novel mechanism-based inhibitors of serine proteases
TRANSCRIPT
NOVEL MECHANISM-BASED INHIBITIORS OF SERINE PROTEASES
A Thesis by
Xiangdong Gan
M.S., Sichuan University, P. R. China, 1994
Submitted to the College of Liberal Arts and Sciencesand the faculty of the Graduate School of
Wichita State University in partial fulfillment ofthe requirements for the degree of
Master of Science
December 2005
ii
NOVEL MECHANISM-BASED INHIBITIORS OF SERINE PROTEASES
I have examined the copy of this thesis for form and content and recommend it be accepted inpartial fulfillment of the requirement for the degree of Master of Science, with a major inChemistry.
___________________________________William C. Groutas, Committee Chair
We have read this thesis
and recommend its acceptance
__________________________________Erach R. Talaty, committee Member
__________________________________Ram P. Singhal, Committee Member
__________________________________Lop-Hing Ho, Committee Member
iii
ACKNOWLEDGEMENTS
I would like to express my sincere heart felt gratitude to my advisor, Dr. William C. Groutas
for his excellent guidance, support and encouragement throughout my studies at Wichita State
University. It is my great pleasure to be one of his students. He is a great teacher, a great
researcher and a great advisor.
I also would like to extend my gratitude to Dr. Erach R. Talaty, Dr. Ram P. Singhal, and Dr.
Lop-Hing Ho for their guidance and for being a member of my thesis committee. Furthermore, I d
like thank Dr. Kevin Alliston for his assistance with the biochemical studies. I am also thankful
to the members of my group and friends for their help and kindness. I sincerely thank all the
faculty and staff of the department of chemistry at Wichita State University.
Finally, I would like to thank my family for their love and support.
iv
ABSTRACT
The design and in vitro biochemical evaluation of two novel classes of mechanism-based
inhibitors of human leukocyte elastase (HLE) that inactivate the enzyme via an unprecedented
enzyme-induced sulfonamide fragmentation cascade is described. The inhibitors incorporate in
their structure either an appropriately-functionalized saccharin scaffold, or a 1,2,
5-thiadiazolidin-3-one-1,1-dioxide scaffold. The inactivation of the enzyme by these inhibitors
was found to be efficient, time-dependent and to involve the active site. Biochemical, HPLC,
and mass spectrometric studies show that the interaction of these inhibitors with HLE results in
the initial formation of a Michaelis-Menten complex and subsequent formation of a tetrahedral
intermediate with the active site serine (Ser-195). Collapse of the tetrahedral intermediate with
tandem fragmentation results in the formation of a highly reactive conjugated sulfonyl imine
which can either react with water to form a relatively stable acyl enzyme and/or undergo a
Michael addition reaction with an active site nucleophilic residue (His-57). The results also
demonstrate convincingly the superiority of the 1, 2, 5-thiadiazolidin-3-one-1,1-dioxide scaffold
over the saccharin scaffold in the design of inhibitors of (chymo)trypsin-like serine proteases.
v
TABLE OF CONTENTS
CHAPTER PAGE
1 INTRODUCTION.................................................................................................................1
1.1 Proteases ............................................................................................................................1
1.2 Terminology and Classification ..........................................................................................2
1.3 Serine Proteases .................................................................................................................4
1.3.1 Catalytic Mechanism .................................................................................................4
1.3.2 Human Leukocyte Elastase ........................................................................................6
1.3.3 Substrate Specificity ..................................................................................................8
1.4 Enzyme Inhibition ..............................................................................................................9
1.4.1 Reversible Inhibition ............................................................................................... 10
1.4.2 Irreversible Inhibition .............................................................................................. 13
1.4.3 Inhibitors of Human Leukocyte Elastase..................................................................17
2 DESIGN RATIONALE AND RESEARCH GOALS .......................................................... 23
2.1 Design Rationale for Inhibitors Derived from Saccharin Scaffold ............................... 24
2.2 Design Rationale for Inhibitors Derived from 1, 2, 5-Thiadiazolidin-3-one 1, 1 Dioxide
Scaffold ...................................................................................................................... 25
2.3 Research Goals ........................................................................................................... 26
3 EXPIREMENTAL .............................................................................................................. 27
3.1 Enzyme Assays and Inhibition Studies: Incubation Method ........................................ 27
3.2 Enzyme Assays and Inhibition Studies: Progress Curve Method ................................. 28
vi
3.3 Hydroxylation Reactivation of Inactivated HLE ......................................................... 29
3.4 Substrate Protection.................................................................................................... 30
3.5 Efficiency of Inactivation (Determination of Partition Ratio) ...................................... 30
3.6 Reactivation of HLE-Inhibitor Complex ..................................................................... 31
3.7 HPLC Studies. Product Analysis and Identification of Inhibitor with HLE..................32
4 RESULTS AND DISCUSSION..........................................................................................33
4.1 Inactivation of HLE by Derivatives of (I) (Compound 8-13)....................................... 33
4.2 Inactivation of HLE by Derivatives of (II) (Compound 4-7)........................................45
5 CONCLUSIONS.................................................................................................................52
REFFERENCES .................................................................................................................... 53
vii
LIST OF TABLES
Table Page
1.1 NC-IUBMB Definition for Subclassifications of Peptidases ........................................2
1.2 Classes of Proteinases According to Catalytic Mechanism (Dunn, 1992) .....................4
1.3 Human Elastases..........................................................................................................7
1.4 Substrate Specificity of Neutropil-Derived Serine Proteases ........................................8
1.5 Templates Employed in Serine Protease Inhibitor Design (Mechanism-based Inhibitors)
................................................................................................................................. 18
4.1 Inhibitory Activity of Compounds 8-13 Towards Human Leukocyte Elastase and Bovine
Trypsin ..................................................................................................................... 33
4.2 Inhibitory Activity of Compounds 4-7 Towards Human Leukocyte Elastase and Bovine
Trypsin ..................................................................................................................... 38
viii
LIST OF FIGURES
Figure Page
1.1 Hydrolysis of Amide Bonds by Proteases. ...................................................................1
1.2 Terminology of Specific Subsites of Proteases and the Complementary Features of the
Substrate.....................................................................................................................3
1.3 Mechanism of Amide Hydrolysis Catalyzed by a Serine Protease ................................6
1.4 Mechanism-based Inactivation of a Serine Protease via an Enzyme-induced Lossen
Rearrangement.......................................................................................................... 19
1.5 Mechanism of the Gabriel-Colman Rearrangement ....................................................20
1.6 Mechanism-based Inactivation of a Serine Protease via an Enzyme-induced
Gabriel-Colman Rearrangement................................................................................ 21
1.7 Mechanism-based Inactivation of a Serine Protease via an Enzyme-induced Formation
of a Michael Acceptor............................................................................................. 17
2.1 General Structures of Inhibitors (I-II)......................................................................... 24
2.2 (a) Spontaneous Fragmentaion of -Amido Sulfonopeptides to Yield a Michael
Acceptor; (b) -Amido Sulfonamide Motif in Phthalimide and Saccharin Derivatives.
................................................................................................................................. 25
4.1 Structures of Compounds 8-13................................................................................... 33
4.2 Time-dependent Loss of Enzymatic Activity. ............................................................ 35
4.3 Kinetics of Inactivation of Human Leukocyte Elastase by Compound 9..................... 36
4.4 Kinetics of Inactivation of Bovine Trypsin by Compound 9....................................... 37
ix
4.5 Postulated Mechanism of Action of Inhibitor (I) ........................................................ 38
4.6 Subtrate Protection. ...................................................................................................39
4.7 Effect of Hydroxylamine on Enzyme Reactivation..................................................... 40
4.8 Inactivation of Human Leukocyte Elastase as a function of the Molar Ratio of Inhibitor
9 to Enzyme.............................................................................................................. 41
4.9 HPLC Analysis of Products Formed by Incubating Human Leukocyte Elastase with
Inhibitor 9................................................................................................................. 43
4.10 Structures of Compounds 4-7................................................................................... 45
4.11 Time-dependent Loss of Enzymatic Activity ........................................................... 46
4.12 Time-dependent Loss of Enzymatic Activity ........................................................... 48
4.13 Postulated Mechanism of Inhibition of Human Leukocyte Elastase by Inhibitor (II) 50
1
CHAPTER 1
INTRODUCTION
1.1 Proteases
Living systems are shaped by an enormous variety of biochemical reactions, nearly all of
which are mediated by a series of remarkable biological catalysts (enzymes). Biochemical
research since Pasteur s era has shown that although enzymes are subject to the same laws of
nature that govern the behavior of other substances, enzymes differ from ordinary chemical
catalysts in several important respects: a) higher reaction rates; b) milder reaction conditions;
c) greater reaction specificity; and d) capacity for regulation.1
Proteases (proteinases) are enzymes that can selectively catalyze the hydrolysis of peptide
bonds (Figure 1.1). Proteolytic enzymes are involved in a great variety of physiological
processes, including blood coagulation, digestion, complement activation, phagocytosis,
protein turnover (tissue remodeling), fibrinolysis, ovulation and fertilization, and hormone
generation and degradation. Poor regulation of the activity of proteases leads to a range of
diseases (cancer metastasis, inflammation, rheumatoid arthritis, muscle degradation, etc.)
HN
ONH
O
OP1
P1' HN
OOH
O
P1
+ H2NO
P1'Protease
Figure 1.1 Hydrolysis of Amide Bonds by Proteases
2
1.2 Terminology and Classification
In chemical terms, the enzymatic cleavage of peptide bonds is considered as hydrolysis,
usually called proteolysis. The enzymes responsible for the catalysis of proteolysis have been
named proteases , a term that originated in the nineteenth century German literature on
physiological chemistry. The International Union of Biochemistry and Molecular Biology
(IUBMB) (1984) has recommended to use the term peptidase for the subset of peptide bond
hydrolases (Subclass E.C.3.4). The widely used term protease is synonymous with peptidase.
Peptidases are further divided into exopeptidases , acting only near a terminus of a
polypeptide chain, and endopeptidases , acting internally in polypeptide chains. The term
proteinase used previously has been replaced by endopeptidase for consistency. In
addition, the EC list specifies different subtypes of exopeptidases and endopeptidases (Table
1.1).
Table 1.1 NC-IUBMB Definition for Subclassifications of Peptidases
Subclasses Activity
Exopeptidases Cleave near a terminus of peptides or proteins Aminopeptidases Remove a single amino acid from the free N-terminus Dipeptidyl peptidases Remove a dipeptide from the free N-terminus Tripeptidyl peptidases Remove a tripeptide from the free N-terminus Carboxypeptidases Remove a single amino acid from the free C-terminus Peptidyl dipeptidases Remove a dipeptide from the free C-terminus Dipeptidases Cleave dipeptides Omega peptidases Remove terminal residues that are substituted, cyclized or Linked by isopeptide bondsEndopeptidases Cleave internally in peptides or proteins Oligopeptidases Cleave preferentially on substrates smaller than proteins
3
To define a common nomenclature on the interaction of a substrate with a peptidase, the
system of Berger and Schechter (1976) has become generally accepted and used (Figure 1. 2).
This system is based on a schematic interaction of amino acid residues of the substrate with
specific binding subsites located on the enzyme. By convention, the subsites on the protease
are called S (for subsites e.g. S3, S2, S1, S1 , S2 , S3 ) and the substrate amino acid residues are
called P (for peptide e.g. P3, P2, P1, P1 , P2 , P3 ). The numbering of the residues is given from
the scissile bond2.
Figure 1.2 Terminology of Specific Subsites of Proteasesand the Complementary Features of the Substrate
Based on the nature of a key catalytic residue located at the active site of the proteases,
proteases are classified as serine, cysteine, aspartic and metallo proteases.
The catalytic nucleophile in serine and cysteine poteinases is the hydroxyl group of the
active site serine and the sulphydryl group of the active site cysteine, respectively. In aspartic
proteinases, two aspartic acid residues directly bind the nucleophilic water molecule. Metallo
proteinases contain a metal ion (typically zinc) that is usually bound by three amino acids.
The nucleophile is a water molecule, as in aspartic proteinases, positioned and possibly
activated by the active site metal ion (Table 1.2) 3.
HNNH
HN
NH
HN
NHP3 P1 P2'
P2O
O
O
O
OP1' P3'
S2 S1' S3'
S3 S1 S2'scissile bond
O
4
Table 1.2 Classes of Proteinases According to Catalytic Mechanism (Dunn, 1992)
Class Catalytic Residue Examples
Serine Ser195 (His57, Asp102)chymotrypsin, trypsin, elastase, thrombin, proteinase 3,
factor Xa, plasmin, cathepsin G
Cysteine Cys25 (His159) Cathepsins B, L, S
Aspartic Asp32, Asp215 pepsin, HIV protease, rennin, cathepsin D
Metallo- Zn2+ Angiotensin converting enzyme (ACE), MMP-2, MMP-9
1.3 Serine Proteases
Serine proteases are known as a diverse and widespread group of proteolytic enzymes.
They are so named because they have a common catalytic mechanism involving a reactive
Ser residue. The serine proteases include digestive enzymes from prokaryotes and eukaryotes,
as well as more specialized proteins that participate in development, blood coagulation
(clotting), inflammation, and numerous other processes. Elastase, trypsin and chymotrypsin
are some of the best studied serine proteases.
1.3.1 Catalytic Mechanism
The classical mechanism of serine peptidases is based on the catalytic triad of Ser195,
His57 and Asp102. After formation of a Michaelis complex, the carbonyl carbon atom of the
scissile bond is attacked by the active site Ser (Figure 1.3). The nucleophilicity of the serine
is enhanced by an adjacent histidine (His57) functioning as a general base catalyst. The
formation of an acyl enzyme complex is achieved through a tetrahedral intermediate which
collapses to yield the acyl enzyme and the cleavage of the peptide bond. Deacylation occurs
5
via the same mechanistic steps. In this case, the nucleophilic attack is performed by a bound
water molecule resulting in the release of the peptide and restoration of the Ser-hydroxyl of
the enzyme. The mechanism requires a binding site for the oxyanion of the tetrahedral
intermediate. This site is formed by the backbone amides of Ser195 and Gly193. The
catalytic contribution of the third member in the catalytic triad, the conserved Asp, has been
controversial. The early suggestion that the Asp accepts a proton to become uncharged in the
transition state has been opposed by newer experimental and theoretical data (Kossiakoff and
Spencer 1981; Warshel et al 1989). Further suggested roles of the Asp include stabilization of
the imidazole orientation (Rogers and Bruice 1974) as well as stabilization of the local
structure around the active site (Lau and Bruice 1999). In addition, it has been proposed that
the hydrogen bond between Asp and His is a low-barrier hydrogen bond which accounts for
the transition state stabilization (Frey et al 1994). 3
The nucleophilicity of the serine is enhanced by an adjacent histidine functioning as a
general base catalyst. The general base catalysis is involved in the formation of the
tetrahedral transition state intermediate, where the imidazole group of His-57 functions as a
general base to abstract the proton from the O of Ser-195 (during the acylation) or from a
water molecule (during the deacylation), facilitating the nucleophilic attack. This newly
protonated imidazole then provides general acid catalysis to assist the collapse of the
tetrahedral transition state intermediate by transferring its proton to the leaving group (the
scissile amide nitrogen during the acylation, and O of Ser-195 during the deacylation). As a
proton shuttle , the imidazole of His-57 is involved in a direct interaction with the substrate,
and a slight movement of the imidazole group may also be required for such function.
6
Therefore, the position and orientation of the imidazole ring is critical for catalysis.
R1 NH
HN
O
OR1'
OP1
P1'
N N
Ser195 Gly193
H H
NN
His57
H O Asp102
O
HO
Ser195
NN
His57
H O Asp102
O
HO
H
H2NR1
O
P1'
R1 NH
OHO
O
P1
NN
His57
H O Asp102
O
HO
HN
Ser195
N
Gly193
H
R1 NH
O
O
P1
HHN N
Ser195 Gly193
O
R1 NH
NHO
OR1'
OP1
P1'
HHN N
Ser195 Gly193
NN
His57
H O Asp102
O
OH
Tetrahedralintermediate
Primed aminoterminal fragment
Acyl-enzyme
Non-primed carboxyterminal fragment
Michaelis complex
E Ser195 E
Ser195 ESer195 E
Figure 1.3 Mechanism of Amide Hydrolysis Catalyzed by a Serine Protease.
1.3.2 Human Leukocyte Elastase
The fibrous protein elastin, which comprises an appreciable percentage of all protein
content in some tissues, such as arteries, some ligaments and the lungs, can be hydrolysed or
otherwise destroyed by a select group of enzymes classified as elastases. Elastases are
derived from many tissues in man, including the pancreas, neutrophils, macrophages,
monocytes, platelets, smooth muscle cells and fibroblasts. Elastases are defined by their
ability to generate soluble peptides from insoluble elastin fibres by a proteolytic process
7
called elastinolysis. Examples of human elastases include human leukocyte elastase (HLE)
(E.C. 3.421.37, Pancreatic elastase II (PE-II) (E.C. 3.4.21.71), and Macrophage
metalloelastase (MMP-12) (E.C. 3.4.24.65) (Table 1.3). HLE and PE are serine proteinases
whereas MMP-12 is a metallo-protease. The catalytic triads of HLE and PE-II are composed
of the residues of His57, Asp102 and Ser195. The catalytic site of active MMP-12 consists of a
catalytic zinc ion that is co-ordinated to three stabilizing histidine residues: His218, His222 and
His228 according to human fibroblast collagenase-1 (MMP-1) numbering system.
Table 1.3 Human Elastases
Enzyme Class Molecular weight(kDa) Catalytic residues
Human leukocyte elastase Serine protease 30 His57, Asp102, Ser195
Pancreatic elastase II Serine protease 26 His57, Asp102 , Ser195
Macrophage metalloelastase Metalloprotease 54 Zn, His218, His222 , His228
Human leukocyte elastase (HLE) is a glycosylated, strongly basic serine protease with a
molecular weight of approximately 30 kDa and is found in the azurophilic granules of human
polymorphonuclear leukocytes (PMN).4 This enzyme is released from PMN upon
inflammatory stimuli and has been implicated as a pathogenic agent in a number of disease
states such as pulmonary emphysema5, 6, rheumatoid arthritis7, adult respiratory distress
syndrome (ARDS)8, glomerulonephiritis9, cystic fibrosis10, 11 and cancer12. Increased
proteolysis, especially elastolysis, may occur in the lung parenchyma as a result of an
imbalance between HLE and its major endogenous inhibitor -PI, because of either an
8
acquired or an inherited deficiency of the protease inhibitor. As replacements to -PI,
synthetic, low molecular weight HLE inhibitors delivered to the site of unregulated PMN
elastase activity can be potentially useful in the treatment of pulmonary emphysema and
related diseases.
1.3.3 Substrate Specificity
The ability of proteases to selectively act upon a small number of targets in a myriad of
potential physiological substrates is necessary for maintaining the fidelity of most biological
functions. While substrate selection by proteases is governed by many factors, a principal
determinant is the substrate specificity of the enzyme s active site. Knowledge of a protease s
substrate specificity can greatly facilitate the elucidation of the physiological substrates of the
protease, which is essential for defining its role in complex biological pathways.
Determination of substrate specificity also can provide the basis for potent and selective
substrates and inhibitors. Table 1.4 summarizes the specificity of the three neutrophil-derived
proteases.
Table 1.4 Substrate Specificity of Neutrophil-Derived Serine Proteases
Enzyme S5 S4 S3 S2 S1 S1' S2' S3'
HLE Hydrophobic AlaAlaVal
ProValLeuNva
Hydrophobic
Cat G Polar AlaValThr
ProMet
Phe (not mapped)
PR 3 Pro Ala ProAlaAbu
Hydrophobic
Peptide P5 P4 P3 P2 P1 P1' P2' P3'
9
The substrate sequential specificity of serine proteases has often been mapped with
chromogenic substrates such as peptidyl p-nitroanilides, which identify favorable Pn-Sn
interactions. The most reliable information about the Pn -Sn interactions is from X-ray crystal
structures of protease-protein inhibitor complexes, which are not available for most serine
proteases.
Over the last few years a number of elegant combinatorial approaches have been
developed to determine the substrate specificity of proteases. All of these combinatorial
methods involve the generation of libraries of potential substrates, proteolysis of favorable
substrates, and identification of the cleaved substrate sequences. Combinatorial substrate
libraries can be divided into two categories depending on the method of generation:
biological and synthetic. Biological library methods include the use of substrate phage
display13, 14, the randomization of amino acids at physiological cleavage sites15, and the use of
in vitro expression cloning of complementary DNA (cDNA) libraries to determine
physiological substrates16. While biological combinatorial methods are very powerful, they
are complicated by the lack of homogeneity in their presentation of potential substrates and
the fact these methods are often time consuming and technically demanding17.
1.4 Enzyme Inhibition
Many substances alter the activity of an enzyme by reversibly combining with it in a way
that influences the binding of substrate and/or its turnover number. Substances that reduce an
enzyme s activity in this way are known as inhibitors. A large part of the modern
10
pharmaceutical arsenal consists of enzyme inhibitors. For example, AIDS is treated almost
exclusively with drugs that inhibit the activity of certain viral enzymes.
Inhibitors act through a variety of mechanisms.
1.4.1 Reversible Inhibition
A. Competitive Inhibition
A substance that competes directly with a normal substrate for an enzyme s
substrate-binding site is known as a competitive inhibitor. Such an inhibitor usually
resembles the substrate so that it specifically binds to the active site but differs from the
substrate in that it cannot react as the substrate does.
The general model for competitive inhibition is given by the following reaction scheme:
E + S ES P + E
+
I
EI + S NO REACTION
k1
k-1
k2
KI
Here it is assumed that I, the inhibitor, binds reversibly to the enzyme and is in rapid
equilibrium with it so that
KI =[ E ][ I ]
[ EI ]
and EI, the enzyme-inhibitor complex, is catalytically inactive. A competitive inhibitor
11
therefore reduces the concentration of free enzyme available for substrate binding.
Competitive inhibition studies are also used to determine the affinity of transition state
analogs for an enzyme s active site. If an enzyme preferentially binds its transition state, then
it can be expected that transition state analogs, stable molecules that geometrically and
electronically resemble the transition state, are potent inhibitors of the enzyme. Hundreds of
transition state analogs for various enzymes have been reported. Some are naturally occurring
antibiotics. Others were designed to investigate the mechanism of particular enzymes or to
act as specific enzyme inhibitors for therapeutic or agriculture use. Indeed, the theory that
enzymes bind transition states with higher affinity than substrates has led to a rational basis
for drug design based on the understanding of specific enzyme reaction mechanism.
B. Uncompetitive Inhibition
In uncompetitive inhibition, the inhibitor binds directly to the enzyme-substrate complex
but not to the free enzyme:
E + S ES P + E
+
I
ESI NO REACTION
k1
k-1
k2
K'I
In this case, the inhibitor binding step has the dissociation constant
12
K'I =[ ES ][ I ]
[ ESI ]
The uncompetitive inhibitor, which need not resemble the substrate, presumably distorts
the active site, thereby rendering the enzyme catalytically inactive. Uncompetitive inhibition
requires that the inhibitor affect the catalytic function of the enzyme but not its substrate
binding. This is difficult to envision for single-substrate enzymes. In actuality, uncompetitive
inhibition is significant only in multisubstrate enzymes.
C. Noncompetitive Inhibition
If both the enzyme and the enzyme-substrate complex bind inhibitor, the following
mode results:
E + S ES P + E
+
I
ESI NO REACTION
k1
k-1
k2
K'I
+
I
EI
KI
This phenomenon is known as noncompetitive inhibition. Presumably, a noncompetitive
inhibitor binds to enzyme sites that participate in both substrate binding and catalysis. The
two dissociation constants for inhibitor binding are not necessarily equivalent.
KI =[ E ][ I ]
[ EI ]and K'I =
[ ES ][ I ]
[ ESI ]
13
1.4.2 Irreversible Inhibition
If an inhibitor binds irreversibly to an enzyme, the inhibitor is classified as an
irreversible inhibitor or an inactivator. Reagents that chemically modify specific amino acid
residues can act as irreversible inhibitors. For example, the compounds used to identify the
catalytic Ser and His residues of serine proteases are irreversible inhibitors of these enzymes.
Irreversible in this context, however, does not necessarily mean that the enzyme activity
never returns, only that the enzyme becomes dysfunctional for an extended (but unspecified)
period of time. A compound that forms a covalent bond with an enzyme may lead to the
formation of an E-I complex that regains activity slowly. There are other cases of irreversible
inhibition in which no covalent bond forms at all, but the compound binds so tightly to the
active site of the enzyme that the rate constant for release of the compound from the enzyme
is exceedingly small. This, in effect, produces irreversible inhibition. Irreversible inhibitors
include affinity labeling agents and mechanism-based inhibitors.
A. Affinity Labeling Agents
Affinity labeling agents are generally reactive (electrophilic) compounds that alkylate or
acylate enzyme nucleophiles. Often more than one enzyme nucleophile reacts with these
compounds, and if a crude system containing more than one enzyme is being used, reactions
with multiple enzymes can occur. The closer that the structure of the affinity labeling agent
resembles that of the substrate for the target enzyme, the greater the specificity that will be
14
attained. A less reactive variation of affinity labeling agents, termed quiescent affinity
labeling agents, has been described. These inactivators contain an electrophilic carbon that is
stable in solution in the presence of external nucleophiles but which can react with an enzyme
active site nucleophile involved in covalent catalysis.
B. Mechanism-based Inhibitor
The name mechanism-based enzyme inactivator conjures up the idea of an enzyme
inactivator that depends on the mechanism of the targeted enzyme. In the broadest sense of
the term any of the inactivators that utilize the enzyme mechanism could be classified as
mechanism-based enzyme inactivators. In fact, Krantz and Pratt have suggested this general
classification for mechanism-based enzyme inhibitors. However, there is a more narrow
definition of mechanism-based enzyme inhibitor that Silverman has invoked. A
mechanism-based enzyme inactivator is an unreactive compound whose structure resembles
that of either the substrate or product of the target enzyme, and which undergoes a catalytic
transformation by the enzyme to a species that, prior to release from the active site,
inactivates the enzyme. A mechanism-based inhibitor requires a step to convert the compound
to the inactivating species by the catalytic machinery of the enzyme. Most often these
inhibitors result in covalent bond formation with the enzyme (Scheme 1.1), and, therefore,
dialysis or gel filtration does not restore enzyme activity. In other words, the inhibition is
irreversible.
15
E + I EI EI* E - I*kon
koff
k2 k3
k4
E + I*
Scheme 1.1
The ratio of product release to inactivation is termed the partition ratio and represents
the efficiency of the mechanism-based enzyme inactivator. When inactivation is the result of
the formation of an E I* adduct, the partition ratio is described by k4/k3. The partition ratio
depends on the rate of diffusion of I* from the active site, its reactivity, and the proximity of
an appropriate nucleophile, radical, or electrophile on the enzyme for covalent bond
formation. It does not depend on the initial inactivator concentration. There are some cases
where the partition ratio has been shown to be 0, that is, every turnover of inactivator
produces inactivated enzyme.
The two principal areas where mechanism-based enzyme inactivators have been most
useful are in the study of enzyme mechanisms and in the design of new potential drugs.
Mechanism-based inhibition is time dependent. Following a rapid equilibrium between
the enzyme and the mechanism-based inhibitor to give the EI complex, there is a slower
reaction that converts the inhibitor to the form that actually inactivates the enzyme. This
produces a time-dependent loss of enzyme activity. Formation of the EI complex occurs
rapidly, and the rate of inactivation is proportional to added inhibitor until sufficient inhibitor
is added to saturate all of the enzyme molecules. Then there is no further increase in rate with
additional inhibitor, that is, saturation kinetics is observed. Mechanism-based enzyme
16
inhibitors act as modified substrates for the target enzymes and bind to the active site.
Therefore, addition of a substrate or competitive reversible inhibitor slows down the rate of
enzyme inactivation. This is referred to as substrate protection of the enzyme.
Most enzyme inhibitor drugs (EID) are noncovalent, reversible inhibitors. The basis for
their effectiveness is the tightness of their binding to the enzyme, so that they compete with
the binding of substrates (S) for the enzyme. The efficacy of the drug continues as long as the
enzyme is complexed with the drug (E EID). Because the enzyme concentration is low and
fixed, the equilibrium between E + EID and E EID will depend on the concentrations of
EID and S. When the concentration of EID diminishes because of metabolism, the
concentration of E EID diminishes and the concentration of E S increases. To maintain the
pharmacological effect of the drug, administration of the drug several times a day, then,
becomes necessary. An effective mechanism-based enzyme inhibitor, however, could form a
covalent bond to the enzyme. This would mean that frequent administration of the drug
would not be necessary. Inactivation of an enzyme, however, induces gene-encoded synthesis
of that enzyme, but this could take hours to days before sufficient newly synthesized enzyme
is present. Although affinity labeling agents also could form a covalent bond to the enzyme,
the reactivity of these compounds renders them generally unappealing for drug use because
of the possibility that they could react with multiple enzymes or other biomolecules, thereby
leading to toxicity and side effects. Mechanism-based enzyme inhibitors, however, are
unreactive compounds, so nonspecific reactions with other biomolecules would not be a
problem. Only enzymes that are capable of catalyzing the conversion of these compounds to
the form that inactivates the enzyme, and also have an appropriately positioned active site
17
group to form a covalent bond, would be susceptible to inactivation. With a sufficiently
clever design it should be possible to minimize the number of enzymes that would be affected.
Provided the partition ratio is zero or a small number, in which case potentially toxic product
release would not be important, then a mechanism-based enzyme inhibitor could have the
desirable drug properties of specificity and low toxicity18.
1.4.3 Inhibitors of Human Leukocyte Elastase
The design of potent, synthetic inhibitors of HLE requires a thorough understanding of
the catalytic mechanism by which serine proteinases degrade proteins. This general
mechanism is illustrated in Figure 1.3. The catalytic triad of serine proteinases must generate
the tetrahedral intermediate in order for the enzyme to complete its task of cleaving
proteins. Synthetic, small molecule inhibitors of HLE may be realized if a proper design
element is built into the inhibitor which may cause an interference with the generation of the
tetrahedral intermediate. Numerous reports have been generated which describe in detail the
chemical strategy in designing potent and selective HLE inhibitors. All of these compounds,
however, can be divided into three subclasses: electrophilic ketones, acylating agents, and
miscellaneous agents.
Mechanism-based inhibitors are included in the miscellaneous agent subclass. They are
very important because mechanism-based inhibitors have some advantages against others. In
general, the design of a mechanism-based inhibitor involves the use of a suitable template to
which appropriate recognition and reactivity elements are appended. A wide range of
templates have been used in the design of mechanism-based inhibitors of serine proteases
18
such as HLE (Table 1.5).
NR1
O
O
OSO2R2 NN
R1
O
O
OSO2R3
R2N
NR1
R2
O
OOSO2R3
N
O
O
R1OSO2R2 X
N
O
Z
L
SN
OR1
LO O
N SN
R1O
R2L
O OS
NR
O
LO O
A B C
D E F
G H
Table 1.5 Templates Employed in Serine Protease Inhibitor Design (Mechanism-based Inhibitors)
The succinimide A19-22, hydantoin B23, dihydrouracil C24, and phthalimide D25, 26
templates, and variants thereof27, were first used in comjunction with the design of
mechanism-based inhibitors of (chymo)trypsin-like proteases that inactive the target enzyme
via an enzyme-induced Lossen rearrangement [Fig. 1.4].
19
N
O
O
OSO2R2
OH N
NH
Ser195
His57
E
N
O
OSO2R2
OHN
NH
Ser195
His57
EO
HN
NH
Ser195
His57
E
N
O
O
O
OSO2R2
N
NH
Ser195
His57
E
N
OO
CO
N
NH
Ser195
His57
EHN
OO
O
Figure 1.4 Mechanism-based Inactivation of a Serine Proteasevia an Enzyme-induced Lossen Rearrangement
As shown in Fig.1.4, these compounds react with the catalytic serine residue to give a
ring-opened species which undergoes a Lossen rearrangement to generate a latent
enzyme-bounded isocyanate. This isocyanate is subsequently attacked by the imidazole ring
of the catalytic histidine residue (His57) to give an enzyme-bound imidazole-N-carboxamide.
The double hit process leading to the formation of the enzyme inhibitor complex is
supported by 13C NMR studies28. The aforementioned approach has been extended to the
phthalimide template (Table 1.5, D). Kerrigan and coworkers29, 30 have demonstrated that the
introduction of hydrophobic and/or chiral substituents through an amide linkage at the
6-position of the phthalimide template enhances both the potency and selectivity of
(sulfonyloxy) phthalimide inhibitors. The mechanism of action of inhibitor D is similar to
that of A [Fig. 1.4]. The succinimide and phthalimide templates, as well as the saccharin
template, have also been used in the synthesis of mechanism-based inhibitors designed to
inactivate a serine protease via an enzyme-induced Gabriel-Colman rearrangement31. This
20
rearrangement involves the reaction of a phthalimido- or saccharino- acetic ester or ketone
with an alkoxide to yield a ring expansion product. The reaction is believed to involve
alkoxide-induced ring opening, followed by a prototropic shift of the resulting imide anion to
the carbanion and subsequent ring closure [Fig. 1.5].
XN
O
Z
RO
XN
Z
O OR
X
OR
O
N
Z
X
OR
O
NH
Z
XNH
ZO OR
XNH
ZO
XNH
ZOH
( X=CO, SO2; Z=COOR )
Figure 1.5 Mechanism of the Gabriel-Colman Rearrangement
It was reasoned that an appropriate saccharin or phthalimide derivative E might undergoes
enzyme-induced ring opening followed by a prototropic shift and loss of leaving group L to
yield a reactive electrophilic species (a Michael acceptor) which, upon further reaction with a
nearby nucleophilic residue, would lead to inactivation of the enzyme, as illustrated in Fig.
1.6.
21
XN
O
ZL
OH N
NH
Ser195
His57
E
XN
Z
O O
HN
NH
Ser195
His57
E
L
X
O
O
N
LZ
N
NH
Ser195
His57
E
X
O
O
HN
N
NH
Ser195
His57
E
X
O
O
HN
Ser195
His57
E
Z
L
ZN NH
X
O
O
HN
Ser195
His57
E
Z
NNH
Figure 1.6 Mechanism-based Inactivation of a Serine Proteasevia an Enzyme-induced Gabriel-Colman Rearrangement
The enzyme induced generation of a reactive Michael acceptor has also been
accomplished by employing various heterocyclic templates, including phthalimide D,
saccharin F32-36, 1,2,5-thiadiazolidin-3-one 1,1 dioxide H (Table 1.5)37, 38. In each instance,
enzyme-induced ring opening is followed by elimination to yield a reactive N-sulfonyl imine
(a Michael acceptor) which, upon further reaction with an active site neucleophilic residue
(His57) leads to irreversible inactivation of the enzyme [Fig. 1.7]39.
22
SN
O OH N
NH
Ser195
His57
E
SN
OHN
NH
Ser195
His57
EO
N
NH
Ser195
His57
E
S
O
O
N
NH
Ser195
His57
E
S
OO
O OL
O OL
NO O
HN
O O
Figure 1.7 Mechanism-based Inactivation of a Serine Proteasevia Enzyme-induced Formation of a Michael Acceptor
23
CHAPTER 2
DESIGN RATIONALE AND RESEARCH GOALS
Chronic obstructive pulmonary disease (COPD) (pulmonary emphysema and chronic
bronchitis) affects more than 16 million Americans and is the fourth most common cause of
death40. The pathogenesis of COPD is currently poorly understood41, 42. COPD is a
multifactorial disorder that is characterized by airway inflammation and the influx of
neutrophils, macrophages and natural killer lymphocytes to the lungs. This is accompanied by
the extracellular release of an array of proteases (serine, cysteine and metallo- proteases),
including the serine endopeptidases elastase, proteinase 3 and cathepsin G43, leading to a
protease/antiprotease imbalance44, 45. The presence of elevated levels of proteases in the lungs
leads to the degradation of lung elastin and other components of the extracellular matrix46, 47;
however, the identity, origin and precise role of the proteases involved in the pathogenesis of
COPD have not been rigorously defined as yet41, 42. The design and utility of novel
mechanism-based (suicide) inhibitors in mechanistic enzymology and drug discovery are
well-documented18. A mechanism-based inhibitor is an inherently unreactive compound that
acts as a substrate and is processed by the catalytic machinery of an enzyme, generating a
highly reactive electrophilic species which, upon further reaction with an active site
nucleophilic residue, leads to irreversible inactivation of the enzyme48. Inhibitors of this type
offer many potential advantages, including high enzyme specificity, since the latent reactivity
in the inhibitor is unmasked following catalytic processing of the inhibitor by the target
enzyme only.
24
SN
O
O OSO2NHR
N SN
O
O OSO2NHR
P1
R2
S1
S2 Sn'
(I) (II)
Figure 2.1 General Structures of Inhibitors (I-II)
2.1 Design Rationale for Inhibitors Derived from Saccharin Scaffold (Fig. 2.1 (I))
The biochemical rationale underlying the design of inhibitor (I) was based on the
following observations: (a) replacement of the carbonyl group in peptides with SO2 yields
-amido sulfonamides which are known to undergo a spontaneous fragmentation reaction, as
illustrated in Figure 2.2 (a)49, 50; (b) in contrast to acyclic -amido sulfonamides,
N-(phthalimidosulfonyl)-L-phenylalanine methyl ester 13 (Figure 2.2 (b), X = CO) and
related compounds have been shown to be stable (8); (c) inhibitors based on the saccharin
scaffold are known to dock to the active site of (chymo)trypsin-like proteases, an event that is
followed by acylation of the active site serine (Ser195)51-55; and, (d) the imidazole ring of
histidine residues located at the active site of enzymes is known to undergo facile Michael
addition reactions with conjugated systems56, 57. Based on these considerations, we
reasoned that an entity such as (I) might inactivate a target serine protease via a sequence of
steps involving the initial formation of a Michaelis-Menten complex, followed by
enzyme-induced ring opening and tandem fragmentation, leading to the release of an amine
or aminoacid ester, sulfur dioxide, and the formation of a Michael acceptor (in this instance,
an N-sulfonyl imine)58 capable of reacting with an active site nearby nucleophilic residue
(His57), ultimately leading to inactivation of the enzyme.
25
NH
NN
O
O
O
H
H
R1 R2
R
NH
N SN
O O
H
H
R1 R2
R
O ONH
N
O
R1
R
+ SO2 + H2NO
R2
a)
b)
XN
O
SO
NH
RO
X=CO,SO2
Figure 2.2 (a) Spontaneous Fragmentation of -Amido Sulfonopeptides to Yield a MichaelAcceptor; (b) -Amido Sulfonamide Motif in Phthalimide and Saccharin Derivatives.
2.2 Design Rationale for Inhibitors Derived from 1, 2, 5-Thiadiazolidin-3-one 1, 1
Dioxide Scaffold (Fig. 2.1 (II))
While the saccharin scaffold has been used in the design of protease inhibitors59, 60, it has
some serious limitations. For instance, structural constraints, such as the lack of a tetrahedral
carbon, preclude its binding to the active site in a substrate-like fashion. Secondly, synthetic
constraints severely limit the ready availability of ring-substituted derivatives. These
constraints make the systematic optimization of potency and enzyme selectivity of potential
inhibitors problematic. In contrast, a scaffold that binds to the active site of a target protease
like a substrate and is capable of orienting recognition elements toward the S and S´ subsites
26
would be expected to exhibit superior characteristics, since such a template would make
possible the exploitation of (a) binding interactions with multiple S and S´ subsites and, (b)
the subtle differences that exist in the various subsites of closely-related proteases. Based on
the above considerations, a versatile heterocyclic template, 1, 2, 5-thiadiazolidin-3-one-1, 1-
dioxide scaffold, (Figure 2.1, structure (II)) has been used in the design of a general class of
mechanism-based inhibitors of HLE61-69.
2.3 Research Goals
With respect to the above two types of compounds (I II), the following objectives were
explored:
1) Do inhibitors (I II) inactivate serine proteases, in particular HLE?
2) Do derivatives of I and II function as mechanism-based inhibitors?
3) What is the mechanism of inactivation of inhibitors (I II)?
4) Are inhibitors based on scaffold (II) more effective than those based on scaffold (I)?
27
CHAPTER 3
EXPERIMENTAL
3.1 Enzyme Assays and Inhibition Studies: Incubation Method
(A) Human Leukocyte Elastase (HLE)
Human leukocyte elastase ([E]f = 0.7 M) was assayed by mixing 10 L of a 70 M
enzyme solution in 0.05 M sodium acetate buffer, pH 5.5, 100 L dimethyl sulfoxide, and
890 L of 0.1 M HEPES buffer, pH 7.25, in a thermostatted test tube. A 100 L aliquot was
transferred to a thermostatted cuvette containing 880 L of 0.1 M HEPES, pH 7.25, and 20
L of a 7.0 mM solution of MeOSuc-Ala-Ala-Pro-Val-p-NA ([S]f = 0.14 M), and the change
in absorbance was monitored at 410 nm for 1 minute. In a typical inhibition run, 10 L of a
21 mM solution of inhibitor 9 ([I]f = 0.21 mM) in dimethyl sulfoxide and 90 L dimethyl
sulfoxide were mixed with 10 L of a 70 M enzyme solution ([E]f = 0.7 M) and 890 L of
M HEPES buffer, pH 7.25, and placed in a constant temperature bath, Aliquots (100 L)
were withdrawn at different time intervals and transferred to a cuvette containing 20 L of a
7.0 mM substrate solution ([S]f = 0.14 mM) and 880 L of 0.1 M HEPES buffer, pH 7.25.
The absorbance was monitored at 410 nm for 1 minute. The kinetics data obtained by using
the incubation method was analyzed by determining the slopes of the semilogarithmic plots
of enzymatic activity remaining vs time using eq 3.1 below, where [E]t/[E]o is the amount of
active enzyme remaining at time t.70
ln([E]t/[E]o) = kobs t (3.1)
28
(B) Trypsin
Bovine trypsin was assayed spectrophotometrically by mixing 10 L of a 0.50 mM
enzyme solution (0.1 M Tris-HCl buffer containing 0.01 M CaCl2, pH 7.2), 20 L dimethyl
sulfoxide, and 970 L 0.025 M sodium phosphate buffer containing 0.1 M NaCl, pH 7.51, in
a thermostated test tube. A 100 L aliquot was transferred to a thermostated cuvette
containing 10 L of a 60 mM solution of N -benzoyl-L-Arg p-nitroanilide and 890 L 0.025
M sodium phosphate buffer, and the change in absorbance was monitored at 410 nm for one
minute. In a typical inhibition run, 20 L of a 5.00 mM inhibitor solution in dimethyl
sulfoxide was mixed with 10 L of a 0.50 mM enzyme solution and 970 L 0.025 M sodium
phosphate buffer containing 0.1 M NaCl, pH 7.51, and placed in a constant temperature bath.
Aliquots (100 L) were withdrawn at different time intervals and transferred to a cuvette
containing 10 L of a 60 mM solution of N -benzoyl-L-Arg p-nitroanilide and 890 L 0.025
M sodium phosphate buffer, and the change in absorbance was monitored at 410 nm for one
minute. The pseudo first-order rate constants (kobs) were obtained by determining the slopes
of the semilogarithmic plots of enzymatic activity remaining vs time using eq 3.1 , where
[Et]/[Eo] is the amount of active enzyme remaining at time t. These are the average of two or
three determinations. The potency of the inhibitors was expressed in terms of the bimolecular
rate constant kobs/[I] M-1 s-1.
3.2 Enzyme Assays and Inhibition Studies: Progress Curve Method
The apparent second-order rate constants kinact/KI of compounds were determined by the
29
progress curve method (23). Thus, in a typical run 5 L of a 2.0 M HLE solution was added
to a solution containing 10 L of inhibitor (0.5 mM solution in dimethyl sulfoxide), 15 L
substrate (7.0 mM MeOSuc-Ala-Ala-Pro-Val p-NA), and 970 L 0.1 M HEPES buffer, pH
7.25, and the absorbance was continuously monitored at 410 nm for 600 s. Control curves in
the absence of inhibitor were linear. The pseudo-first order rate constants, kobs, for the
inhibition of HLE as a function of time were determined according to eq 2 below, where A is
the absorbance at 410 nm, vo is the reaction velocity at t = 0, vs is the final steady-state
velocity, kobs is the observed first-order rate constant, and Ao is the absorbance at t = 0. This
involved fitting by non-linear regression analysis the A ~ t data into eq 3.2 (SigmaPlot, Jander
Scientific) to determine kobs. The second order rate constants (kinact/KI) were determined in
duplicate or triplicate by calculating kobs/[I] and then correcting for the substrate
concentration and Michaelis constant using eq 3.3.
A = vst + {(vo vs) (1 e-kobs t)/kobs} + Ao (3.2)
kobs/[I] = kinact/KI {1 + [S]/Km} (3.3)
3.3 Hydroxylamine Reactivation of Inactivated HLE.
A solution containing 980 L 0.1 M HEPES buffer, pH 7.25, 10 L 21.9 mM inhibitor
([I]f = 219 M) in DMSO, and 10 L 21.9 M HLE ([E]f = 219 nM) was incubated for 30
minutes. The enzyme was totally inactivated, as shown by withdrawing an aliquot and
assaying for remaining enzyme activity. Excess hydroxylamine (100 L of a 0.50 M solution
in water) was then added to the fully-inactivated enzyme. Aliquots (100 L) were removed at
various time intervals (from one minute to 24 h) and assayed for remaining enzyme activity
30
by mixing with 20 L of a 3.86 mM solution of MeOSuc-Ala-Ala-Pro-Val p-nitroanilide ([S]f
= 77.2 M), and 880 L 0.1 M HEPES buffer, pH 7.25), and monitoring the absorbance at
410 nm. Enzyme activity was determined by comparing the activity of an enzyme solution
containing no inhibitor (control) with the activity of an enzyme solution containing inhibitor
at the same time point.
3.4 Substrate Protection
In separate experiments, the kobs/[I] values were determined by incubating HLE with
inhibitor 9 in the absence and presence of substrate. In the former case, 10 L of HLE ([E]f
= 219 nM) was incubated with 10 L of inhibitor ([I]f = 21.9 M) dissolved in DMSO and
0.1 M HEPES buffer (980 L), pH 7.25, in a thermostatted cuvette. Aliquots (100 L) were
withdrawn at different time intervals and added to a thermostatted cuvette containing 0.M
HEPES buffer (880 L), pH 7.25, and MeOSuc-Ala-Ala-Pro-Val pNA ([S]f = 77.2 M). The
absorbance was monitored at 410 nm and the kobs/[I] M-1s-1 value was then determined. The
kobs/[I] value was also determined by repeating the experiment in the presence of substrate:
HLE ([E]f = 219 nM) was incubated with inhibitor ([I]f = 21.9 M), and
MeOSuc-Ala-Ala-Pro-Val pNA ([S]f =231.6 M) in 0.1 M HEPES buffer (974 L, pH 7.25)
in a thermostatted cuvette. Aliquots (100 L) were withdrawn at different time intervals and
added to a thermostatted cuvette containing MeOSuc-Ala-Ala-Pro-Val pNA ([S]f = 77.2 M)
and 0.1 M HEPES buffer (880 L), pH 7.25.
3.5 Efficiency of Inactivation (Determination of Partition Ratio)
31
Ten microliters of inhibitor ([I]f = 3.5-42 M) in DMSO was incubated with 10 L HLE
([E]f = 0.70 M) and 980 L of 0.1 M HEPES buffer, pH 7.25, for 15 minutes. At the end of
the 15-minute incubation period enzyme activity was assayed by transferring an aliquot (100
L) to a cuvette containing 20 L MeOSuc-Ala-Ala-Pro-Val p-NA ([S]f = 0.14 M) and 880
L of 0.1 M HEPES buffer, pH 7.25, and monitoring the absorbance at 405 nm. The partition
ratio was calculated as described by Knight and Waley by plotting the fraction of remaining
enzyme activity ([E]t/[E]o) versus the initial ratio of inhibitor to enzyme ([I]/[E]o).
3.6 Reactivation of the HLE-Inhibitor Complex
Forty L of a 70.0 M solution of human leukocyte elastase was incubated with excess
inhibitor (10 L of a 25.2 mM solution in dimethyl sulfoxide), 40 L DMSO and 410 L 0.1
M HEPES buffer, pH 7.25 at 25.0 oC. After the solution was incubated for 30 minutes, a 25
L was removed and assayed for enzymatic activity (the enzyme was found to be completely
inhibited). Excess inhibitor was removed via Centricon-10 filtration by centrifuging at
14,000g for 45 minutes at 25.0 oC. Buffer (500 L 0.1 M HEPES buffer, pH 7.25) was added
to the HLE-inhibitor complex and the centrifugation was repeated at 14,000g for 1 h at 25.0
oC. The HLE-inhibitor complex was dissolved in 2.0 mL buffer and aliquots (100 L) were
withdrawn at different time intervals and added to a cuvette containing 20 L of 7.0 mM
MeOSuc-Ala-Ala-Pro-Val p-nitroanilide, 880 L of 0.1 M HEPES buffer, pH 7.25, and
monitoring the absorbance at 410 nm. The amount of active enzyme was determined by
comparing the activity of an enzyme solution containing no inhibitor (control) with the
activity of an enzyme solution containing inhibitor at the same time point.
32
3.7 HPLC Studies. Product Analysis and Identification from the Incubation of Inhibitor
with HLE.
A solution of HLE (50 L) containing 1 mg/mL of enzyme was added to a test tube
containing 300 L 0.1 M HEPES buffer, pH 7.25, containing 0.5 M NaCl and 0.2 mM
inhibitor. A second sample containing 0.1 N HEPES buffer, pH 7.25 with 0.5 M NaCl, and
0.2 mM inhibitor but no enzyme was used as a control. Both samples were incubated in a
water bath at 23 oC for 24 h. Fifty microliters of a saturated dansyl chloride solution (18
mg/mL) in methanol was added to each test tube and after 1 h the samples were analyzed by
HPLC. Both samples were stored at 4 oC for 72 h after which the clear solutions were
re-injected sequentially along with compound 14 (N-dansyl-L-Phe-OCH3) (used as a
standard).
N
O2S NHCOOCH3(L)
14
Each sample (20 L) and dansyl standard 14 (5 L) were sequentially injected into a 250 x
4.6 mm C18 Luna(2) column (Phenomenex) with a flow rate of 1.5 mL/min. Mobile phase: A
10% acetonitrile, 25% methanol, 65% water; B 10% acetonitrile, 90% methanol using a
gradient of 55% B to 100% B over 30 min using two 510 pumps, a 717+ refrigerated
autosampler, and a 474 fluorescence detector (excitation 380 emission 470), all controlled
33
by Millenium software (Waters). The fraction eluting from 10 to 11.5 min was collected and
analyzed by electrospray ionization (ESI) and collision-induced dissociation (CID) mass
spectrometry using a Finnigan LCQ-DeccaTM ion-trap mass spectrometer (Thermoquest).
34
CHAPTER 4
RESULTS AND DISCUSSION
4.1 Inactivation of HLE by Derivatives of (I) (Compounds 8-13)
SN
O
O OSO2 NHR
( I )
R= CH2CHPh2 (8) CH (CH2Ph ) COOCH3 (9)
( L - isomer ) CH (CH2Ph ) COOCH3 (10)
( D - isomer ) benzyl (11) phenethyl (12)
N
O
SO2 NHO
Ph
COOCH3
(13)
Figure 4.1 Structures of Compounds 8-13
(A) Relationship of Structure to Inhibitory Potency and Specificity
The inhibitory activity of compounds 8-13 toward HLE was evaluated using the
incubation method. The potency of the inhibitors was expressed in terms of the bimolecular
rate constants (kobs/[I] M-1 s-1), and these are listed in Table 4.1.
Table 4.1 Inhibitory Activity of Compounds 8-13 Towards Human Leukocyte Elastase and Bovine Trypsin
kobs/[I] (M-1s-1)Compound
Elastase Trypsin
8 270 80
9 870 290
10 870 320
11 90 Inactive
12 60 Inactive
13 Inactive
35
As shown in Table 4.1, with the exception of phthalimide derivative 13, the rest of the
compounds were also found to inhibit HLE. It is evident that inhibitory activity is
dependent on the nature of R. Assuming that R is oriented toward the S subsites, the spatial
requirements observed for R may simply reflect the inability of the saccharin template to bind
to the active site of the enzyme in a strictly substrate-like fashion. Furthermore, in the case of
compound 12, for example, the short alkyl chain (one methylene group) may not be sufficient
to place the phenyl ring close enough to Phe-41 of HLE for an optimal hydrophobic
-stacking interaction. The fact that phthalimide derivative 13 is devoid of any inhibitory
activity suggests that the interaction of the saccharin and phthalimide templates with the
active site involves a poorly-understood delicate interplay of binding and electronic
interactions that affect potency.
Among compounds 8-13, inhibitor 9 is the most potent one. The incubation of HLE with
inhibitor 9 led to rapid time-dependent loss of enzymatic activity (Figure 4.2). The enzyme
slowly regained virtually all of its activity after 24 h. Kitz and Wilson analysis70 of the data
(Figure 4.3) yielded a bimolecular rate constant kobs/[I] of 870 M-1 s-1. Interestingly, the
potency of the corresponding D isomer 10 was comparable to that of the L-isomer (kobs/[I]
870 M-1 s-1).
36
Time (hours)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 24.0
Perc
ent R
emai
ning
Act
ivity
(%)
0
20
40
60
80
100
120
Figure 4.2 Time-dependent Loss of Enzymatic Activity. a) Excess inhibitor 9 ([I]f = 0.21mM) wasincubated with human leukocyte elastase ([E]f = 0.70 M) in 0.1 M HEPES buffer, pH 7.25, 25 oC, aliquotswere withdrawn at different time intervals, and assayed for enzymatic activity using MeOSuc-Ala-Ala-Pro-Valp-nitroanilide ([S]f = 0.14 mM) (solid circles); b) A 300-fold excess of inhibitor 9 was incubated with bovinetrypsin ([E]f = 0.84 M) in 0.1 M Tris buffer, pH 7.51, containing M CaCl2, aliquots were withdrawn at differenttime intervals, and assayed for enzymatic activity using N-p-Tosyl-Gly-Pro-Lys p-nitroanilide ([S]f = 0.1 mM)(open circles).
37
Time (min)
0 2 4 6 8
Log
(%R
emai
ning
Act
ivity
)
0.0
0.5
1.0
1.5
2.0
I/E=50
I/E=200
I/E=400
I/E=600
Figure 4.3 Kinetics of Inactivation of Human Leukocyte Elastase ([E]f = 0.70 M) byCompound 9. Inhibitor 9 was incubated with human leukocyte elastase (the [I]/[E] ratio varied between 50 to600) in 0.1 M HEPES buffer, pH 7.25, at 0 oC. Aliquots were withdrawn periodically and assayed for remainingenzyme activity using MeOSuc-Ala-Ala-Pro-Val p-nitroanilide.
The specificity of compounds 8-12 was briefly investigated using bovine trypsin. Thus,
incubation of compound 9 with the enzyme led to rapid inactivation of the enzyme (Table
4.1), followed by gradual and full regain of enzyme activity after 24 h. The regain in
enzymatic activity was somewhat faster when compared to the analogous interaction with
HLE. Replotting of the data yielded a kobs/[I] value of 290 M-1 s-1 (Figure 4.4).
38
Time (min)
0 2 4 6 8
Log
(%R
emai
ning
Aci
tivity
)
0.0
0.5
1.0
1.5
2.0
I/E=300
I/E=500
I/E=800
I/E=1000
Figure 4.4 Kinetics of Inactivation of Bovine Trypsin by Compound 9. Excess inhibitor(the [I]/[E] ratio varied between 300 to 1000) was incubated with bovine trypsin ([E]f = 0.84 M) in 0.1 M Trisbuffer, pH 7.51, containing 0.021 M CaCl2. Aliquots were withdrawn at different time intervals and assayed forremaining enzyme activity using N-p-Tosyl-Gly-Pro-Lys p-nitroanilide ([S]f = 0.1 mM).
The results of the studies (Table 4.1) clearly indicate that (a) inhibitor recognition by
these enzymes is critically dependent on interactions with the S subsites of the enzyme, and
(b) potent inhibitors of trypsin-like enzymes can, in principle, be designed that primarily
exploit S interactions only and do not incorporate in their structure a Lys or Arg side chain.
Since the presence of the latter is associated with poor pharmacokinetics and low selectivity,
optimized derivatives of (I) may offer several distinct advantages.
39
(B) Mechanism of Inactivation
SN
O
O OSO2
HO Ser195 EHis57
NHR
( I )
SN
O OSO2 NHR
Ser195 EHis57-O O
SN
O
O O
OSer195 E
His57
SNH
O
O O
OSer195
His57E
H2O
SHN
O
O O
OSer195 E
His57
OH SNH2
O
O O
OSer195 E
His57
HO Ser195 EHis57
+ Low molecularweight product
( III ) ( IV )
( V )
Figure 4.5 Postulated Mechanism of Action of Inhibitor (I)
The proposed tentative mechanism of action of (I) (Figure 4.5) was probed as follows: a
substrate protection experiment was carried out in order to demonstrate that the interaction of
(I) with HLE involves the active site. This is clearly evident in Figure 4.6
40
Time (min)
0 2 4 6 8 10
Log
(% R
emai
ning
Act
ivity
)
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Figure 4.6 Substrate Protection. HLE (219 nM) was incubated with inhibitor 9 (21.9 M)in 0.1 M HEPES buffer, pH 7.25, aliquots were taken at different time intervals, and assayed for remainingenzyme activity using MeOSuc-Ala-Ala-Pro-Val p-nitroanilide (77.2 M). The experiment was repeated byincubating HLE (219 nM), inhibitor 9 (21.9 M) and MeOSuc-Ala-Ala-Pro-Val p-nitroanilide (0.232 mM) in 0.1 M HEPES buffer, pH 7.25. Aliquots were taken at different time intervals andassayed for remaining enzyme activity using MeOSuc-Ala-Ala-Pro-Val p-nitroanilide (77.2 M). The data wasthen analyzed using the method of Kitz and Wilson .
where the presence of substrate in the incubation mix led to a decrease in kobs/[I] from 320 to
220 M-1 s-1. Next, the possible formation of an HLE-inhibitor acyl enzyme complex (or
complexes) was investigated by adding excess hydroxylamine (0.50 M) in 0.1 M HEPES
buffer, pH 7.25) to HLE that had been fully inactivated with a 100-fold excess of inhibitor 9,
and the regain in enzymatic activity was monitored over a 24 h period (Figure 4.7).
41
Time (hour)
0.0 0.5 1.0 1.5 2.0 25.0
Per
cent
Rem
aini
ng A
ctiv
ity
0
10
20
30
40
50
60
70
80
90
100
Figure 4.7 Effect of Hydroxylamine on Enzyme Reactivation. Human leukocyte elastase(219 nM) was totally inactivated by incubating with a 100-fold excess of inhibitor 9 for thirty minutes in 0.1 MHEPES buffer, pH 7.25. Excess hydroxylamine was added (0.045 mM final concentration, closed circles), andaliquots were removed at different time intervals, and assayed for remaining enzyme activity usingMeOSuc-Ala-Ala-Pro-Val p-nitroanilide (77.2 M).
The data suggest that the regain in enzymatic activity arises from the possible
presence of a labile acyl linkage that leads to active enzyme upon treatment with
hydroxylamine. Thus, it can be reasonably assumed that the interaction of the inhibitor with
HLE leads to acylation of the active site serine, however, further structural studies are needed
to arrive at a definitive conclusion regarding the precise structure of the acyl enzyme complex.
The partition ratio, a parameter that corresponds to the number of molecules of inhibitor
necessary to inactivate a single molecule of enzyme, and thus describes how efficiently a
mechanism-based inhibitor inactivates an enzyme, was determined by plotting the fraction of
42
remaining enzyme activity after a 15-minute incubation period versus the initial ratio of
inhibitor to enzyme (Figure 4.8).
0.00
20.00
40.00
60.00
80.00
100.00
0 10 20 30 40 50 60 70 80 90 100
[I] / [E]
Perc
ent R
emai
ning
Act
ivity
Figure 4.8 Inactivation of Human Leukocyte Elastase as a Function of the Molar Ratio ofInhibitor 9 to Enzyme. HLE (70 M) and various amounts of inhibitor 9 (0.35-4.20 M in 0.1 M HEPESbuffer, pH 7.25, were incubated for fifteen minutes, aliquots were withdrawn at the end of the incubation period,and assayed for remaining enzyme activity using MeOSuc-Ala-Ala-Pro-Val p-nitroanilide. Thefractional activity remaining is proportional to the molar ratio of inhibitor to enzyme.
The extent of inactivation was found to be linearly dependent on the inhibitor to enzyme
molar ratio. Extrapolation of the linear part of the curve to the line of complete inactivation
yielded a partition ratio of 40 for inhibitor 9, attesting to a moderately efficient inhibitor.
The reactivation of the complex formed between HLE and inhibitor 9 was also
investigated. Thus, HLE was totally inactivated using excess inhibitor 9. The excess inhibitor
was then removed be Centricon-10 filtration, and the regain in enzymatic activity was
monitored. The experiment was repeated using inhibitor 10. In both cases total regain of
43
enzymatic activity was observed, suggesting the likely formation of one enzyme-inhibitor
complex. The deacylation rate constants (kdeacyl) for the HLE-inhibitor complexes derived
from inhibitors 9 and 10 were determined by replotting the data according to eq 3.1. These
were found to be 0.0028 and 0.0041 s-1, respectively.
To establish that the interaction of 9 with HLE produces an N-sulfonyl imine that arises
from an enzyme-induced fragmentation process, the products formed by incubating inhibitor
9 with HLE were identified using HPLC and mass spectrometry. Initial HPLC analysis of the
assay solution showed that a trace of (L) Phe-OCH3 was observed in this assay, possibly
arising from the slow hydrolysis of inhibitor 9, which was labeled by dansyl chloride
resulting in a small background. Therefore, a control given identical treatment in each step of
the experiment, except for the addition of HLE, was used in order to subtract out this
background. HPLC analysis of the two samples, after performing the product identification
procedure described under EXPERIMENT, showed that a peak with an identical retention
time to compound 14 standard s peak (10.3 min) was observed in both samples. The sample
including HLE had an area 2.7 times greater than the control, demonstrating that this
compound is a product arising from the HLE-inhibitor 9 reaction (Figure 4.9). The fractions
eluting from 10 to 11.5 min of both the standard 14 (Figure 4.9A) and the HLE-inhibitor
reaction (Figure 4.9C) were collected and analyzed by mass spectrometry. ESI revealed
identical protonated molecule peaks of 413 amu, and CID of the 413 peak of each sample
gave identical gave identical fragmentation patterns, with a principal peak at 301 amu.
44
A
B
C
D
Figure 4.9 HPLC Analysis of Products Formed by Incubating Human Leukocyte Elastasewith Inhibitor 9. (a) Standard: compound 14 (N-Dansyl-L-Phe-OCH3); (b) Control: 20 Linjection of assay composed of 300 L of 0.1 M HEPES buffer, pH 7.25, containing 0.5 M NaCl, and 0.2 mMcompound 9 after incubation for 24 h at 23 C, addition of 50 L saturated dansyl chloride in methanol, and 72h incubation at 4 oC.; (c) Compound 9 / HLE reaction: 20 L injection of assay composed of 300 L of 0.1 MHEPES buffer, pH 7.25, with 0.5 M NaCl, 0.2 mM compound 9, and 50 L HLE (1 mg/mL) after incubation for24 h at 23 C, addition of 50 L saturated dansyl chloride in methanol and 72 h incubation at 4 oC; (d) Overlayof panels B and C: the peaks with 10.3 min retention times were one compound and had identical molecularion peaks and fragmentation patterns, corresponding to standard 14.
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
220.00
240.00
260.00
280.00
300.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
10.3
01
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
220.00
240.00
260.00
280.00
300.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
10.3
43
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
220.00
240.00
260.00
280.00
300.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
6.95
0 10.2
95
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
220.00
240.00
260.00
280.00
300.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00
45
Therefore, the product arising from the inhibition of HLE by inhibitor 9 is confirmed to be (L)
Phe-OCH3. The chemical competence of the cascade steps outlined in Figure 4.5 was readily
established by stirring inhibitor 9 with excess sodium methoxide in methanol at room
temperature which led to rapid disappearance of the inhibitor. NMR product analysis revealed
the presence of a roughly 1:1 mixture of (o-carboxymethyl)benzene-sulfonamide and
Phe-OCH3. Taken together, these results suggest that the binding of (I) to the active site of
HLE leads to acylation of the enzyme with concomitant sulfonamide fragmentation resulting
in the release of L-Phe-OCH3, SO2 and the formation of N-sulfonyl imine (III) (Figure 4.5).
The available evidence suggests that (III) ultimately leads to the formation of a stable acyl
enzyme whose structure can be tentatively represented by structure (V). The slow deacylation
rate observed with (V) may be the result of a conformational change in the enzyme that
perturbs the positions of the catalytic residues and/or H-bonding between the SONH2 group
of the tethered inhibitor and the imidazole ring of His-57, thereby impairing the ability of
His-57 to function in general base catalysis. Lastly, while the initial design of inhibitor (I) and
postulated mechanism of action invoked the likely involvement of a double hit mechanism
leading to the formation of species (IV), the available data can be adequately explained by the
formation of a stable acyl enzyme species, particularly in light of earlier high-field NMR
studies62 which demonstrated the formation of formaldehyde from species (IV). In previous
studies using saccharin derivatives of the type Saccharin-CH2X, where X is a good leaving
group (halide, carboxylate, etc.), a similar mechanism involving the formation an N-sulfonyl
imine (structure (III), Figure 4.5) was proposed for the inactivation of HLE51, 36. It should be
noted, however, that compounds represented by (I) are intrinsically more stable chemically
46
and employ a sulfonamide fragmentation reaction as the driving force for the formation of the
N-sulfonyl imine.
4.2 Inactivation of HLE by Derivatives of (II) (Compounds 4-7)
R2N
P1
SN
O
O O
S NHCOOR
O O
( II )
P1= isobutylR2= benzylR= CH3 (L, L) (4)
(L, D) (5)benzyl (L, L) (6)H (L, L) (7)
Figure 4.10 Structures of Compounds 4-7
(A) Relationship of Structure to Inhibitory Potency and Specificity
All four derivatives of (II) were found to inactivate HLE rapidly and in a time-dependent
fashion (Table 4.2).
Table 4.2 Inhibitory Activity of Compounds 4-7 Toward Human Leukocyte Elastase and Bovine Trypsin.
Compound HLEa Trypsinb
4 (L,L) 6,700 (870)c 480
5 (L,D) 38,800 (870)c 500
6 (L,L) 5,900 350
7(L,L) 9,600 1200akinact/KI M-1 s-1 values (progress curve method) are the average of several runs fit to equation 2 with R2 > 0.999;bkobs/[I] M-1 s-1 values (incubation method) and are the average of two or three determinations;cvalues in parentheses are for the corresponding saccharin derivatives (ref 19).
47
The incubation of inhibitor 4 with HLE led to rapid and time-dependent inactivation of
the enzyme (Figure 4.11).
Figure 4.11 Time-dependent Loss of Enzymatic Activity. Excess inhibitor 4 ([I]f = 14 M)was incubated with human leukocyte elastase ([E]f = 0.7 M) in 0.1 M HEPES buffer, pH 7.25, 25 oC, aliquotswere withdrawn at different time intervals, and assayed for enzymatic activity using MeOSuc-Ala-Ala-Pro-Val p-nitroanilide.
The enzyme regained its activity slowly but completely within 24 h (half-life for enzyme
reactivation ~ 3 h). The kinact/KI M-1 s-1 values for inhibitors 4-7 are listed in Table 4.2, along
with the values for the corresponding saccharin derivatives for compounds 4 and 5 (in
parentheses). It is clearly evident from these results that the 1, 2, 5-thiadiazolidin-3-one 1,1
dioxide scaffold is superior to the saccharin scaffold, yielding highly potent inhibitors of HLE.
This is in agreement with the design that derivatives of (II) can bind to the enzyme in a more
Time (h)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 24.0
Per
cent
Rem
aini
ng A
ctiv
ity
0
20
40
60
80
100
120
48
substrate-like fashion by varying the P1 to satisfy the enzyme specificity.
Surprisingly, the most potent inhibitor was found to be diastereomer 5, and this was
significantly more potent than diastereomer 4, suggesting that the binding of 5 to the active
site leads to a more effective - stacking interaction between Phe-41 and the phenyl group in
the inhibitor. Furthermore, because the S and S´ subsites of HLE are fairly hydrophobic, the
enzyme shows a strong preference for hydrophobic substrates and inhibitors. Thus, the
relatively high potency of inhibitor 7, despite the presence of a polar group, is also surprising.
Clearly, further structural studies are needed in order to gain a better understanding of these
observations. Taken together, these findings suggest that inhibitors having optimized potency
and pharmacokinetics can be realized using this type of mechanism-based inhibitor.
Previous studies have shown that the attachment of various leaving groups (halogen,
carboxylate, heterocyclic sulfide, sulfone, etc.) to the 1, 2, 5-thiadiazolidine-3-one 1,1 dioxide
scaffold yields highly potent inhibitors of HLE62-69. Compared to those inhibitors,
compounds 4-7 are less potent by one to two orders of magnitude, however, sulfonamide
derivatives 4-7 are intrinsically more stable chemically and appear to strike a better balance
between potency and chemical stability.
An intriguing observation, previously made with the corresponding saccharin derivatives
(19), was their observed inhibitory activity toward bovine trypsin. Thus, despite the fact that
those compounds lacked a basic side chain (the primary substrate specificity residue P1 for
trypsin is Lys or Arg), the compounds did exhibit inhibition toward trypsin, suggesting that
favorable binding interactions with the S´ subsites are sufficient for bestowing inhibitory
activity, and that the presence of a basic side chain is not necessary for inhibitory activity.
49
Based on those considerations, the inhibitory activity of compounds 4-7 toward trypsin was
investigated. All four compounds were found to be time-dependent inhibitors of trypsin. For
example, incubation of compound 7 with trypsin led to rapid and efficient time-dependent
loss of enzyme activity (Figure 4.12).
Figure 4.12 Time-dependent Loss of Enzymatic Activity. Excess inhibitor 7 (at the indicated inhibitor toenzyme molar ratios) was incubated with 10 L bovine trypsin ([E]f = 5.0 M) in 0.025 phosphate buffercontaining M NaCl, pH 7.51, 25 oC, aliquots were withdrawn at different time intervals, and assayed forenzymatic activity using N -benzoyl-L-Arg p-nitroanilide.
Only partial regain of enzymatic activity was observed after 24 h, and the extent of
reactivation was found to be dependent on the inhibitor/enzyme ratio used. Replotting of the
data according to equation 1 yielded the observed rate constant of enzyme inactivation (kobs)
and the potency of the inhibitors was then expressed as the bimolecular rate constant, kobs/[I]
M-1 s-1 (values are listed in Table 4.2). Assuming that these compounds bind to the active
site of trypsin, then the results suggest that favorable binding interactions with the S´ subsites
Time (h)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 24.0
Perc
ent R
emai
ning
Act
ivity
(%)
0
20
40
60
80
100
120
I/E=5
I/E=20
50
can be exploited and used to guide the design of potent inhibitors of trypsin and trypsin-like
serine proteases that lack a basic side chain. The presence of a basic side chain in thrombin
inhibitors has been shown to affect adversely oral bioavailability. Compounds 4-7 were
inactive toward papain, a prototypical cysteine protease, when incubated with the enzyme at
an [I]/[E] ratio of 250 for 0.5 h.
(B) Mechanism of Inactivation
Derivatives of scaffold (II) were envisioned to inactivate HLE by the mechanism shown
in Figure 4.13, where binding to the active site of the enzyme is followed by nucleophilic
attack by Ser195 to form a tetrahedral adduct. Collapse of the adduct via a sulfonamide
fragmentation-driven process as anticipated to generate a highly reactive electrophilic species
(an N-sulfonyl imine) which could be trapped by the active site histidine (His57) to form
enzyme-inhibitor adduct (A) (Figure 4.13, path (a)), or react with water to form a stable acyl
enzyme (B) (Figure 4.13, path (b)). It should also be noted that, since the P1 group2 in
inhibitor (II) serves as the primary specificity residue that is accommodated at the primary
specificity subsite (S1) of a target enzyme, enzyme selectivity can be attained by simply
varying the nature of P1. In the present case, since HLE shows a strong preference for
medium size hydrophobic side chains (such as those of Val and Leu), (L) Leu was choosen as
P1. Furthermore, in order to enhance the binding interactions of (II) with the S´ subsites of the
enzyme, an aromatic residue (Phe) was linked to the heterocyclic scaffold for a favorable
-stacking interaction with Phe-41 (located close to the S2´ subsite). Interactions with the S´
subsites were also probed by investigation of the corresponding inhibitor derived from D-Phe
51
methyl ester. Lastly, in order to optimize aqueous solubility without compromising potency,
the presence of a polar group was also investigated.
P1
SN
O
O OSO2
HO Ser195 EHis57
NH
( II )
SN
O OSO2 N
H
Ser195 EHis57-O O
SN
O
O O
OSer195 E
His57
SNH
O
O O
OSer195
His57E
H2O
R2 SNH2
O
O O
OSer195 E
His57
R2
COOR
Ph
P1
R2
COOR
Ph
P1
R2
path (b)
path (a)P1
R2
P1
( A )
( B )
Figure 4.13 Postulated Mechanism of Inhibition ofHuman Leukocyte Elastase by Inhibitor (II).
The complete recovery of enzymatic activity (Figure 4.11) suggests that the interaction
of (II) with HLE leads to the eventual formation of a relatively stable acyl enzyme (Figure
4.13, structure B). It s not intuitively obvious what the determinants of acyl enzyme (B)
stability are, although it s tempting to speculate that the SO2NH2 may be involved in a
52
H-bonding interaction with His-57, impeding its ability to function in general base catalysis.
Subtle differences in the make up of the active sites of HLE and trypsin may account for the
differences in the observed deacylation rates for the two enzymes.
53
CHAPTER 5
CONCLUSIONS
In conclusion, two novel classes of mechanism-based inhibitors of serine proteases that
incorporate in their structure either an appropriately-functionalized saccharin scaffold, or a 1,
2, 5-thiadiazolidin-3-one 1, 1 dioxide scaffold capable of generating a highly reactive
Michael acceptor by an enzyme-induced sulfonamide fragmentation process. The inactivation
of the enzyme by these inhibitors was found to be efficient, time-dependent and to involve
the active site. Biochemical studies show that the interaction of these inhibitors with HLE
results in the initial formation of a Michaelis-Menten complex and subsequent formation of a
tetrahedral intermediate with the active site serine (Ser-195). Collapse of the tetrahedral
intermediate with tandem fragmentation results in the formation of a highly reactive
conjugated sulfonyl imine which can either react with water to form a relatively stable acyl
enzyme and/or undergo a Michael addition reaction with an active site nucleophilic residue
(His-57). The results also demonstrate convincingly the superiority of the 1, 2,
5-thiadiazolidin-3-one-1, 1-dioxide scaffold over the saccharin scaffold in the design of
inhibitors of (chymo)trypsin-like serine proteases.
54
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