Towards subunit specific proteasome inhibitors Linden, W.A. van der

Towards subunit specific proteasome inhibitors

Linden, W.A. van der

Citation

Linden, W. A. van der. (2011, December 22). Towards subunit specific proteasome inhibitors. Retrieved from https://hdl.handle.net/1887/18273

Version: Corrected Publisher’s Version

License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden

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Towards subunit specific proteasome inhibitors

PROEFSCHRIFT ter verkrijging van

de graad van Doctor aan de Universiteit Leiden, op gezag van Rector Magnificus prof. mr. P. F. van der Heijden,

volgens besluit van het College voor Promoties te verdedigen op donderdag 22 december 2011

klokke 15:00 uur

door

Wouter Alexander van der Linden

Geboren te Rotterdam in 1983

Promotie commissie

Promotor: Prof. Dr. H. S. Overkleeft Co-promotores: Prof. Dr. A. F. Kisselev

Dr. B. I. Florea

Overige leden: Prof. Dr. C. A. van Boeckel Prof. Dr. M. Kaiser

Prof. Dr. J. Brouwer

Prof. Dr. G. A. van der Marel Prof. Dr. M. Groll

The printing of this thesis was financially supported by the J. E. Jurriaanse stichting.

Typeset in L

TEX

Printed by Wöhrman Print Service

Van wie moet dat dan?

Table of Contents

List of Abbreviations vi

1 General Introduction 1

1.1 Introduction . . . . 1

1.2 Ubiquitin Proteasome System . . . . 1

1.3 Structure and function of the proteasome . . . . 2

1.4 Proteasome inhibitors . . . . 4

1.5 Natural occurring proteasome inhibitors . . . . 4

1.6 Synthetic proteasome inhibitors . . . . 9

1.7 Therapeutic implications of proteasome inhibition . . . 14

1.8 Subunit selective proteasome inhibitors . . . 16

1.9 Aim and outline of this Thesis . . . 21

2 Subunit selectivity of a proteasome inhibitor is influenced by the electrophile 23 2.1 Introduction . . . 23

2.2 Results and Discussion . . . 25

2.3 Conclusion . . . 31

2.4 Experimental . . . 31

3 Proteasome selectivity towards Michael acceptor containing oligopeptide-based inhibitors 39 3.1 Introduction . . . 39

3.2 Results and Discussion . . . 39

3.3 Conclusion . . . 44

3.4 Experimental . . . 44

4 Syringolin ureido-peptide moiety containing inhibitors show tunable subunit selectivity 63 4.1 Introduction . . . 63

4.2 Results and Discussion . . . 65

4.3 Conclusion . . . 73

4.4 Experimental . . . 74

5 Two-step bioorthogonal activity-based proteasome profiling using copper-free click reagents: a comparative study 83 5.1 Introduction . . . 83

5.2 Results and Discussion . . . 84

5.3 Conclusion . . . 87

iv

5.4 Experimental . . . 88

6 Basicity at P1 and P3 induces β2 selectivity in proteasome inhibitors 91 6.1 Introduction . . . 91

6.2 Results and Discussion . . . 93

6.3 Conclusion . . . 100

6.4 Experimental . . . 101

7 Summary and Future Prospects 109 7.1 Experimental . . . 120

References 131

Samenvatting 147

List of Publications 150

Curriculum Vitae 152

Nawoord 153

v

List of Abbreviations

ABP activity-based probe

ABPP activity-based protein profiling

Ac acetyl

Ac₂O acetic anhydride AcCl acetyl chloride AcOH acetic acid

Ada 1-adamantyl acetic acid Ahx 6-aminohexanoic acid AMC 7-amino-4-methyl coumarin APT attached proton test

aq. aqueous

ATP adenosine triphosphate

BM biotinylated molecular weight marker Boc tert-butoxycarbonyl

Boc₂O tert-butoxycarbonic anhydride BODIPY 4,4-difluoro-4-bora-3a,4a-diaza-s-

indacene

BSA bovine serum albumin

Bu butyl

calcd. calculated cat. catalytic

Cbz benzyloxycarbonyl

d doublet

δ chemical shift

Da Dalton

DBU diazabicyclo[5.4.0]undec-7-ene DC dual colour molecular weight marker DCM dichloromethane

dd double doublet ddd double double doublet DiPEA N,N-diisopropylethylamine DMF N,N-dimethylformamide DMSO dimethylsulfoxide dq double quartet dt double triplet DTT dithiothreitol

DUB deubiquitinating enzyme EA ethyl acetate

EDTA ethylenediaminetetraacetate

EK epoxyketone

EL4 murine lymphoid cell line emPAI exponentially modified protein

abundance index equiv. molar equivalent ER endoplasmatic reticulum

EtOH ethanol

FDA U.S.A. food and drug administration Fmoc (9H-fluoren-9-yl)methoxycarbonyl GlbA glidobactin A

HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3- tetramethyluronium

hexafluorophosphate

HCTU 2-(6-chloro-1H-benzotriazole-1-yl)- 1,1,3,3-tetramethyluronium hexafluorophosphate

HEK293T human embryonic kidney cell line HMB 3-hydroxy-2-methylbenzamide HOBt N-hydroxybenzotriazole

hr. hour(s)

HRMS high resolution mass spectometry HWE Horner-Wadsworth-Emmons

Hz Hertz

J coupling constant

LC/MS liquid chromatography/ mass spectrometry

LMP2 low molecular weight protein 2, β1i LMP7 low molecular weight protein 7, β5i

M molar

m multiplet

m/z mass to charge ratio

Me methyl

MECl1 multicatalytic endopeptidase complex-like-1, β2i MeCN acetonitrile

MeOH methanol

MHC I major histocompatibility complex class I

min. minute(s)

MS (ESI) mass spectrometry (electronspray ionization)

NMR nuclear magnetic resonance

o/n overnight

vi

OAc acetate

PAI protein abundance index PBS phosphate buffered saline Pd/C palladium on charcoal PE petroleum ether PEG polyethylene glycol

Ph phenyl

ppm parts per million

pv. pathovar

q quartet

quant. quantitative R_t retention time

RAJI human lymphocyte cell line derived from Burkitt’s lymphoma RAW264.7 mouse leukaemic monocyte

macrophage cell line rcf relative centrifugal force RT room temperature

s singlet

Sacc. Pier Andrea Saccardo sat. saturated

SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate

polyacrylamide gel electrophoresis

sp. species

SPPS solid phase peptide synthesis subsp. subspecies

SylA syringolin A

T temperature

t triplet

t, tert tertiary tBuONO tert-butyl nitrite TBDMS tert-butyl dimethylsilyl

TBTA tris-(benzyltriazolylmethyl)amine td triple doublet

TEA triethyl amine TFA trifluoroacetic acid THF tetrahydrofuran TIS triisopropyl silane TLC thin layer chromatography TMR tetramethylrhodamine

Tol. toluene

Tris 2-amino-2-(hydroxymethyl)-1,3- propanediol

Ub ubiquitin

UPS ubiquitin proteasome system VS vinyl sulfone

Z benzyloxycarbonyl

Note: The one or three letter codes for the amino acids follow the recommendations of IUPAC. J. Biol.

Chem. 1968, 243, 3557-3559 and J. Biol. Chem. 1972, 247, 977-983.

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