Characterization of binding pocket flexibility of aldose reductase [Elektronische Ressource] / vorgelegt von Matthias Zentgraf

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C haracterization of Bin ding P ocket Flexibility of Aldose ReductaseD ISSE RTATIO NZURE RLANG UNG DE S D O K TO RG RADE SDE R NATURWISSE NSCHAFTE N(D R. RE R. NAT.)demFachbereich Pharm azie de rPHILIPPS-UNIVE RSITÄT MARBURGvorgelegt vonMatthias Z entgrafaus FuldaMarburg a n der Lahn, 2006Vom Fachbereich Pharm azie de r PHILIPPS-UNIVE RSITÄT MARBURG als Dissertationangenomm en am : 15.11.2006E rstgutachter: Prof. D r. G. KlebeZ weitgutachter: Dr. C. S otriffe rTag de r mündlichen Prüfung: 16.11.2006ITable of ContentsAldose Reductase and the sorbitol pathway.................................................................1Introduction..............................................................................................................1Structure of AR and related enzymes.......................................................................1Mechanism and kinetics of the AR catalyzed reaction............................................2The sorbitol pathway and its influences on metabolism..........................................4Aldose Reductase inhibitors.....................................................................................5Why consider protein flexibility in structure-based drug design?................................9Addressing protein flexibility and ligand selectivity by "in-situ cross-docking".......21Introduction..........................................................................................................

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C haracterization of Bin ding P ocket Flexibility of Aldose
Reductase
D ISSE RTATIO N
ZUR
E RLANG UNG DE S D O K TO RG RADE S
DE R NATURWISSE NSCHAFTE N
(D R. RE R. NAT.)
dem
Fachbereich Pharm azie de r
PHILIPPS-UNIVE RSITÄT MARBURG
vorgelegt von
Matthias Z entgraf
aus Fulda
Marburg a n der Lahn, 2006Vom Fachbereich Pharm azie de r PHILIPPS-UNIVE RSITÄT MARBURG als Dissertation
angenomm en am : 15.11.2006
E rstgutachter: Prof. D r. G. Klebe
Z weitgutachter: Dr. C. S otriffe r
Tag de r mündlichen Prüfung: 16.11.2006I
Table of Contents
Aldose Reductase and the sorbitol pathway.................................................................1
Introduction..............................................................................................................1
Structure of AR and related enzymes.......................................................................1
Mechanism and kinetics of the AR catalyzed reaction............................................2
The sorbitol pathway and its influences on metabolism..........................................4
Aldose Reductase inhibitors.....................................................................................5
Why consider protein flexibility in structure-based drug design?................................9
Addressing protein flexibility and ligand selectivity by "in-situ cross-docking".......21
Introduction............................................................................................................21
Results and Discussion...........................................................................................22
Summary and Conclusion......................................................................................28
Supporting Information A: Computational Methods.............................................28
Protein setup and grid calculations. ..................................................................28
Ligand setup......................................................................................................29
Docking ............................................................................................................30
Supporting Information B: Docking to trypsin and thrombin................................31
Sting Information C: Docking to multiple conformers by grid-based
averaging................................................................................................................32
Extending charted space: comparative MD simulations of Aldose Reductase...........35
Introduction............................................................................................................35
Materials and Methods...........................................................................................38
Crystal structure analysis...................................................................................38
MD simulations.................................................................................................41
Generation of 2D rms plots...............................................................................42
Docking with AutoDock....................................................................................42
Results and Discussion...........................................................................................43
Comparative crystal structure analysis..............................................................43
Comparative MD simulations analysis..............................................................63
Simulations of AR complexes......................................................................64
MD of AR in complex with sorbinil.............................................................64
MD of AR in complex with tolrestat............................................................67
MD of AR in complex with idd 594.............................................................70
MD of AR in complex with the Pfizer compound........................................73
MD of AR in complex with the zopolrestat compound................................76
MD of AR in complex with the 47d compound...........................................78
MD of AR in complex with the JFD com...........................................81
Simulations of the holo enzyme...................................................................83
Summarizing the results....................................................................................89
Implications for the binding pocket.................................................................104
Addressing the new binding pocket conformations........................................108
Summary and Conclusions...................................................................................120II
Evaluating MM-PBSA in case of a flexible binding pocket: the AR test case.........122
Introduction..........................................................................................................122
Methodology....................................................................................................123
Setup and workflow.........................................................................................124
Literature overview.........................................................................................129
The test system: Aldose Reductase.................................................................135
Materials and Methods.........................................................................................138
Data set............................................................................................................138
ITC measurements...........................................................................................138
Molecular Dynamics Simulations ..................................................................140
MM-PBSA calculations...................................................................................141
Calculation of Predictive Indices (PI).............................................................143
Results and Discussion.........................................................................................144
MD trajectories................................................................................................144
Sampling the reference state............................................................................146
MM-PBSA results...........................................................................................151
Summary and Conclusions...................................................................................159
Expect the Unexpected while working with AR.......................................................163
Introduction..........................................................................................................163
Results and Discussion.........................................................................................165
Conclusions..........................................................................................................176
Summary and Outlook..............................................................................................178
Zusammenfassung.....................................................................................................185
Appendix A...............................................................................................................193
The Molecular Dynamics Database (MDDB)......................................................193
Introduction.....................................................................................................193
Database and program architecture.................................................................194
Data content.....................................................................................................194
Using MDDB...................................................................................................195
Appendix B...............................................................................................................197
Additional crystal structures for MD simulations................................................197
Structure Determination..................................................................................197
The sorbinil complex structure........................................................................198
The tolrestat complex structure.......................................................................199
Usage of crystal structures from collaboration partners...........................................202
References.................................................................................................................203
Publications arising from this work..........................................................................223
Articles.................................................................................................................223
Oral Communications..........................................................................................224
Posters..................................................................................................................224
Acknowledgments.....................................................................................................226
Curriculum vitae.......................................................................................................228
Erklärung...................................................................................................................229Aldose Reductase and the sorbitol pathway 1
Aldose Reductase and the sorbitol pathway
Introduction
Aldose Reductase (AR) is a cytosolic NADPH-dependent enzyme that catalyzes the reduc-
tion of various aldoses and aldehydes to the corresponding alcohol s. It was first described
1
in 1956 by Hers et al. as a glucose-reducing activity. The authors dem onstrated that the
conversion of bl ood gl ucose to fruc tose was used as an energy source for s perm cells.
Today it is known that AR is the first and rate-lim iting enzyme of the sorbitol pathway.
Via this pathway gl ucose is first re duced to sorbitol by A R. In a second step sorbitol is then
+
re-oxi dized to fructose by an enzyme caled sorbitol dehydroge nase (SDH) using NAD as
cofa ctor. Galactose is an even be tter substrate for A R, but the corresponding product galac-
2;3
tiol is not further m etabolized by SDH . The net reactions of the sorbitol pathway for glu-
cose are conversions of gl ucose to fruc tose as wel as of N ADPH to NADH.
4;5
Both enzymes are expressed in alm ost all hum an tissues . However, the ratio of AR to
6
SDH di ffe rs in diffe rent tissues . The kidney, for e xam ple, is one of t he tissues showing the
7
highest concentrations of A R .
Under physiological conditions glucose is usualy phosphoryl ated by the enzyme hexo-
kinase to glucose-6-phosphat which is then further m etabolized using glycolysis or the
pentose phosphate m etabolism . Under raised blood glucose levels, as found in people suf-
fe ring from diabetes m elitus, up to one third of the available glucose is processed via the
8;9
sorbitol pathway . The accum ulation of sorbitol on the one hand and the consum ption of
NADPH on the other have been postulated to be responsible for some of the late-onset
10-16complications of di abetes melitus .
Structure of AR and related enzymes
AR belongs to the superfa m ily of the aldo-ke to reductases (AK R). It comprises 315 am ino
acids and fol ds to an (β/α)-TIM barrel structure with the catalytic site deeply buried at the 8
17center of the barrel . The protein core is composed of eight paralel β-strands. Adjacent 2 Aldose Reductase and the sorbitol pathway
strands are connected by eight paralel α-helices running anti-paralel to the β-sheets. The
active site is located at the C-term inal end of the barrel. The bottom of the barrel is closed
by two short anti-paralel β-strands near the N-term inus. Three large loops partialy cover
the top of t he ba rrel. Two a dditional α-helices are found out side the ba rrel structure pre ced-
ing two of three additional loop regions in sequence. The cofa ctor NADPH binds at the
18;19bottom of the active site, with its nicotinam ide m oiety pointing towards the active site .
The shape and properties of the binding pocket of AR as wel as the binding m odes of dif-
fe rent inhibitors in complex with AR will be discussed in great detail in the chapter on
Com parative Crystal Structure Analysis (see page 43).
Structuraly related enzymes of the AK R fa m ily which also catalyze the pyridine nu-
cleotide-dependent reduction of carbonyl functions are abundant in m ost organism s. How-
ever, the accepted substrates diffe r between the single enzymes. AR and the very closely
related enzyme aldehyde reductase reduce aldo and keto sugars as wel as aromatic and
20-22 23
aliphatic aldehyde s . Hydrosteroid dehydroge nase accepts steroids as substrates , and
the AR like prostaglandin synthase is responsible for the production of prostaglandin f2 al-
24
pha . However, no strict rules can be applied for AR since substrate promiscuity is very
pronounced for this enzyme. A broad variety of diffe rent substrates is accepted. Am ong
25;26 27 28
these substrates are glutathiolated , phospholipid , saturated, and unsaturated aldehy-
28 29;30des as wel as 4-hydroxya lkenals and steroids . The best known physiological substrate
31-33
for AR is the 2-oxoa ldehyde m ethyl glyoxya l . In general, m any AR substrates are m ore
hydrophobi c than expected for a n enzyme involved i n sugar metabolism .
Mechanism and kinetics of the AR catalyzed reaction
The reaction m echanism of AR fol lows a sequential binding of the involved m olecules.
The cofa ctor, NADPH, binds first and forms a binary complex with AR. Upon binding
34
large m ovem ents in a protein region caled the 'cofa ctor safe ty belt' are induced . Thus, the
cofa ctor becomes deeply buried in the protein. Subsequently, the ternary complex of sub-
strate, cofa ctor and protein, is formed. The substrate is kept properly aligned with respect
to the cofa ctor by a network of hydroge n bonds with residues around the catalytic pocket.
Then the hydride transfe r takes place reducing the aldehyde to the corresponding alcoho-Aldose Reductase and the sorbitol pathway 3
late. To finalize the reaction a proton needs to be transfe rred from the protein to the prod-
uct. Three residues around the active site are at suitable distances to act as proton donor:
35 36 37Cys 298, His 110, and Tyr 48. K inetic , computational , as wel as therm odynam ic stud-
ies fa vor Tyr 48 as the source of the proton. After the product has been released, exchange
of the cofa ctor is necessary to enable the next catalytic cycle. Therefore , the conformation-
al change which occurred during binding of the cofa ctor needs to be reversed. Several ki-
38-40
netic studies suggest that rem oving the cofa ctor from its binding site after the reaction
is indeed the rate lim iting step of t he whole proc ess.
Com pared to other substrates, glucose is a rather poor substrate of AR having K values m
41;42
in the range of 50-200 m M which is m uch higher than physiological concentrations .
However, less than 0.1% of glucose exists in the acyclic carbonyl form which is m andatory
for binding to AR. It is, thus, conceivable that the enzyme processes significant am ounts of
glucose only if the glucose concentrations are pathologicaly high. Surprisingly, it has been
shown that AR catalyzes the reduction of saturated and unsaturated m edium - to long-chain
3 4(C-6 to C-18) aldehydes, which are generated during lipid peroxidation, with 10 to 10 -
28;43;44
fol d hi gher e fficiency than gl ucose .
Several studies present results on post-translational m odifications of AR to regulate the
45-49activity of the enzyme . These m odifications are in part held responsible for the reduced
12;47
efficiency of hydantoine-based AR inhibitors such as sorbinil . Furtherm ore, it was ob-
served that depending on the presence or absence of reducing agents the kinetic param eters
50;51of AR diffe red significantly . The sam e effe cts were encountered upon in vitro thiol
43;52-55 55 42;53
m odifications using diffe rent agents . O xidative m odifications and glutathiolation
of Cys 298, which is located directly at the binding site are deem ed responsible for these
effe cts. Thus, Cys 298 is considered to be a m odulator for AR activity. Depending on the
conditions of the reactions, AR becomes either S-thiolated (inactivated) or S-nitrosated
(activated). Therefore , nitric oxide (NO ) is thought to be the physiological regulator of AR
56;57
activity .
If NADPH is bound, the Cys 298 side chain is less prone to oxidative m odifications.
Due to the high binding affinity of NADPH to AR m ost of the enzyme in the cel wil be in
the complexed state. E xchange of the cofa ctor wil only take place if substrate concentra-4 Aldose Reductase and the sorbitol pathway
tions have re ached a level where the catalytic re action can occur. Thus, t he switch for re gu-
lating the AR activity by m odifyi ng the Cys 298 side-chain wil only be available at situa-
tions of hi gh substrate concentration.
The sorbitol pathway and its influences on metabolism
Several hypotheses have been discussed with respect to the question how the activation of
the sorbitol pathway m ay induce diabetic complications. O xidative stress is one of the m a-
jor issues in this context. The sorbitol pathway contributes to oxidative stress in diffe rent
ways:
• The increased activity of AR depletes its cofa ctor NADPH. A suffi cient concentra-
tion of NADPH is required in cells due to its role as cofa ctor of glutathione reduc-
tase (G R). G R is responsible for several critical reductive m etabolic steps, such as
the detoxification of reactive oxyge n species (RO S) and hydrope roxides. Thus, by
consum ing NADPH, AR weakens the ability of cels to protect them selves from
oxidative stress.
• In the second step of the pathway sorbitol is oxidized to fructose by SDH. Thereby
+
NAD is reduced to NADH. However, NADH is the substrate of NADH oxidase,
58which i n turn produces additional ROS .
• The conversion of glucose to fructose is also problem atic in itself, since fructose
and its m etabolites are m ore potent non-enzymatic glycation agents than glucose.
The formed glycation products of these reactions are known to cause oxidative
stress within cells.
The accum ulation of sorbitol within cels and the resulting increase of osm otic stress is
the second hypothesis discussed as putative explanation for the occurrence of diabetic
complications. Since sorbitol can be oxidized by SDH to fructose, which then re-enters the
glycolytic pathway, accum ulation of sorbitol occurs prim arily in cells and tissues where
only low levels of SDH are present. For diabetic cataract it could be dem onstrated that os-
59;60
m otic stress is the m ain reason for this complication . However, it rem ains unclear
whether this mechanism can be transfe rred to ot her di abetic pathological phe nomena.