Theoretical investigation into the inhibition of cystein proteases [Elektronische Ressource] / vorgelegt von Milena Mladenovic

Milena Mladenovic

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Julius-Maximilians-Universität
Würzburg

Fakultät für Chemie und Pharmazie



Theoretical Investigation into the Inhibition of
Cysteine Proteases


Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrads
der Julius-Maximilians-Universität Würzburg



vorgelegt von

Milena Mladenovic

aus Niš



Würzburg 2007









Eingereicht am: _________________________________________________________
bei der Fakulät für Chemie und Pharmazie.



1. Gutachter:
2. Gutachter:
der Dissertation.




1. Prüfer: _________________________________________________________
2. Prüfer:
3. Prüfer:
des öffentlichen Promotionskolloquiums.



Tag des öffentlichen Promotionskolloquium: __________________________________



Doktorurkunde ausgehändigt am:















Die vorliegende Arbeit wurde unter Anleitung von Prof. Dr. Bernd Engels von Juli 2003 bis
November 2007 am Institut für Organische Chemie der Julius-Maximilians-Universität
Würzburg angefertigt.

Teilergebnisse dieser Arbeit waren Gegenstand von Publikationen sowie von Postern und
Kurzvorträgen.















Hiermit erkläre ich an Eides statt, dass ich die Dissertation

“Theoretical Investigations into the Inhibition of Cystein Proteases”

selbständig angefertigt habe und keine anderen als die von mir angegebenen Quellen und
Hilfsmittel benutzt habe.

Ich erkläre weiterhin, dass diese Dissertation weder in gleicher oder andere Form bereits in
einem anderen Prüfungsverfahren vorgelegen hat.

Ich habe früher außer den mit dem Zulassungsgesuch urkundlich vorgelegten Graden keine
weiteren akademischen Grade erworben oder zu erwerben versucht.

Würzburg, 14.11.2007



Milena Mladenovic















I
Table of Contents
Chapter 1 Introduction ...................................................................................................... 1
Chapter 2 Background ....................................................................................................... 5
2.1 Cysteine Proteases and Their Inhibitors...................................................................................... 5
2.1.1 Occurrence and structure of cysteine proteases ............................................................ 6
2.1.2 Mechanism of proteolysis............................................................................................. 9
2.1.3 Inhibitors of cysteine proteases................................................................................... 10
2.1.4 Inhibition mechanism.................................................................................................. 13
2.2 Background on Theoretical Methods Used in This Work......................................................... 15
2.2.1 Molecular modeling (MM) methods........................................................................... 15
2.2.1.1 Energy terms ............................................................................................... 16
2.2.2 Quantum mechanical (QM) methods.......................................................................... 21
2.2.2.1 The basics.................................................................................................... 21
2.2.2.2 Semi-empirical methods.............................................................................. 26
2.2.2.3 Density functional theory (DFT)................................................................. 28
2.2.3 Combined quantum mechanics / molecular modeling (QM/MM) methods................ 30
2.2.3.1 General principles ....................................................................................... 30
2.2.3.2 Treating covalent bonds between the QM and MM regions ....................... 33
2.2.3.3 Coupling of the QM and MM regions......................................................... 35
2.2.4 Molecular Dynamics (MD)......................................................................................... 38
Chapter 3 Results and discussion.................................................................................... 43
3.1 QM Calculations ....................................................................................................................... 47
3.1.1 The method ................................................................................................................. 47
3.1.2 The role of His199 in protonation of the inhibitor...................................................... 49
3.1.3 Substitution at the N-atom of aziridine ....................................................................... 53
3.2 QM/MM and MD Calculations................................................................................................. 65
3.2.1 The method 65
3.2.2 QM/MM treatment of inhibitor protonation by the His199 residue............................ 69
3.2.3 QM/MM calculations clarifying the inhibition potency of epoxide- and aziridine-
based inhibitors ......................................................................................................................... 80
II
3.2.4 QM/MM calculations explaining the regiospecificity of the reaction with epoxide- and
aziridine-based inhibitors.......................................................................................................... 98
3.2.5 Investigations into the stereospecificity of the cysteine protease inhibition reaction
with epoxysuccinyl derivatives............................................................................................... 115
3.2.6 Using MD Simulations to correct the existing X-ray data ........................................ 131
3.3 QM/MM MD Calculations...................................................................................................... 137
3.3.1 The method ............................................................................................................... 137
+ -3.3.2 Explaining the stability of the active site HisH /CysS ion-pair in cysteine proteases
138
Chapter 4 Summary ....................................................................................................... 157
Chapter 5 Zusammenfassung ........................................................................................ 165
Chapter 6 References...................................................................................................... 173

Chapter 1 Introduction
Chapter 1 Introduction
The most challenging field in computational chemistry of today, and probably its most
promising field of the future, is the calculation of large molecular entities. Proteins are by far
the most interesting macromolecules, due to their astonishing range of structure and function,
and particularly their vital role in nature.
Proteolytic activity is essential for the normal functioning of an organism and failures in its
control mechanisms can result in a vast number of diseases. For many illnesses resulting from
excess proteolysis no medications exist, making research into the physiological roles of
proteases and development of substances to inhibit them of highest importance to science and
the pharmaceutical industry.
1 Chapter 1 Introduction


Infectious diseases - worldwide casue of death No. 1.

10%
Parasitic infections5%
30% Cardiovascular diseases7%
Cancer
Perinatal
Respiratory diseases
11% Other

28%


Figure 1.1.1. Worldwide death causing diseases in percent (Source: WHO). According to the
WHO in 1997 50 million people worldwide died of infectious diseases.

Papain-like lysosomal cysteine proteases are processive and digestive enzymes found in a
1wide variety of living organisms, from bacteria to humans. For many years these enzymes
were believed to be responsible for redundant “house-keeping” activities and as such were
considered to have little, if any, value as drug targets. However, recent progress in identifying
new members of this class, as well as a better understanding of their physiological roles, has
altered this perception. It has become clear that lysosomal cysteine proteases fulfill specific
functions in extracellular matrix turnover, antigen presentation, and processing events. As
such they are involved in many diseases and represent promising drug targets for
2 3 [4] 2osteoporosis, arthritis, cancer, Alzheimer’s disease and a vide variety of parasitic
5 2infections such as malaria and African trypanosomiasis (“sleeping sickness”) . According to
the investigations of the world health organization (WHO), each year 50 million people die
worldwide from parasitic infectious diseases alone (Figure 1.1.1). A large part of these deaths
2