Mimicking Dominant Negative Inhibition of Prion Replication Through Structure-Based Drug Design

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Mimicking Dominant Negative Inhibition of Prion Replication Through Structure-Based Drug Design Mimicking dominant negative inhibition of prion replication through structure-based drug design Ve´ ronique Perrier*†‡, Andrew C. Wallace‡§¶ʈ, Kiyotoshi Kaneko*†**, Jiri Safar*†, Stanley B. Prusiner*†§, and Fred E. Cohen*§¶†† *Institute for Neurodegenerative Diseases and Departments of †Neurology, §Biochemistry and Biophysics, and ¶Molecular and Cellular Pharmacology, University of California, San Francisco, CA 94143-0518 Contributed by Stanley B. Prusiner, March 10, 2000 Recent progress determining the structure of the host-encoded sensitive, might enhance clearance of PrPSc (Fig. 1) (8, 19–22). prion protein (PrPC) and the role of auxiliary molecules in prion Compounds that bind to PrPSc and prevent it from serving as a replication permits a more rational approach in the development of template for the replication of nascent PrPSc could be effective. therapeutic interventions. Our objective is to identify a new class This scenario has been suggested as the mechanism of action of of lead compounds that mimic the dominant negative PrPC mu- the amyloid-binding dye, Congo red (23). However, Congo red Sc tants, which inhibit an abnormal isoform (PrP ) formation. A is effective in rodents only if it is administered close to the day computational search was conducted on the Available Chemicals of scrapie injection, and its highly anionic structure is likely to Directory for molecules that mimic both the spatial orientation and interfere with transport across the blood–brain barrier (24). basic polymorphism of PrP residues 168, 172, 215, and 219, which Another strategy might be to prevent the formation of molecular confer dominant negative inhibition. The search revealed 1,000 complexes by interfering with the interactions between protein potential candidates that were visually analyzed with respect to X, PrPC, and PrPSc (Fig. 1). Disrupting the interaction between the structure of this four-residue epitope on PrPC. Sixty-three PrPC and PrPSc with a small molecule is likely to be difficult, as compounds were tested for inhibition of PrPSc formation in scrapie- the PrPSc binding site on the surface of the PrP(90–231) NMR infected mouse neuroblastoma cells (ScN2a). Two compounds, Cp-60 (2-amino-6-[(2-aminophenyl)thio]-4-(2-furyl)pyridine-3,5-di- structure appears to span a rather large area (25). Moreover, carbonitrile) and Cp-62 (N؅1-({5-[(4,5-dichloro-1H-imidazol-1-yl)m- biochemical data suggest that this interaction is of high affinity C͞ Sc ethyl]-2-furyl}carbonyl)-4 methoxybenzene-1-sulfonohydrazide), and thus, small molecules are unlikely to disrupt PrP PrP C inhibited PrPSc formation in a dose-dependent manner and dem- binding. Blocking the interaction between protein X and PrP onstrated low levels of toxicity. A substructure search of the seems to be the best approach for several reasons. The protein C Available Chemicals Directory based on Cp-60 identified five re- X binding site on PrP maps to a small area primarily involving lated molecules, three of which exhibited activities comparable to residues Q168, Q172, T215, and Q219 (14, 26). This region is of Cp-60. Mimicking dominant negative inhibition in the design of special interest as polymorphisms at codon 171(Q3R) in Suf- drugs that inhibit prion replication may provide a more general folk sheep and codon 219(Q3K) in the Japanese population approach to developing therapeutics for deleterious protein– were reported to prevent disease naturally (27–31). When mouse MEDICAL SCIENCES protein interactions. Prnp genes carrying these polymorphisms were expressed in scrapie-infected neuroblastoma (ScN2a) cells, not only did they Sc rions cause fatal neurodegenerative diseases in mammals, not form PrP , but they also blocked formation of wild-type Sc Pincluding Creutzfeldt-Jakob disease (CJD) in humans, bo- PrP , presumably by sequestering protein X (14). The existence vine spongiform encephalopathy (BSE), and scrapie in sheep of a dominant negative phenotype argues that the protein (1). Although significant advances have been made in under- X͞PrPC interaction is the rate-limiting step in PrPSc formation. standing the molecular and cellular basis of prion diseases, these The ability of protein X to be recycled during PrPSc formation efforts have not yet led to promising therapeutic interventions contends that this is a relatively low-affinity interaction that (2). The emergence of more than 50 cases of a new variant CJD could be blocked by a small molecule. in humans that seems to be caused by BSE prions from cattle has We present a structure-based approach to the identification of heightened the urgency for development of effective therapeu- small heterocyclic molecules that were designed to mimic the tics (3–10). More importantly, lessons from the development of dominant negative inhibition of prion replication by polymorphic inhibitors of prion replication are likely to be relevant to the variants of PrP. Although most structure-based drug design focuses creation of therapeutics for other neurodegenerative syndromes. on filling cavities on the surface of a molecule to inhibit an At the molecular level, the prion diseases are characterized by interaction or on optimizing the efficiency of small molecule whose Sc the accumulation of an abnormal isoform (PrP ) of the host- detailed interactions with its molecular target are visualized crys- C encoded prion protein (PrP ) (11, 12). The recent progress in tallographically, we followed a distinctly different route. We sought understanding the tertiary structure of PrPC and the role of auxiliary molecules e.g., protein X, in PrPSc replication permit a more rational approach in the development of therapeutic Abbreviations: PrP, prion protein; PrPC, host-encoded PrP; PrPSc, abnormal isoform of PrP; agents. From a consideration of many transgenic and cellular SHa, Syrian hamster; N2a, mouse neuroblastoma cells; ScN2a, scrapie-infected mouse C Sc neuroblastoma cells; TRF, time-resolved fluorescence; MoPrP, mouse PrP; Cp-60, 2-amino- transfection studies, a model of the conversion of PrP into PrP 6-[(2-aminophenyl)thio]-4-(2-furyl)pyridine-3,5-dicarbonitrile; Cp-62, NЈ1-({5-[(4,5- Sc has emerged (Fig. 1) (13, 14). This model suggests that PrP dichloro-1H-imidazol-1-yl)methyl]-2-furyl}carbonyl)-4 methoxybenzene-1-sulfonohydra- accumulation can be blocked at five logical sites. One approach zide; Cp-18, purine riboside; Cp-32, 2Ј3Ј-di-O-acetyladenosine. C involves the inhibition of PrP synthesis by antisense oligonu- ‡V.P. and A.C.W. contributed equally to this work. cleotides or ribozymes targeted to PrP mRNA and is supported ʈPresent address: Genome Therapeutics Corp., Waltham, MA 02453. by work on knockout MoPrP mice demonstrating that these **Present address: National Institute of Neuroscience, Tokyo 187-8502, Japan. animals are resistant to prion disease (15–17). A second ap- ††To whom reprint requests should be addressed. C proach would be to stabilize the PrP molecule and make the The publication costs of this article were defrayed in part by page charge payment. This conformational change energetically less favorable (18). Alter- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. natively, drugs that destabilize PrPSc, rendering it protease §1734 solely to indicate this fact. PNAS ͉ May 23, 2000 ͉ vol. 97 ͉ no. 11 ͉ 6073–6078 Downloaded by guest on September 26, 2021 the DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl- ammonium methylsulfate) liposomal transfection reagent (Boehringer Mannheim). Compounds at a final concentration of 10 ␮M were applied 24 h after the transfection directly in the cell culture medium over 3 days. Confluent cells then were lysed by addition of 500 ␮l of lysis buffer (10 mM Tris⅐HCl, pH 8͞100 mM NaCl͞0.5% NP-40͞0.5% deoxycholate). The protein concentra- tion of each sample was measured with the BCA reagent (Pierce). Samples of equal protein amounts and volumes then were digested with 20 ␮g͞ml proteinase K at a ratio of 1:25 protease to protein for1hat37°C. Digestions were stopped by 2 mM PMSF, and the samples were ultracentrifuged. Pellets then were resuspended in 20 ␮l of lysis buffer and 20 ␮lofSDS loading buffer and boiled 5 min before loading on 12% SDS͞ PAGE precast Novex gel. Immunoblot analysis was performed according to a protocol described elsewhere (35). The 3F4 mAb was used to detect the MHM2PrP protein on the membrane. Fig. 1. Model of PrPSc formation. Five potential strategies for drug discovery are suggested by this model: 1) block PrPC synthesis, 2) stabilize PrPC,3) Quantification of PrPSc by Immunoassay Using Time-Resolved Fluo- enhance PrPSc clearance, 4) interfere with binding of PrPC to PrPSc, and 5) rescence (TRF) Spectroscopy. Different concentrations of Cp-60 prevent binding of protein X to PrPC. were applied to transiently transfected ScN2a cells according to the same protocol described in the screening procedure. Cells were trypsinized, collected in tubes, and washed twice with 5 ml to mimic the surface of a macromolecule by using a small molecule. of 1ϫ PBS solution. Each sample was lysed with 1 ml of PBS To the extent that our computational algorithm identifies appro- containing 2% sarkosyl. The crude cell extract was sheared with priate mimetics of the PrPC epitope that specifies the dominant Ϫ Sc a syringe and stored overnight at 80°C. Protein concentration negative phenotype, PrP formation will be blocked. Our studies was measured with the BCA reagent and samples were digested demonstrate that it is possible to identify plausible mimetics of a C with proteinase K as described (21). The reaction was stopped by localized epitope on the surface of PrP and that a subset of these a mixture of protease inhibitors (5 mM PMSF, aprotinin, and molecules inhibits prion replication in cultured cells. leupeptin at 4 ␮g͞ml each). Samples were precipitated with a Materials and Methods solution of 4% sodium phosphotungstate (NaPTA) and 34 mM MgCl2 to obtain a final concentration of 0.3% NaPTA.
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