Patent Publication Number: US-8541166-B1

Title: Peptide sequences for binding infectious prions

Description:
RELATED APPLICATIONS 
     This application claims priority to U.S. Provisional Patent Application Ser. No. 61/101,447, filed Sep. 30, 2008 the contents of which are incorporated herein by reference in their entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to novel peptide sequences that specifically bind infectious prion protein. 
     BACKGROUND OF THE INVENTION 
     Transmissible spongiform encephalopathies are incurable, fatal neurodegenerative diseases characterized by the accumulation of abnormal prion protein (PrPsc), neuronal cell death and vacuolation of brain tissue (1). The PrPsc protein is extractable from diseased tissue and is distinguished from endogenous PrPc by partial protease resistance and detergent insolubility (2). The transmissible agent is the PrPsc protein and it serves as a template for the molecular conversion of endogenous host PrPc into the abnormal PrPsc structural isoform (3, 4). Host expression of PrPc is necessary for disease transmission, as ablation of the PrPc gene prevents disease (5) whereas the over expression of PrPc followed by PrPsc challenge accelerates disease (6, 7). The molecular events that mediate neuronal PrPc to PrPsc conversion, not simply accumulated PrPsc, appears to be the initiating factor mitigating the neurodegenerative disease process (8, 9). Bovine Spongiform Encephalopathy (BSE) is one example of many prion diseases that include Scrapie in sheep and goats, chronic wasting disease (CWD) in deer and elk, and Kuru and Creutzfeldt-Jakob disease (CJD) in humans. Prions are thought to propagate in infected hosts by a novel mechanism that occurs when normal prion protein (PrPc) becomes “misfolded” into an abnormal infectious form (PrPsc), in a template-driven process. Unlike conventional microbes, prions do not require an agent-specific nucleic acid in order to multiply. The protein nature of prions makes them difficult to detect and nearly impossible to treat and control. 
     Current technologies inadequately address the inherent problems in prevention of PrPsc transmission, early detection and decontamination from animals and environmental by-products. Fundamentally, these issues reflect the fact that both PrPc and PrPsc have identical primary structures, differing in three-dimensional configuration only. To date no unique antibodies or surrogate markers have been identified that can distinguish between PrPc and PrPsc. This has created an extremely difficult and challenging dilemma in regard to the detection, vaccination and treatment of PrPsc (5). 
     Definitive diagnosis of PrPsc in animals requires assessment of clinical symptoms and post-mortem evaluation of brain tissue for spongiform degeneration. Pre-clinical assessment of animal status via non-invasive sampling has proven difficult and unreliable. The most definitive method to confirm the presence of infectious PrPsc requires an arduous animal bioassay. Biochemical assays all utilize a complex sequence of steps applied to tissue samples that exploit the resistance of PrPsc, relative to PrPc, to enzymatic degradation by Proteinase K (PK). Confirmation of PrPsc is defined by the detection of remaining PK resistance protein by a PrPc antibody (5-6). These methods detect only PK-resistant PrPsc, are prone to variability, and are inadequate for detection of low level PrPsc in contaminated samples such as blood. 
     SUMMARY OF THE INVENTION 
     Method of identifying peptides that specifically interact with intact prion isoforms (PrPsc) or native PrPc proteins is disclosed herein. 
     Identification of protein and peptide sequences that specifically bind to PrPsc, PrPc or both are also disclosed herein. 
     Methods of detecting prions in potentially infected animal and environmental samples are also disclosed. 
     Methods for diagnostic detection of prion diseases in biological samples using identified protein and peptides markers as surrogate analytes. 
     Methods for therapeutic intervention in prion diseases using identified protein and peptides as biological targets for pharmaceutical drugs, immunomodulation, and other chemical agents. 
     Methods for the development of novel drugs or reagents that modulate identified protein and peptide targets for the detection and treatment of prion diseases. 
     Methods for the capture, isolation, enrichment and inactivation of prions from biological and environmental samples. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 . is a picture of the isolation and enrichment of prion in detergent resistant membranes (drm) from hamster brain homogenate. 
         FIG. 2  is a photo of specific prion binding to WW domain peptide. 
         FIG. 3  is a photo of specific prion binding to PDZ peptides. 
         FIG. 4  is a photo of specific prion binding to PDZ peptides. 
         FIG. 5  is a photo of specific prion binding to SH3 peptides. 
         FIG. 6  is a photo of specific prion binding to SH3 peptides. 
         FIG. 7  is a photo of specific prion binding to SH2 peptides. 
         FIG. 8  is a diagram of the assay strategies for detection of infectious prions using selective binding to synthetic peptides of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The terminology used in the description of the invention herein is for describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. 
     Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth as used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated, the numerical properties set forth in the following specification and claims are approximations that may vary depending on the desired properties sought to be obtained in embodiments of the present invention. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical values, however, inherently contain certain errors necessarily resulting from error found in their respective measurement. 
     Herein is described a novel approach to identify peptides that specifically interact with intact PrPsc or PrPc protein isoforms. 
     We isolated both PrPc and PrPsc biochemically within detergent resistant membranes (DRMs) ( FIG. 1 ), used this material to probe peptide libraries that correspond to defined functional domains of whole proteins, and identified novel peptides that bind specifically to PrPsc and PrPc ( FIGS. 2-7 ). The peptides spotted in these arrays correspond to the specific functional domains of known proteins. These families of functional domains mediate protein-protein interaction within cells and are involved in intracellular trafficking and signal transduction. Importantly, the binding specificity observed in our data likely reflects the three-dimensional conformational differences between PrPsc and PrPc. Moreover, the conformational endpoint of a specific ‘strain’ of infectious prion would likely show a distinct peptide-binding fingerprint useful in identification. 
     An embodiment is unique peptide sequences that specifically bind to PrPc, PrPsc or both using peptide arrays on PVDF membranes. 
     Another embodiment of the invention is the use of unique peptide sequences as arrays that specifically bind PrPsc and isoforms thereof in detection assays of potentially infected animal and environmental samples. 
     Peptides with selective binding for PrPsc, PrPc or both would be used to develop a sample evaluation array allowing for rapid detection of infectious PrPsc and strain identification. Variations of these arrays can be employed as: 1) spots on PVDF membrane, 2) peptide bound ELISA plates, 3) affinity columns, and 4) labeled molecular probes ( FIG. 8 ). Peptide affinity columns could be used to enrich for PrPsc from low level samples such as body fluids. Direct labeled peptide probes could be used in concert with prion antibodies to identify PrPsc. 
     These assays may be independent or combined with current methodologies used for the detection of infectious prions. Moreover, utilization of specific qualitative binding differences would allow for capture and enrichment of PrPsc from samples (i.e. body fluids) from pre-clinical animals with low-level contaminant (5-50 fmol) below current assay detection limits. 
     Method of identifying peptides that specifically interact with intact prion isoforms (PrPsc) or native PrPc proteins is disclosed herein. PrPc derived sequences are available in bacterial and mammalian cell expression vectors and recombinant protein can be produced and purified resulting in source of PrPc protein and peptide for interaction. As PrPsc is a conformational isoform of PrPc and cannot be produced by recombinant technology. Some work to produce isoforms of PrPsc in vitro have been demonstrated { }. Here we outlined a methodology to enrich and purify both PrPc and PrPsc from tissue. This method uses the detergent resistant properties of PrPsc and its association with specialized membrane microdomains. Disruption of tissue in detergent at 4 C is followed by high speed centrifugation in a discontinuous sucrose gradient 5-40%; a process that allows for the isolation of PrPc and PrPsc with detergent resistant membranes (DRMs). This method allows for the enrichment of both native PrPc and infectious PrPsc effectively removing &gt;99.75% of contaminating proteins. PrPc, but not PrPsc, can be proteolytically digested in DRMs by addition of proteinase-K (15 ug/ml 1 h at 60 C) with only PK-resistant PrPsc remaining in the sample. The interaction of peptide or protein domains with either PrPc, PrPsc or both can be determined by differential comparison. Briefly, DRMs from normal tissue containing only PrPc, DRMs from infected tissue digested with PK containing only PrPsc are incubated with protein or peptide sequences (ie. attached to membranes or microwell plates) allowed to interact then probed with antibodies against the PrP protein and positive interactions determined (ie. horseradish peroxidase [HRP] conjugated antibodies followed by chemiluminescent detection [ECL]). Comparative analysis of positive interactions would allow for the identification of specific prion interactions (ie. PrPc negative but PrPsc positive). 
     Identification of protein and peptide sequences that specifically bind to PrPsc, PrPc or both are also disclosed herein. Using the methodology describe above, recombinant peptides corresponding to known functional domains spotted on PVDF membranes (Panomics) and specific interactions determined. The following sequences were identified: 
     Methods of detecting prions in potentially infected animal and environmental samples are also disclosed. Prion specific peptides can be readily adapted to current protein detection strategies. These sequences can be directly or indirectly labeled (enzymatic or dyes) and used as diagnostic probes functioning in a manner similar that of an antibody. Prion specific sequences could be attached to a micro-well plate to capture prion from biological or environmental samples and then probed with prion specific reagents (peptides or antibodies) for detection. 
     Methods for diagnostic detection of prion diseases in environmental or biological samples using identified protein and peptides markers as surrogate analytes. Prion specific proteins and peptides may be either physiological or non-physiological. Physiological prion specific protein interactions maybe used as surrogate biomarkers for the detection of prions. For example a complex formed between specifically between a prion and unique peptides may allow the peptide to serve as a surrogate biomarker for the indirect detection of prions via antibody based detection of the surrogate peptide. If the peptide sequence in not physiological (ie not found in the sample) it could be added to either a biological or environmental sample, allowed to interact with prion, then detected with an antibody that binds to the peptide sequence. Antibodies against prion specific peptides in conjunction with antibodies against the prion protein could be combined with current detection methods (ELISA) to provide a multi-labeled detection strategy. Prion and PrPc specific peptides could be added in proprietary multi-valent combinations along with corresponding antibodies for detection prion isoforms. Direct labeling of prion specific peptides or indirect targeting using surrogate reagents (ie antibodies) could be used for clinical detection of prion disease by imaging. 
     Methods for therapeutic intervention in prion diseases using identified protein and peptides as biological targets for pharmaceutical drugs, immunomodulation, and other chemical agents. Physiological proteins or peptides that specifically interact with prion could be direct targets of pharmaceutical drugs. Disruption of the physiological interaction of prions or PrPc with associated proteins or peptides may be used to disrupt, cure or alter prion diseases. Protein or peptides sequences specific for PrPc or Prion may be modified as to serve as targeted carrier of therapeutic drugsor chemical agents (quiocrine, amphotericin D, beta-sheet breakers or death domains) that disrupt, destroy or impede the propagation of prions in those inflicted or exposed to prions. Vaccination or antibody production against prion specific peptide and prion-peptide complex maybe used to generate a therapeutic immune response for treatment or cure of disease. 
     Methods for the development of novel drugs or reagents that modulate identified protein and peptide targets for the detection and treatment of prion diseases. Prion specific peptide complexes may serve as molecular targets for the generation of specific antibodies. These antibodies may then be used alone or with the addition of peptide to biological or environmental samples for the detection of prions. Antibodies generated against specific prion-peptide complexes may be used as reagents for diagnosis of disease and detection of prion in biological and environmental samples. Specific physiological prion-peptide complexes associated with disease may be used as targets for the development of therapeutic drugs. 
     Methods for the capture, isolation, enrichment and inactivation of prions from biological and environmental samples. Prion specific sequences may be used in the capture and enrichment of prion from biological or environmental samples. These peptides maybe covalently attached to a matrix (filter) and samples applied in small or large volumes to deplete prion from sample. Moreover this approach can be used to enrich prions from samples for detection following elution. Prion specific peptides may be bound as spots to membranes (PVDF or Nitrocellulose) or attached to micro-well plates to generate an array of protein interactors capable of distinguishing prion isoforms by distinct pattern of interactions from biological and environmental samples. Specific peptide sequences may bind and inactivate the conformational conversion of PrPc to PrPsc effectively inactivating prions. These peptides may be used directly or with modification for direct treatment of prion diseases.