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Screening molecules with anti-prion activity: kits, methods and screened moleculesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test StripScreening molecules with anti-prion activity: kits, methods and screened molecules description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070031821, Screening molecules with anti-prion activity: kits, methods and screened molecules. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a Continuation-In-Part of U.S. application Ser. No. 10/531,594, filed Nov. 28, 2005, which is the national phase of PCT/FR03/003101 filed Oct. 20, 2003, which claims priority of French Applications FR02/13022 filed Oct. 18, 2002 and FR03/08289 filed Jul. 7, 2003, which are hereby incorporated by reference in their entireties and are relied upon. DRAWINGS [0002] FIG. 1 relates to the feasibility of the screen. [0003] FIG. 2 illustrates the screening protocol. [0004] FIG. 3 relates to the isolation of the Kastellpaolitines, phenanthridine and to their structure/activity relationship. [0005] FIG. 4 relates to the determination of the activity of the phenanthridine derivatives. [0006] FIG. 5 shows the results of the liquid curing tests. [0007] FIG. 6 relates to the secondary screen based on the [URE3] prion. [0008] FIG. 7 demonstrates the validation of the test with chlorpromazine, quinacrine, and verapamil. [0009] FIG. 8 shows the results of the effect of KP1 on the mammal prion in an in vitro model. [0010] FIG. 9 relates to a structure/activity study carried out on the molecule of general formula (II). [0011] FIG. 10 concerns an immunoblot (a) and a filter retardation assay (b) showing a dose dependent decrease of the soluble (a) and aggregated (b) pathogenic fragment of Huntingtin, Htt48, in cells treated with 6-aminophenanthridine (6AP). [0012] FIG. 11 relates to an immunoblot (a) showing a reduced accumulation of the N-terminal fragment of Huntingtin, T73 in NG108-15 cells upon treatment with 6AP and a quantification of the signal (b) corresponding to T73 in 6AP treated cells. DETAILED DESCRIPTION [0013] The present invention relates to screening of molecules with anti-prion activity. It relates more particularly to kits for screening molecules with anti-prion activity, methods of screening, and a family of molecules with anti-prion activity revealed using the screen according to the invention. [0014] Prions are infectious proteins responsible for certain neuro-degenerative diseases of spongiform encephalopathy type in mammals, such as Creutzfeldt-Jakob's disease in humans or also the so-called "mad cow disease" in bovines or "scrapie" in ovines. These different diseases are caused by unconventional infectious agents: unlike traditional infectious agents (bacteria, viruses for example), they contain no nucleic acids. Professor Stanley Prusiner formulated the "protein-only" hypothesis, according to which the infectious agent would be constituted only by a protein. This protein exists naturally in cells in a normal (or PrP.sup.c) form, i.e. soluble, essentially in the form of an .alpha. helix and non-aggregated, therefore functional. Under certain still unknown conditions, this protein can be converted to a prion (or PrP.sup.sc) form. In this prion form, the protein forms insoluble aggregates, essentially in the form of .beta. sheets. The infectious character of this PrP.sup.sc prion conformation would result from the fact that, apart from the characteristics indicated previously, the protein in prion form also gains the ability to catalyze the passage from the normal Prp.sup.c cell form to the PrP.sup.sc prion form in a "snowball"-type mechanism. [0015] Baker's yeast Saccharomyces cerevisiae contains several proteins that behave like prions (Fernandez-Bellot and Cullin, 2001). Since as long ago as the 1960s, two unconventional genetic mechanisms have been described. In 1994, the corresponding [PSI+] and [URE3] phenotypes were proposed as resulting from the autocatalytic inactivation of the Sup35p and URE2p proteins respectively. These prion proteins therefore have .alpha. priori a mechanistic analogy with mammal systems deleterious to public health. Like the PrP protein, the "normal" Sup35p protein passes from a soluble state to an insoluble and aggregated state as soon as the protein is in contact with another Sup35p protein in prion form. This aggregated state is verified both by centrifugation experiments and by intracellular localization experiments. Yeast prions can be eliminated ("cured") by a strong dose (1 to 5 mM) of guanidium chloride. As a result of such a treatment (which must applied to at least six to ten generations), the protein aggregates generated by the presence of the prions disappear and the protein in question (Sup35p, for example) is found in a normal, soluble, functional form but having retained the capability of being converted to a prion form should it again come into contact with another Sup35p protein in such a state. [0016] The Sup35p protein, in a heterodimeric complex with the Sup45p protein, forms a translation termination factor. This factor recognizes the opal stop codons (UGA). In its normal cell form (soluble and active) in the [psi-] strains, Sup35p, in combination with Sup45p effectively terminates translation at the level of these opal codons. In a [PSI+] strain where the Sup35p protein is in prion form, it is mostly present in the form of insoluble aggregates. Being unable to bind to Sup45p, it is thus non-functional in the translation termination. A small fraction of all of the cellular Sup35p proteins however remains soluble in these [PSI+] cells where it makes it possible, in a complex with Sup45p, to ensure a "minimum translation termination service", a service essential to the survival of the yeast. A colorimetric system making it possible to detect, in an indirect fashion, the form in which the Sup35p protein is present: normal or prion, has been produced from these findings. This system, which has been described for a long time (see the article on synthesis by Fernandez-Bellot and Cullin, 2001), is based on the use of the adel-14 allele of the ADE1 gene, coding for an enzyme of the adenine biosynthesis route: SAICAR synthetase. This enzyme catalyzes the formation of 4-(N-succinocarboxamide)-5-aminoimidazole ribonucleotide (SAICAR) from 4-carboxy-5-aminoimidazole ribonucleotide (CAIR). The adel-14 allele contains an opal codon in the reading frame of the ADE1 gene. In a [psi-] strain, Sup35p in combination with Sup45p will therefore stop the translation of the ADE1 gene at the level of this stop codon. The protein adel-14p thus translated will be truncated and therefore non-functional. As a result the substrates upstream of the Ade1p enzyme will accumulate, in particular the 5-aminoimidazole ribonucleotide (AIR). The AIR being oxidized to a red-coloured compound, the colonies formed by the [psi-] cells will be red in colour. Moreover, these cells will be auxotrophic for adenine. Conversely, in a [PSI+] strain, the protein Sup35p is essentially present in the form of aggregates therefore incapable of being combined with Sup45p in order to stop translation at the level of the opal codon of the adel-14 allele of the ADE1 gene. As a result, the ribosomes will pause at the level of this stop codon before resuming their translation activity (readthrough). A certain quantity of functional Ade1p protein will therefore be synthesized, the cells will be autotrophic for adenine and will form white to pink-coloured colonies. [0017] In an article which appeared in P.N.A.S, Prof. Stanley Prusiner's team discloses a test for detecting molecules with anti-prion activity (Korth et al., 2001). This test is carried out on a mammal model (murine neuroblastomas infected with PrP.sup.sc). The safety conditions (P3 laboratory) and cell culture conditions (significant handling) do not allow high-throughput screening to be carried out. [0018] The Application WO 98/30909 also describes a process for screening molecules with anti-prion activity carried out on rodents infected with an unconventional transmissible agent. This screening method has the same limits as the method described in P.N.A.S. [0019] The inventors' work has led them to produce a high-throughput screening system in order to detect molecules possessing an anti-prion activity, based on the colorimetric reporter system of the protein Sup35p, described above. [0020] The present invention therefore relates to a kit for screening molecules with an anti-prion activity, characterized in that it comprises in combination a yeast of phenotype [PSI+], an antibiogram and a prion curing agent in sub-effective doses, said yeast having the adel-14 allele of the ADE1 gene as well as an inactivated ERG6 gene. [0021] Although based on yeast prions, the kit according to the invention makes it possible to isolate molecules active against mammal prions. Example 7 below shows that the most active molecules isolated by Prof. Prusiner also have an activity in the screen according to the invention. Continue reading about Screening molecules with anti-prion activity: kits, methods and screened molecules... Full patent description for Screening molecules with anti-prion activity: kits, methods and screened molecules Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Screening molecules with anti-prion activity: kits, methods and screened molecules patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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