A High-Throughput Drug Screen Targeted to the 5'Untranslated Region of Alzheimer Amyloid Precursor Protein mRNA

Abstract

The authors employed a novel approach to identify therapeutics effective in Alzheimer disease (AD). The 5'untranslated region (5'UTR) of the mRNA of AD amyloid precursor protein (APP) is a significant regulator of the levels of the APP holoprotein and amyloid beta (Aβ) peptide in the central nervous system. The authors generated stable neuroblastoma SH-SY5Y transfectants that express luciferase under the translational control of the 146-nucleotide APP mRNA 5'UTR and green fluorescent protein (GFP) driven by a viral internal ribosomal entry site. Using a high-throughput screen (HTS), they screened for the effect of 110,000 compounds obtained from the library of the Laboratory for Drug Discovery on Neurodegeneration (LDDN) on the APP mRNA 5'UTR-controlled translation of the luciferase reporter. This screening yielded several nontoxic specific inhibitors of APP mRNA 5'UTR-driven luciferase that had no effect on the GFP expression in the stable SH-SY5Y transfectants. Moreover, these compounds either did not inhibit or inhibited to a much lower extent the expression of the luciferase reporter regulated by a prion protein (PrP) mRNA 5'UTR, used as an alternative mRNA structure to counterscreen APP mRNA 5'UTR in stably transfected SH-SY5Y cell lines. The hits obtained from this robust, specific, and highly quantitative HTS will be characterized to identify agents that may be developed into useful future therapeutic agents to limit APP translation and Aβ production for AD.

Keywords

Alzheimer disease APP mRNA 5'UTR HTS luciferase drugs inhibitors

References

Selkoe DJ , Schenk D : Alzheimer's disease: molecular understanding predicts amyloid-based therapeutics. Annu Rev Pharmacol Toxicol 2003;43: 545-584.

Fillit HM , Refolo LM : Advancing drug discovery for Alzheimer's disease. Curr Alzheimer Res 2005;2 (2):105-107.

Selkoe DJ : Normal and abnormal biology of the beta-amyloid precursor protein. Annu Rev Neurosci 1994;17:489-517.

Lichtenthaler SF , Haass C : Amyloid at the cutting edge: activation of α-secretase prevents amyloidogenesis in an Alzheimer disease mouse model. J Clin Invest 2004;113 (10):1384-1387.

Selkoe DJ : Alzheimer's disease: genes, proteins, and therapy. Physiol Rev 2001;81 (2):741-766.

Glenner GG , Wong CW : Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984;120 (3):885-890.

Furukawa K , Sopher BL , Rydel RE , Begley JG , Pham DG , Martin GM , et al: Increased activity-regulating and neuroprotective efficacy of α-secretase-derived secreted amyloid precursor protein conferred by C-terminal heparin-binding domain. J Neurochem 1996;67:1882-1896.

Lahiri DK , Ge YW , Maloney B : Characterization of the APP proximal promoter and 5'-untranslated regions: identification of cell type specific domains and implication in APP gene expression and Alzheimer's disease. FASEB J 2005;19 (6):653-655.

Zuker M : On finding all suboptimal foldings of an RNA molecule. Science 1989;244 (4900):48-52.

10.

Rogers JT , Leiter LM , McPhee J , Cahill CM , Zhan SS , Potter H , et al: Translation of the Alzheimer amyloid precursor protein mRNA is up regulated by interleukin-1 through 5'-untranslated region sequences. J Biol Chem 1999;274 (10):6421-6431.

11.

Lahiri DK , Nall C : Promoter activity of the gene encoding the beta-amyloid precursor protein is up regulated by growth factors, phorbol esters, retinoic acid and interleukin-1. Brain Res Mol Brain Res 1995;32 (2):233-240.

12.

Lahiri DK , Chen D , Vivien D , Ge YW , Greig NH , Rogers JT : Role of cytokines in the gene expression of amyloid β-protein precursor: identification of a 5'-UTR-binding nuclear factor and its implications in Alzheimer's disease. J Alzheimer Dis 2003;5 (2):81-90.

13.

Rogers JT , Randall JD , Cahill CM , Eder PS , Huang X , Gunshin H , et al: An iron-responsive element type II in the 5' untranslated region of the Alzheimer's amyloid precursor protein transcript. J Biol Chem 2002;277 (47):45518-45528.

14.

Maloney B , Ge YW , Greig N , Lahiri DK : Presence of a “CAGA box” in the APP gene unique to amyloid plaque-forming species and absent in all APLP-1/2 genes: implications in Alzheimer's disease. FASEB J 2004;18 (11):1288-1290.

15.

Novac O , Guenier AS , Pelletier J : Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucleic Acids Res 2004;32:902-915.

16.

Werstuck G , Green MR : Controlling gene expression in living cells through small molecule-RNA interactions. Science 1998;282 (5387):296-298.

17.

Maurel MC , Biard B , Moulinier C , Braz D , Nugier J , Chaumas I , et al: RNA-acting antibiotics: in-vitro selection of RNA aptamers for the design of new bioactive molecules less susceptible to bacterial resistance. J Pharm Pharmacol 2002;54:1019-1031.

18.

Lind KE , Du Z , Fujinaga K , Peterlin BM , James TL : Structure-based computational database screening in vitro assay, and NMR assessment of compounds that target TAR RNA. Chem Biol 2002;9:185-193.

19.

Hamy F , Felder ER , Heizmann G , Lazdins J , Aboul-ela F , Varani G , et al: An inhibitor of the Tat/TAR RNA interaction that effectively suppresses HIV-1 replication. Proc Natl Acad Sci U S A 1997;94:3548-3553.

20.

Rogers JT , Jeffrey DR , Eder PS , Huang X , Bush AI , Tanzi RE , et al: Alzheimer's disease drug discovery targeted to the APP mRNA 5'untranslated region. J Mol Neurosci 2002;19 (1-2):77-82.

21.

Payton S , Cahill C , Randall J , Gullans S , Rogers JT : Drug discovery targeted to the Alzheimer's APP mRNA 5'-untranslated region: the action of paroxetine and dimercaptopropanol. J Mol Neurosci 2003;20 (3):267-275.

22.

Morse LJ , Payton SM , Cuny GD , Rogers JT : FDA preapproved drugs targeted to the translational regulation and processing of the amyloid precursor protein. J Mol Neurosci 2004;24 (1):129-136.

23.

Tucker S , Ahl M , Bush A , Westaway D , Huang X , Rogers JT : Pilot study of the reducing effect on amyloidosis in vivo by three FDA pre-approved drugs via the Alzheimer's APP 5' untranslated region. Curr Alzheimer Res 2005;2 (2):249-254.

24.

Kretzschmar HA , Stowring LE , Westaway D , Stubblebine WH , Prusiner SB , Dearmond SJ : Molecular cloning of a human prion protein cDNA. DNA 1986;5 (4):315-324.

25.

Liao YC , Lebo RV , Clawson GA , Smuckler EA : Human prion protein cDNA: molecular cloning, chromosomal mapping, and biological implications. Science 1986;233 (4761):364-367.

26.

Hsiao K , Baker HF , Crow TJ , Poulter M , Owen F , Terwilliger JD , et al: Linkage of a prion protein missense variant to Gerstmann-Straussler syndrome. Nature 1989;338 (6213):342-345.

27.

Venti A , Giordano T , Eder P , Bush AI , Lahiri DK , Greig NH , et al: The integrated role of desferrioxamine and phenserine targeted to an iron-responsive element in the APP-mRNA 5'-untranslated region. Ann N Y Acad Sci 2004;1035:34-48.

28.

Lipinski CA , Lombardo F , Dominy BW , Feeney PJ : Experimental and computational approaches to estimate solubility and permeability in compound discovery and development settings. Adv Drug Deliv Rev 2001;46 (1-3):3-26.

29.

Bakshi P , Liao YF , Gao J , Ni J , Stein R , Yeh L-A , et al: A highthroughput screen to identify inhibitors of amyloid β-protein precursor processing. J Biomol Screen 2005;10 (1):1-12.

30.

Liu Y , Lashuel HA , Choi S , Xing X , Case A , Ni J , et al: Discovery of inhibitors that elucidate the role of UCH-L1 activity in the H1299 lung cancer cell line. Chem Biol 2003;10 (9):837-846.

31.

Case A , Ni J , Yeh LA , Stein RL : Development of a mechanism-based assay for tissue transglutaminase—results of a high-throughput screen and discovery of inhibitors. Anal Biochem 2005;338:237-244.

32.

Ahn JS , Musacchio A , Mapelli M , Ni J , Scinto L , Stein R , et al: Development of an assay to screen for inhibitors of tau phosphorylation by cdk5. J Biomol Screen 2004;9 (2):122-131.

33.

Zhang JH , Chung TD , Oldenberg KR : A simple statistical parameter for use in evaluation and validation of high-throughput screening assays. J Biomol Screen 1999;4 (2):67-73.

34.

Lundholt BK , Scudder KM , Paliaro L : A simple technique for reducing edge effect in cellbased assays. J Biomol Screen 2003;8 (5):566-570.

35.

Hegde RS , Tremblay P , Groth D , DeArmond SJ , Prusiner SB , Lingappa VR : Transmissible and genetic prion disease share a common pathway of neurodegeneration. Nature 1999;402 (6763):822-826.

36.

Collinge J , Sidle KC , Meads J , Ironside J , Hill AF : Molecular analysis of prion strain variation and aetiology of “new variant” CJD. Nature 1996;383 (66020):685-690.

37.

Mahal SP , Asante EA , Antoniou M , Collinge J : Isolation and functional characterization of the promoter region of the human prion protein gene. Gene 2001;268 (1-2):105-114.

38.

Luck R , Steger G , Riesner D : Thermodynamic prediction of conserved secondary structure: application to the RRE element of HIV, the tRNA like element of CMV and the mRNA of prion protein. J Mol Biol 1996;258 (5):813-826.

39.

Jackson GS , Murray I , Hosszu LL , Gibbs N , Waltho JP , Clarke AR , et al: Location and properties of metal-binding sites on the human prion protein. Proc Natl Acad Sci U S A 2001;98:8531-8535.

40.

Purdey M : Ecosystems supporting clusters of sporadic TSEs demonstrate excesses of the radical-generating divalent cation manganese and deficiencies of antioxidant co factors Cu, Se, Fe, Zn. Does a foreign cation substitution at prion protein's Cu domain initiate TSE? Med Hypotheses 2000;54 (2):278-306.

41.

Dedeoglu A , Cormier K , Payton S , Tseitlin KA , Kremsky JN , Lai L , et al: Preliminary studies of a novel bifunctional metal chelator targeting Alzheimer's amyloidogenesis. Exp Gerontol 2004; 39 (11-12): 1641-1649.