprising at least the mutated motif (MRR), d) determination of the affinity of said mutated motif (MRR) of said recombinant polypeptide for said IRES sequence or one of its fields; e) selection of recombinant polypeptides whose mutated motif (MRR) has an affinity greater than or equal to that of the motif (MRR) of said protein which naturally binds RNA.  2. Method according to claim 1, characterized in that said steps b, c and d are carried out using phage display technology.  3. Method according to claims 1 or 2, characterized in that said IRES sequence is the IRES sequence of a viral RNA. <Desc / Clms Page number 24>

4. Method according to claim 3, characterized in that said viral RNA comes from RNA virus selected from the group of Flaviviridae, Picornaviridae, Coronaviridae.  5. Method according to claim 4, characterized in that said Flaviviridae is chosen from the hepatitis C virus, bovine diarrhea virus, swine fever virus.  6. Method according to claim 5, characterized in that said Flaviviridae is the hepatitis C virus.  7. Method according to claim 6, characterized in that said IRES sequence of said hepatitis C virus has the sequence SEQ ID Null.  8. Method according to claim 6, characterized in that said domain of the IRES sequence of the hepatitis C virus is chosen from loop IIIa of sequence SEQ ID NO2, loop IIIb of sequence SEQ ID NO3, loop IIIc of sequence SEQ ID N 4.  9. Method according to one of the preceding claims, characterized in that said protein which binds RNA naturally is selected from the group consisting of RNA processing proteins and factors which regulate translation. <Desc / Clms Page number 25>

10. The method of claim 9, characterized in that said RNA processing protein is selected from the group consisting of UlsnRNP and eIF3pno.  11. Method according to claim 10, characterized in that said recognition pattern

 of RNA (MRR) of said protein eIF3pno is the amino acid sequence 175 to 283 of the sequence SEQ ID NO 5, or one of its fragments or their natural variants. 12. Method according to claim 11, characterized in that said fragment of said MRR is the amino acid sequence 185 to 270 of the sequence SEQ ID NO 5.  13. Method according to claims 11 or 12, characterized in that said MRR eIF3pllo or one of its fragments, or their variants is (are) mutated (s) to au

 at least one of the following positions of the sequence SEQ ID NO 5: - valine in position 188, - glutamate in position 224 and 225, - aspartate in position 226 and 251, - glycine in position 227 and 252, - la lysine in position 228 and 248 - tyrosine in position 232 and 253, - phenylalanine in position 234, - asparagine in position 249, - alanine in position 250.  14. Recombinant polypeptide capable of being obtained by the method according to any one of claims 1 to 13. <Desc / Clms Page number 26>

15. The polypeptide according to claim 14, characterized in that said polypeptide comprises at least one minimal motif of functional RNA recognition (MRR).  16. A polypeptide according to claims 14 or 15, characterized in that the sequence of said polypeptide comprises the amino acid sequence 185 to 270 of the sequence SEQ ID NO 5 mutated at at least one of the following positions: valine in position 188, glutamate in position 224 and 225, aspartate in position 226 and 251, glycine in position 227 and 252, lysine in position 228 and 248 tyrosine in position 232 and 253, phenylalanine in position 234, - asparagine in position 249, - alanine in position 250.  17. A polypeptide according to any one of claims 14 to 16 as a medicament.  18. The polypeptide according to claim 17 as a medicament for the treatment of viral pathology involving a virus which during its cycle of viral replication implements a CAP-independent translation step of an RNA sequence.  19. A polypeptide according to claim 18, characterized in that said virus is the hepatitis C virus. <Desc / Clms Page number 27>

20. Use of a polypeptide according to claims 14 to 19 for the preparation of a medicament intended to inhibit CAP-independent translation of a coding region of an RNA.  21. Use of the polypeptide according to claims 14 to 20 for the preparation of a medicament intended for the treatment of viral pathologies involving viruses which during their cycle of viral replication implement a step of CAP-independent translation of a sequence RNA.  22. Use according to claim 21, characterized in that said virus selected from Flaviviridae, Picornaviridae, Coronaviridae.  23. Use according to claim 22, characterized in that said Flaviviridae is chosen from the hepatitis C virus, bovine diarrhea, calf diarrhea, human diarrhea, swine fever, gastric Communicable enteritis of piglets, foot and mouth disease, encephalomyocarditis, hepatitis A, mouse hepatitis, colds, avian bronchitis.  24. Use according to claim 23, characterized in that said Flaviviridae is the hepatitis C virus.  25. Pharmaceutical composition for the preventive and curative treatment of a pathology chosen from <Desc / Clms Page number 28>  hepatitis C, bovine diarrhea, foot-and-mouth disease, encephalomyocarditis, hepatitis A, colds, swine fever, characterized in that it contains a therapeutically effective amount of a polypeptide according to claims 14 to 19 and a pharmaceutically acceptable vehicle.