Discovery and characterization of novel drugs for treatment of Alzheimer's disease from a high

179 Pages·2014·32.25 MB·English

by  Honarnejad, Kamran

LUDWIG-MAXIMILIANS-UNIVERSITÄT MÜNCHEN
GRADUATE SCHOOL OF SYSTEMIC NEUROSCIENCES

PHD THESIS

DISCOVERY AND CHARACTERIZATION OF NOVEL DRUGS
FOR TREATMENT OF ALZHEIMER’S DISEASE FROM A HIGH-
THROUGHPUT COMPOUND SCREEN

KAMRAN HONARNEJAD

2014

DISCOVERY AND CHARACTERIZATION OF NOVEL DRUGS
FOR TREATMENT OF ALZHEIMER’S DISEASE FROM A HIGH-
THROUGHPUT COMPOUND SCREEN

Dissertation
an der Graduate School of Systemic Neurosciences
der Ludwig–Maximilians–Universität München

vorgelegt von

Kamran Honarnejad
aus Teheran

München, 2014

Erstgutachter: Prof. Dr. Jochen Herms
Zweitgutachter: Prof. Dr. Jacek Kuźnicki

Tag der mündlichen Prüfung: 05.08.2014

TABLE OF CONTENTS

1 SUMMARY ............................................................................................................................... 1
2 INTRODUCTION .................................................................................................................... 5
2.1 ALZHEIMER’S DISEASE .......................................................................................................... 5
2.2 CALCIUM SIGNALING IN HEALTH AND DISEASE ................................................................... 7
2.3 IMPAIRMENT OF CALCIUM HOMEOSTASIS IN ALZHEIMER’S DISEASE ................................ 7
2.4 APP PROCESSING AND AMYLOID PATHOLOGY IN AD ......................................................... 8
2.5 THE INTERPLAY BETWEEN CALCIUM AND AMYLOID PATHOLOGY IN AD ........................ 11
2.6 CONNECTION BETWEEN CALCIUM AND TAU PATHOLOGY IN AD ..................................... 12
2.7 MITOCHONDRIAL DYSFUNCTION IN ALZHEIMER’S DISEASE ............................................ 13
2.8 THE THERAPY OF ALZHEIMER’S DISEASE .......................................................................... 15
2.8.1 SYMPTOMATIC THERAPY .................................................................................................... 15
2.8.2 DISEASE-MODIFYING THERAPY .......................................................................................... 16
2.8.2.1 AMYLOID-TARGETED THERAPIES .................................................................................... 16
2.8.2.2 TAU-TARGETED THERAPIES ............................................................................................. 18
2.8.2.3 ALTERNATIVE THERAPIES ............................................................................................... 19
2.9 THE DEVELOPMENT STATUS OF CALCIUM SIGNALING-TARGETED THERAPIES FOR AD . 19
2.9.1 PHARMACOLOGICAL MODULATION OF EXTRACELLULAR CALCIUM FLUX ......................... 19
2.9.1.1 RECEPTOR-OPERATED CALCIUM CHANNELS (ROCC) ..................................................... 19
2.9.1.2 VOLTAGE-GATED CALCIUM CHANNELS (VGCC) ............................................................ 20
2.9.2 PHARMACOLOGICAL MODULATION OF ER CALCIUM SIGNALING ....................................... 20
2.10 CALCIUM IMAGING ............................................................................................................ 21
3 PRESENILINS: ROLE IN CALCIUM HOMEOSTASIS ................................................. 23
3.1 ABSTRACT ............................................................................................................................. 24
3.2 INTRODUCTION ..................................................................................................................... 25
3.3 STRUCTURE .......................................................................................................................... 25
3.4 EXPRESSION, ACTIVATION AND TURNOVER ....................................................................... 26
3.5 ROLE IN CALCIUM HOMEOSTASIS ....................................................................................... 26
3.6 PS AS A THERAPEUTIC TARGET FOR ALZHEIMER’S DISEASE TREATMENT ...................... 30
4 AIM OF THE STUDY ........................................................................................................... 33
5 INVOLVEMENT OF PRESENILIN HOLOPROTEIN UPREGULATION IN
CALCIUM DYSHOMEOSTASIS OF ALZHEIMER’S DISEASE ........................................ 35
5.1 ABSTRACT ............................................................................................................................. 36
5.2 INTRODUCTION ..................................................................................................................... 37
5.3 MATERIALS AND METHODS ................................................................................................ 38
5.3.1 CELL CULTURE AND CELL LINES ......................................................................................... 38
5.3.2 CALCIUM IMAGING ............................................................................................................. 38
5.3.3 TREATMENT WITH Γ-SECRETASE INHIBITORS ..................................................................... 39
5.3.4 WESTERN BLOT .................................................................................................................. 39
5.3.5 HUMAN SUBJECTS .............................................................................................................. 40
5.4 RESULTS ............................................................................................................................... 40
5.4.1 EFFECT OF PS1 HOLOPROTEIN OVEREXPRESSION ON CALCIUM RELEASE FROM ER.......... 40
5.4.2 EFFECT OF Γ-SECRETASE INHIBITORS ON CALCIUM RELEASE FROM ER ............................ 41
5.4.3 EFFECT OF PEN-2 KNOCKDOWN ON ER CALCIUM RELEASE .............................................. 42
5.4.4 PS1 HOLOPROTEIN IN BRAINS OF FAD-PS1 PATIENTS ....................................................... 43
5.5 DISCUSSION .......................................................................................................................... 43
6 DEVELOPMENT AND IMPLEMENTATION OF A HIGH-THROUGHPUT
COMPOUND SCREENING ASSAY FOR TARGETING DISRUPTED ER CALCIUM
HOMEOSTASIS IN ALZHEIMER’S DISEASE ..................................................................... 55
6.1 ABSTRACT ............................................................................................................................ 56
6.2 INTRODUCTION .................................................................................................................... 57
6.3 MATERIALS AND METHODS ................................................................................................ 58
6.3.1 CELL CULTURE AND CELL LINES ........................................................................................ 58
6.3.2 COMPOUND LIBRARY ......................................................................................................... 58
6.3.3 HIGH-THROUGHPUT CALCIUM IMAGING ASSAY AND AUTOMATED IMAGE ANALYSIS ....... 58
6.3.4 DATA MINING ..................................................................................................................... 60
6.3.5 CYTOTOXCITY ASSAY ........................................................................................................ 60
6.3.6 AΒ MEASUREMENTS ........................................................................................................... 60
6.3.7 SAPPΑ AND SAPPΒ MEASUREMENTS ................................................................................. 61
6.3.8 AMPK ACTIVITY ASSAY .................................................................................................... 62
6.3.9 STATISTICAL DATA ANALYSIS ............................................................................................ 62
6.4 RESULTS ............................................................................................................................... 63
6.4.1 FAD-PS1 MUTATIONS ENHANCE THE AMPLITUDE OF CCH-INDUCED CALCIUM RELEASE
AND THE NUMBER OF RESPONSIVE CELLS ...................................................................................... 63
6.4.2 HIGH-THROUGHPUT COMPOUND SCREENING ASSAY ENABLES THE DISCOVERY NOVEL
LEAD STRUCTURES ......................................................................................................................... 63
6.4.3 EFFECT OF BEPRIDIL ON CCH-EVOKED CALCIUM RELEASE FROM ER ............................... 66
6.4.4 EFFECT OF BEPRIDIL ON APP PROCESSING AND AΒ GENERATION .................................... 67
6.4.5 EFFECT OF BEPRIDIL ON AMPK ACTIVITY ........................................................................ 67
6.5 DISCUSSION .......................................................................................................................... 68
7 IDENTIFICATION OF TETRAHYDROCARBAZOLES AS NOVEL
MULTIFACTORIAL DRUG CANDIDATES IN THE TREATMENT OF ALZHEIMER’S
DISEASE ...................................................................................................................................... 91
7.1 ABSTRACT ............................................................................................................................ 92
7.2 INTRODUCTION .................................................................................................................... 93
7.3 MATERIAL AND METHODS .................................................................................................. 94
7.3.1 CELL CULTURE AND CELL LINES ........................................................................................ 94
7.3.2 AUTOMATED HIGH-THROUGHPUT FRET-BASED CALCIUM IMAGING AND IMAGE ANALYSIS
94
7.3.3 MITOCHONDRIAL MEMBRANE POTENTIAL TMRM ASSAY ................................................ 96
7.3.4 AΒ MEASUREMENTS ........................................................................................................... 96
7.3.5 SAPPΑ AND SAPPΒ MEASUREMENTS ................................................................................. 97
7.3.6 STATISTICAL DATA ANALYSIS ............................................................................................ 97
7.4 RESULTS ............................................................................................................................... 98
7.4.1 DISCOVERY OF A NOVEL LEAD STRUCTURE FROM A HIGH-THROUGHPUT COMPOUND
SCREEN TARGETING DISRUPTED ER CALCIUM HOMEOSTASIS ....................................................... 98
7.4.2 TETRAHYDROCARBAZOLES ATTENUATE THE FAD-PS1 MEDIATED EXAGGERATED ER
CALCIUM RELEASE .......................................................................................................................... 99
7.4.3 TETRAHYDROCARBAZOLES INCREASE THE MITOCHONDRIAL MEMBRANE POTENTIAL ..... 99
7.4.4 TETRAHYDROCARBAZOLES LOWER AΒ PEPTIDE PRODUCTION ........................................ 100
7.5 DISCUSSION ........................................................................................................................ 101
7.6 SUPPLEMENTARY MATERIAL ............................................................................................ 122
8 GENERAL DISCUSSION ................................................................................................... 123
8.1 THE ROLE OF PRESENILIN HOLOPROTEIN UPREGULATION IN AD-ASSOCIATED CALCIUM
DYSHOMEOSTASIS ....................................................................................................................... 123
8.2 DEVELOPMENT OF A HIGH-THROUGHPUT FRET-BASED CALCIUM IMAGING ASSAY FOR
DRUG SCREENING IN AD ............................................................................................................. 125
8.3 HIGH-THROUGHPUT SCREENING OF COMPOUND LIBRARIES, DISCOVERY AND
CHARACTERIZATION OF NOVEL DRUGS FOR TREATMENT OF AD ........................................... 128
9 REFERENCES ..................................................................................................................... 131
10 ACKNOWLEDGMENTS .................................................................................................. 159
11 CURRICULUM VITAE .................................................................................................... 161