Nε-THIOACETYL-LYSINE AS A MULTIFACETED TOOL FOR ENZYMATIC
PROTEIN LYSINE Nε-DEACETYLATION
A Thesis
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
David G. Fatkins
August, 2007 Nε-THIOACETYL-LYSINE AS A MULTIFACETED TOOL FOR ENZYMATIC
PROTEIN LYSINE Nε-DEACETYLATION
David G. Fatkins
Thesis
Approved: Accepted:
______Advisor Dean of the College Dr. Weiping Zheng Dr. Ronald F. Levant
______Faculty Reader Dean of the Graduate School Dr. Kim C. Calvo Dr. George R. Newkome
______Department Chair Date Dr. Kim C. Calvo
ii ABSTRACT
The histone deacetylase (HDAC) family of enzymes catalyze the specific lysine Nε- deacetylation of proteins such as the core histone proteins, various transcription factors, alpha-tubulin, and acetyl-coenzyme A synthetase that are respectively involved in transcriptional, cytoskeletal, and metabolic control. HDAC-catalyzed reactions represent an integral component for an emerging intracellular signaling mechanism defined by the protein posttranslational reversible lysine Nε-acetylation and deacetylation, and the
enzymes involved have been targeted for developing novel therapies for metabolic and
age-related diseases and cancer. In this study, we disclose the synthesis and
characterization of Nε-thioacetyl-lysine as a multi-faceted tool for enzymatic protein
lysine Nε-deacetylation. In specific, by evaluating multiple peptides containing Nε- thioacetyl-lysine, i) we developed the first spectrophotometric assay selective for
HDAC8 that holds potential for rapid inhibitor screening and selective determination of
HDAC8 activity; ii) we identified potent and selective peptide-based inhibitors for
SIRT1, SIRT2, and SIRT3 that are the only bona fide human class III protein deacetylase enzymes with known physiological substrates. All these results have far-reaching impact on promoting our fundamental understanding and pharmacological exploitation of
HDAC-catalyzed reactions.
iii DEDICATION
I would like to dedicate this work to my parents, Becky and George and my best friend, Laura. To my parents, thank you for all of the sacrifices you made and the opportunities you have given me to become successful in life. To Laura, thank you for always supporting me and helping me overcome any difficulties in life.
iv ACKNOWLEDGMENTS
I would like to thank a number of people who have made my thesis work possible.
The person I would like to thank the most is my advisor, Dr. Weiping Zheng. Thanks to his ideas, suggestions, hard work, and inspiration, our research goals were realized. I am also grateful for the financial support from the James and Martha J. Foght Endowment,
The University of Akron Research Foundation, and The University of Akron Faculty
Research Fellowship. I thank Prof. Tony Kouzarides (University of Cambridge, UK) for the GST-SIRT1 plasmid. Finally, I also thank Prof. Chrys Wesdemiotis and his research group at The University of Akron for the assistance with mass spectrometric analysis.
v TABLE OF CONTENTS
Page
LIST OF TABLES...... ix
LIST OF FIGURES ...... x
LIST OF SCHEMES...... xii
CHAPTER
I. EMPLOYING Nε-THIOACETYL-LYSINE TO DEVELOP A SPECTROPHOTOMETRIC ASSAY FOR HUMAN HISTONE DEACETYLASE 8...... 1
Overview of protein deacetylases ...... 1
Structural aspects of classical protein deacetylases...... 3
Biological functions of classical protein deacetylases...... 8
Regulation of classical protein deacetylases...... 10
Existing assays for human classical protein deacetylases...... 11
Statement of the Problems and Objectives ...... 14
Design Rationale...... 15
Results and Discussion ...... 16
DTNB-based spectrophotometric assay condition establishment...... 20
Assay with recombinant HDAC8 ...... 20
Assay with HeLa nuclear extracts...... 22
Conclusion ...... 25
vi Experimental Section...... 26
Synthesis of Nα-Fmoc-Nε-thioacetyl-lysine ...... 27
Peptide synthesis and purification ...... 28
Kinetic analysis of the reaction between DTNB and the commercially available thioacetic acid...... 30
HPLC-based enzymatic assay...... 30
DTNB-based enzymatic assay ...... 31
II. EMPLOYING Νε-THIOACETYL-LYSINE TO DEVELOP POTENT AND/OR SELECTIVE INHIBITORS FOR HUMAN CLASS III PROTEIN DEACETYLASES...... 32
Overview of class III protein deacetylases ...... 32
Structural aspects of sirtuins ...... 32
Biological functions of sirtuins...... 33
Regulation of sirtuins...... 34
Existing inhibition strategies and inhibitors for human class III protein deacetylases ...... 36
Statement of the Problems and Objectives ...... 38
Design Rationale...... 39
Results and Discussion ...... 40
Conclusion ...... 48
Experimental Section...... 49
Peptide synthesis and purification ...... 49
Synthesis of Nα-acetyl-Nε-thioacetyl-lysine...... 50
Human sirtuin inhibition assays...... 50
Initial SIRT1 assay...... 51
vii SIRT2 & SIRT3 assays...... 52
Human HDAC8 assay...... 53
REFERENCES ...... 55
END NOTES ...... 67
viii LIST OF TABLES
Table Page
1. Overview of classical and non-classical protein deacetylases...... 2
2. Inhibition of SIRT1...... 44
3. Sirtuin inhibitor evaluation ...... 47
ix LIST OF FIGURES
Figure Page
1. The lysine Nε-acetylation and deacetylation reactions catalyzed respectively by protein acetyltransferases and protein deacetylases...... 1
2. A schematic representation of classical HDACs’ primary structures. The black boxes denote the conserved catalytic domains; the open box denotes the pseudo-catalytic domain found in HDAC10...... 6
3. Chemical structures of MAL & Z-MAL...... 13
4. Structural comparison of Nε-acetyl lysine and Nε-thioacetyl lysine...... 15
5. Analytical RP-HPLC profiles for the purified peptides used in this study. Peptide sequences are H2N-KKGQSTSRHKXLMFKTEG-COOH with X = Lys (peptide 1c), Nε-acetyl lysine (peptide 1b), and Nε-thioacetyl lysine (peptide 1a)...... 16
6. Representative RP-HPLC chromatograms from HDAC8-catalyzed de(thio)acetylation of peptides 1a & 1b, parts A and B and non-enzymatic reactions of peptides 1a & 1b, parts C and D...... 18
7. Reaction scheme for the DTNB-based spectrophotometric assay developed in this study. Peptide sequence: H2N-KKGQSTSRHKXLMFKTEG-COOH with X = Lys (peptide 1c), Nε-thioacetyl-lysine (peptide 1a)...... 19
8. Kinetic analysis of DTNB reacted with the commercially available thioacetic acid. Each data point corresponded to an average of the duplicate measurements that agreed with each other within 10%. Concentrations of thioacetic acid used are: ○, 10 μM; ●, 25 μM; ■, 50 μM; ♦, 75 μM; ▲, 100 μM...... 21
9. Standard curve generated with the commercially available thioacetic acid. Each data point corresponded to an average of the duplicate measurements that agreed with each other within 20%...... 22
x 10. Comparative analyses of enzymatic dethioacetylation of peptide 1a to form peptide 1c (product). HPLC: HPLC-based assay quantifying the released peptide 1c; UV-Vis: DTNB-based spectrophotometric assay quantifying the released thioacetate...... 23
11. HPLC chromatograms showing only intact peptide 1a & AcNH- RH(ThAcK)(ThAcK)-CONH2 were recovered from HPLC-based assay with HeLa nuclear extracts. All assays were performed in duplicates and identical results were obtained. A minor impurity in the purified peptide 1a sample was present in the chromatogram at tR~29.5 min, rather than the dethioacetylated product, peptide 1c...... 24
12. A brief representation of the proposed sirtuin chemical mechanism...... 40
13. Representative HPLC assay chromatograms for SIRT1 assay with peptides 1a & 1b...... 42
14. Peptides used in this study. The following peptide templates were used in this study: Peptide1a-c, SIRT1 substrate human p53 tumor suppressor protein (372-389); Peptide 1d, p53 (380-384); Peptide 2, SIRT2 substrate human α- tubulin (36-44); Peptide 3a-c, SIRT3 substrate human Acetyl-coenzyme A synthetase 2 (AceCS2) (633-652)...... 45
15. Representative HPLC chromatograms from HDAC8 assays with peptide 1b, 2, and 3a. All assays were performed in duplicate and essentially the same HPLC chromatograms were obtained for duplicates. The small peak with tR~27 min in the second chromatogram was from a minor impurity in the purified peptide 2 sample, rather than the dethioacetylated product ...... 48
xi LIST OF SCHEMES
Scheme Page
1. Chemical synthesis of Nα-Fmoc- Nε thioacetyl lysine...... 17
2. Chemical synthesis of Nα-Acetyl-Nε thioacetyl lysine...... 44
xii CHAPTER I
EMPLOYING Nε-THIOACETYL-LYSINE TO DEVELOP A
SPECTROPHOTOMETRIC ASSAY FOR HUMAN HISTONE DEACETYLASE 8
Overview of protein deacetylases
Protein posttranslational lysine Nε-acetylation occurs in many intracellular
proteins such as core histone proteins and non-histone proteins (e.g. p53, α-tubulin, and
acetyl coenzyme A synthetase) that are respectively involved in gene transcriptional, cytoskeletal, and metabolic control.1-3 Protein posttranslational Nε-acetylation is a
reversible modification. Protein acetyltransferases and protein deacetylases catalyze the
Nε-acetylation and the Nε-deacetylation reactions, respectively (Figure 1).4-6
H3C O
HN H3N
Protein deacetylases H H Protein acetyl- N transferases N H O H O Acetylated protein Deacetylated protein
Figure 1. The lysine Nε-acetylation and deacetylation reactions catalyzed respectively by protein acetyltransferases and protein deacetylases. 1 To date, eighteen protein deacetylases have been discovered and have been
categorized into four different classes based on homology with yeast transcriptional
repressors, phylogenetic analysis, and different cofactor requirements (Table 1).5 Class
I, II and IV protein deacetylase enzymes are collectively known as the classical protein deacetylases. These enzymes require a catalytic zinc (Zn2+) for activity, while the class
III protein deacetylases, otherwise known as sirtuins, all require the coenzyme NAD+ for activity.5,7
Table 1.a Overview of classical and non-classical protein deacetylases.5
Protein deacetylase Amino Acids Chromosomal location Class I HDAC1 482 1p34.1 HDAC2 488 6q21 HDAC3 428 5q31.1-5q31.3 HDAC8 377 Xq21.2-Xq21.3
Class II HDAC4 1084 2q37 HDAC5 1122 17q21 HDAC6 1215 Xp11.23 HDAC7 952 12q13.1 HDAC9 1011 7p15-p21 HDAC10 669 22q13.31-13.33
Class IV HDAC11 347 3p25.1
Class III (sirtuins) SIRT1 747 10q22.2 SIRT2 389 19q13 SIRT3 399 11p15.5 SIRT4 314 12q SIRT5 310 6p22.3 SIRT6 355 19p13.3 SIRT7 400 17q a HDAC, histone deacetylase; SIRT, sirtuin.
2 Structural aspects of classical protein deacetylases
The classical protein deacetylases consist of classes I, II, and IV. These enzymes
are classified based on sequence similiarity.8,9 Class I protein deacetylases consist of
HDAC 1, 2, 3, and 8. These four enzymes belong to the yeast reduced potassium dependency 3 (Rpd3)-like class I HDACs. Class I HDACs are further divided into an
HDAC1/HDAC2 and an HDAC3 subclass based on phylogenetic analysis.7 HDAC1 and
HDAC2 enzymes possess a high level of sequence homology. In mammals, these two
enzymes have around 82 % sequence identity and a nearly indistinguishable genomic
organization.10,11
The structural organization of the class I HDACs are similar in some ways, but
have a few different regions present that enable their unique functional roles. HDAC1
and 2 are nuclear enzymes containing 482 and 488 amino acids with a molecular mass of
~55 kDa.12,13 HDAC1 is composed of three important functional domains. The first
domain is known as HDAC association domain (HAD) and is located at the N-terminus
within residues 1-53. This domain is necessary for HDAC1 homodimerization, and its
association with HDAC2 and other proteins.14 The second domain is found in a central region of the protein (residues 25-303). Here the central zinc-binding catalytic domain known as HDAC consensus motif is found. Several conserved histidine and aspartate residues are present within the active site pocket of the enzyme in this domain.14,15 The third domain is located at the C-terminal region and contains a lysine rich area with a core nuclear localization sequence (NLS).14
The third enzyme, HDAC3 is more analogous in structural organization to
HDAC1, 2 than HDAC8. HDAC3 is a nuclear and cytoplasmic enzyme and has 428
3 amino acid residues with a molecular mass of 49 kDa.15,16 The sequence variations of
HDAC3 with HDAC1 occur at both the N terminal and C terminal ends of the protein.
At the far N terminus, HDAC3 does not contain a small segment present in HDAC1, 2,
and 8. As for the C-terminus, HDAC3 does not contain the same regions as those in
HDAC1 (residues 399-482) and HDAC2 (residues 400-488) and the final thirty-four
residues are not related to any known proteins. It is believed that the two ends of
HDAC3 have distinctive functions for this enzyme compared to other class I HDACs.
However, the remainder of the HDAC domain (residues 4-316) in HDAC3 is conserved
and has homology to other class I, II, and IV HDACs.15-17
The final member of Class I HDACs, HDAC8 is a unique member. HDAC8
contains 377 amino acids with a molecular mass of 45 kDa and is found primarily in the
cytoplasm.18-20 The uniqueness of HDAC8 stems from the structural differences between
HDAC8 and HDAC1-3. HDAC8 is most similar to HDAC3, but only has 34 % sequence similarity. The main differences arise at the N and C terminal domains. At the N- terminus, the first 34 amino acids are very different from those of HDAC1-3. As for the
C-terminus, the final 30 amino acids of HDAC8 are distinct from HDAC1-3.18-20 Also,
HDAC8 is deficient in a 50 to 111 amino acid C-terminal domain that arises from the catalytic domain of class I HDACs.21 However, HDAC8 does possess a greater sequence
conservation in the catalytic domain and contains nine conserved blocks of amino acids
and two histidine residues, His142 and His143 that are vital for catalysis.18-20
Class II HDACs are composed of six classical protein deacetylases and possess
significant sequence homology to yeast histone deacetylase 1 (Hda1). The six Hda1-like
proteins in mammals are HDAC4, 5, 6, 7, 9, and 10. These six enzymes can be further
4 divided in two subgroups composed of class IIa formed by HDAC4, 5, 7, and 9 and class
IIb composed of HDAC 6 and 10. Class IIa enzymes have sequence similarity in their
catalytic domains and the extended long N and C-terminal domains.22 These enzymes
possess a large, functionally important non-catalytic N-terminal domain which is
responsible for recruiting class IIa HDACs to particular promoters and also shuttling the
enzymes between the nucleus and cytoplasm. These two functions classify class IIa
HDACs as signal-dependent repressors involved with specific genes,23 while a class IIb
member, HDAC6 possess two deacetylase domains and a zinc finger motif. The other
class IIb member, HDAC10 contains a catalytic domain on its N-terminus, a nuclear
export sequence (NES), and a pseudo-catalytic domain on its C-terminus.10,24
An examination of the structure of the final class of HDACs will complete our
understanding of the classical protein deacetylases’ structures. The final member is
HDAC11 and is found in a class by itself. Based on phylogenetic analysis, HDAC11 is related to HDAC3 and HDAC8 more than any of the other HDACs. A schematic
representation of the primary structures for all classical protein deacetylases is
represented in Figure 2.10,24 HDAC11 has a catalytic domain at the N-terminus and
contains 347 amino acid residues.10 A better understanding of HDAC11 and how it relates to the rest of the classical protein deacetylases is needed.
The catalytic domain of classical protein deacetylases is a highly conserved
region. Crystal structures exist in which the catalytic domain is described. The x-ray
crystal structures of HDAC8 in complex with different HDAC inhibitors such as
trichostatin A have been solved.21,25 A single globular domain composed of thirteen
alpha-helices enclosing an eight-stranded parallel beta-sheet is present in the structure of
5 HDAC8. The secondary structural elements are linked together by many loops. The loops present on the C-terminal side of the beta-strands make up the active site. The active site is composed of a long narrow tunnel that contains the catalytic center at the end cavity. The most highly conserved residues in HDACs are present in the active site and the hydrophobic core. A catalytic zinc ion is coordinated by two aspartates and a histidine.21,25
Class I HDACs
28 321 3315 HDAC1 482 HDAC3 428 29 322 16 324 HDAC2 488 HDAC8 377
Class II HDACs
653 994 HDAC4 1084 682 1025 HDAC5 1122 519 829 HDAC7 952 633 974 HDAC9 1011 84 404 480 796 HDAC6 1215
1 315 482 666 HDAC10 669
Class IV HDACs 17 321 HDAC11 347
Figure 2. A schematic representation of classical HDACs’ primary structures. The black boxes denote the conserved catalytic domains; the open box denotes the pseudo-catalytic domain found in HDAC10.
Another crystal structure involving a HDAC like protein of a hyperthermophilic bacterium gives a better understanding of the catalytic mechanism. In the apo form, a water molecule is present next to the acetyl-lysine side chain believed to imitate the
6 hydroxamate group in coordinating the zinc ion. In the proposed catalytic mechanism, the carbonyl carbon of the acetyl group is attacked by a nucleophile, the water molecule.
Also a charge relay system is involved where aspartates and histidines are involved in the hydrogen bonding network. Overall, a simple hydrolysis reaction is achieved where the products are the deacetylated lysine and acetate.26
Crystal structures are also available that model class II protein deacetylases. In both studies, a histone deacetylase-like amidohydrolase from Bordetella Alcaligenes strain FB188 (FB188 HDAH) enzyme is used. FB188 HDAH enzyme has high sequence and functional homology to human class II HDACs. Three crystal structures involving either the reaction product acetate or the hydroxamate inhibitors, suberoylanilide hydroxamic acid (SAHA) and cyclopentyle-propionyle hydroxamic acid (CypX) in complex with the enzyme have been solved. The major conclusions from these structural studies are that a catalytic zinc ion is found in the active site of FB188 HDAH and this enzyme also has a canonical fold similar to that found in class I HDACs. However differences between FB188 HDAH and class I HDACs arose in the loop regions. More differences occurred in the region by the active site entrance. These structural differences suggested that class I and II HDACs bind differently to acetylated proteins.27
Recently, the first crystal structure of a nonhydroxamate HDAC inhibitor complexed with an enzyme was solved.28 This structure also serves as a good model for class II HDACs. In specific, the crystal structure of FB188 HDAH complexed with an inhibitor (9,9,9-trifluoro-8-oxo-N-phenylnonanamide) was reported. Overall, the structures of FB188 HDAH complexed with 9,9,9-trifluoro-8-oxo-N-phenylnonanamide and that complexed with SAHA revealed that both inhibitors were bound in comparable
7 ways. However, differences between the two structures arose when the active site of
FB188 HDAH was bound. The crystal structures of FB188 HDAH in complex with
either the hydroxamate inhibitors or a nonhydroxamate inhibitor revealed molecular details of the inhibitor-enzyme interactions.28
Biological functions of classical protein deacetylases
Over forty years ago, the reversible acetylation of histone proteins was
discovered.29 Vince Allfrey proposed acetylation was involved in regulating gene
expression. However, the exact role in gene expression was not known and remained
unclear until 1996. During this year, the first acetyltransferase, GCN5 and first histone
deacetylase, HDAC1 were discovered.30,12 Since then many other protein deacetylases
have been identified.
In the early 1990s, the role of HDACs in the regulation of transcription came to light. The way DNA is packaged influences transcription in eukaryotic cells. When a
cell is at rest, DNA is tightly packed so transcription machinery can not get access to the
DNA sequence. DNA is packaged and organized into different levels. The first level
involves a DNA-protein complex packaged as a nucleosome. The nucleosome is the
fundamental subunit present in chromatin. The nucleosome is made up of an octamer of
four core histones, H2A, H2B, H3, and H4 with 146 base pairs of DNA. When gene
transcription activation occurs, the inaccessible and compact DNA becomes available to
DNA binding proteins following the structural changes of the nucleosome and chromatin.
Posttranslational modifications (PTM) of histones play a significant role in this process.
Methylation of lysine and arginine side chains, phosphorylation of serine and threonine
8 side chains, and acetylation of lysine side chains are a few major PTMs found in
modified histone proteins. In terms of acetylation, all core histones are able to be
acetylated in vivo. When core histones are hyperacetylated, transcriptional activity
increases. However, if core histones are hypoacetylated, gene expression is
repressed.31,10 From the mechanistic standpoint, acetylation neutralizes the positive
charges of histone tails. The histones then possess less affinity for negatively charged
DNA, which promotes the formation of an “open” structure for proteins to bind to
DNA.10 Furthermore, acetylation creates a specific docking site for the bromodomain, and thus recruiting bromodomain-containing proteins most of which are chromatin- binding proteins.1,32 Therefore histone acetylation and deacetylation can function as an
essential switch that turns gene transcription on and off.
Classical HDACs are involved in the control of gene transcription and
cytoskeletal organization. Besides accepting histone proteins as endogenous substrates,
these enzymes can also accept non-histone proteins (e.g. the major human tumor
suppressor protein p53 and alpha-tubulin) as their endogenous substrates, thus
modulating their structures and functions. p53 acetylation is involved in transcriptional
regulation. Acetylation can stimulate the DNA binding of this and other transcription
factors.33,34 Furthermore, HDAC6 is able to deacetylate alpha-tubulin acetylated at the
K40 position,34,35 thus modulating cytoskeletal organization.
Many HDACs are able to deacetylate nucleosomal histones. For example,
HDAC1 is able to deacetylate all four core histones in vitro and prefers lysine residues
found in histone H4.36 HDAC1 can also function inside the cell to deacetylate histones
H3 and H4.37 Just like HDAC1, HDAC3 is also able to deacetylate histone H4 but in a
9 more efficient way.15 HDAC3 is able to deacetylate all four core histone, but has a
preference to deacetylate H2B and H2A.38
Among the various nonhistone proteins, HDAC1 has been shown to be able to
recognize and deacetylate such transcription factors as p5339,33, E2F140,41, Ying Yang 1
(YY1)42, and proliferating cell nuclear antigen (PCNA). As mentioned above, HDAC6 has been shown to be able to deacetylate the microtubule protein alpha-tubulin. These
few examples are just a highlight of non-histone proteins substrates for classical HDACs.
Many other non-histone proteins have been identified that are recognized by various
HDACs. However, we are far from having a complete understanding of these processes.
For example, no known physiological substrates have been identified for HDAC8.
Regulation of classical protein deacetylases
The subcellular localization of HDACs is important for regulating their functions.
Class I HDACs are present mainly in the nucleus. HDAC1 and HDAC2 are only found
in the nucleus because these enzymes lack a NES.10 On the other hand, HDAC3 contains
a NLS and NES. HDAC3 is thus able to be localized in the cytoplasm and nucleus. The
final member of class I HDACs, HDAC8 is primarily present in the cytoplasm, and
expressed in smooth muscle cells.43
Class II HDACs can move in and out of the nucleus due to various cellular
signals. HDAC6 is found in the cytoplasm. While a class IV HDAC, HDAC11 is
present in the nucleus. Two other HDACs, HDAC9 and HDAC10 are found in either the
nucleus or the cytoplasm. The final three members of class II HDACs, HDAC4, 5, and 7
10 involve a regulated course of action where the enzymes shuttle between the cytosol and
nucleus.10
The 14-3-3 proteins are involved in the regulation of nucleocytoplasmic shuttling involving many signaling proteins. The 14-3-3 proteins bind to phosphorylated serine or threonine residues in the target proteins. The binding of the 14-3-3 proteins with the target proteins occurs in the cytoplasm. In class IIa HDACs, a number of potential 14-3-
3 interacting motifs are present at the N-terminal domains.44 HDACs 4, 5, 7, and 9 contain a sequence with a phosphorylated serine which the 14-3-3 proteins interact with and cause the protein complexes to remain in the cytoplasm.44-48
Another type of posttranslational modification besides phosphorylation involved
in the regulation of HDACs is sumoylation. Sumoylation involves the conjugation of
HDACs 4, 9, 1, and 6 by a small-ubiquiting-like modifier (SUMO) catalyzed by E3
ligase.49 Sumoylation is thought to target sumoylated proteins to the nuclear pore
complex and nuclear bodies. SUMO could involve the interaction of HDAC4 with an
unknown cofactor. Sumoylation has the potential to be an important regulatory
mechanism of class I and II HDACs.49
Existing assays for human classical protein deacetylases
Radioactive, fluorescent, and HPLC-based assays are the three currently available
assays that have been employed for measuring activities and inhibition of the human
classical HDACs. In this section, the advantages and disadvantages for each assay
format will be discussed.
11 The traditional way to measure HDAC activity involved a radioactive substrate.
In this assay format, histones are radiolabeled using either chicken blood or cultures to create a labeled substrate. Also [3H]-acetylated oligopeptides can be synthesized
involving 8 to 24 amino acid residues derived from histone amino acid sequences. The
assay monitors the release of tritiated acetic acid from radiolabeled histones and histone
peptide fragments using extraction and scintillation counting. This technique is very exact, but it is laborious and offers dangers to the user and environment. Another disadvantage is the amount of time it takes to acquire the data which means this technique cannot be used as a high-throughput assay. Overall, the substrates are difficult to standardize during incubation and thus the specific activities vary from batch to batch.
Another format of this assay uses biotin labeled tritiated acetyl-histone peptide fragments that are used in a scintillation proximity assay. This radioactive assay format is also very exact. However, the disadvantages outweigh the advantages offered and are not used often.50
Assays using non-isotopic substrates and fluorescence have been developed,
improved, and commercialized over the years to monitor HDAC activity. Initially,
fluorescein-labeled oligopeptides were constructed and analyzed by HPLC with
fluorescence detection techniques as an alternative to radioactive substrates. However,
these fluorescein-labeled substrates were poor substrates and still had a low throughput.
They did offer the benefit of examining lysine selectivity for different deacetylases.
Also a non-isotopic substrate involving an acetylated lysine derivative, Boc(Ac)-Lys-
AMC, also called MAL was made. This substrate is made via a one-step synthesis and
can be bought commercially. Initially, the deacetylated fluorescent product was
12 monitored via an extraction/HPLC protocol. However, this protocol has been improved, and can now be read via a plate reader. MAL can also be used in two other assay formats. The first assay involves a histone deacetylase assay – homogeneous (HDASH) where the fluorescent product is reacted with naphthalene dicarboxaldehyde (NDA) which stops the fluorescence. NDA allows for the detection of the amine substrate left in the reaction mixture. Another assay with MAL involves a trypsin assay where the acetyl- lysine peptides contain a C-terminal coumarinylamide. First, the acetyl-lysine peptides are deacetylated and then trypsin is used on the deacetylated substrates to release the fluorophore, 7-amino-4-methylcoumarin. The MAL analogue has been optimized to give a better substrate involving MAL and a benzyloxycarbonyl (Z) moiety termed Z-MAL, where substrate turnover is faster than MAL. The chemical structures of MAL and Z-
MAL are in Figure 3. The Z-MAL acetylated lysine derivative is also a good substrate for class III protein deacetylases unlike MAL. The HPLC method can be used to monitor the Z-MAL products. The disadvantage of an HPLC based assay is the time it takes to analyze the enzymatic reaction products. While improvements have been made for these different non-isotopic substrates, limitations are still present in the analyses of the products.50
H R H N O O O N N CH3 O H MAL: R=-Boc CH 3 Z-MAL: R=-Z
Figure 3. Chemical structures of MAL & Z-MAL.
13 Besides these different assays, various kits are available from companies such as
BIOMOL International, L.P., Cayman Chemical, and BPS Bioscience Inc. These kits involve a fluorophore in assessing the HDAC activity present in various protein deacetylase-catalyzed reactions.51-53 The BIOMOL kit involves a larger peptidic
substrate made up of an acetyl-lysine side chain. This kit involves a two step reaction
where the deacetylated substrate is produced and then a fluorophore is generated which is
excited at 360 nm and emitted at 460 nm. The product is detected on a fluorometric plate
reader. The kit can be used with all protein deacetylases, but the product monitored can
be low for various protein deacetylases.51
Statement of the Problems and Objectives
The classical protein deacetylase-catalyzed reaction has been targeted for
developing novel therapies for cancer.54,55 Recently, suberoylanilide hydroxamic acid
was approved by the US Food and Drug Administration to treat cutaneous T cell
lymphoma (CTCL). Also many other small molecule inhibitors for the classical protein deacetylases are in various stages of clinical trials right now to primarily examine their anti-cancer activity. Therefore having a convenient assay platform for protein
deacetylase activities could aid in the drug discovery process. However, the currently
available assay formats all have some drawbacks to them and we were interested in
developing a more convenient, environmentally friendly, and cost-effective assay format
which could be useful not only for inhibitor screening but also for reporting HDAC activity under (patho)physiological conditions.
14 Design Rationale
The overall strategy applied to the research objective as stated in the last section is to develop and utilize chemical tools for answering biochemical and biological questions.
In particular, an old “chemical trick” for studying protease and peptidase-catalyzed
hydrolytic reactions, where the peptide bond is replaced with a thioamide bond in the
substrates, has been used to study protein deacetylase-catalyzed deacetylation reactions.
Figure 4 shows the structural comparison of Nε-acetyl lysine and Nε-thioacetyl lysine.
We hypothesized that the thioacetyl group would be a close structural mimic for
the acetyl group, but would also be able to reveal its own functional uniqueness that can
be exploited to develop novel means for studying and understanding protein deacetylase-
catalyzed deacetylation reactions.
If the thioacetyl group could serve as a functional mimic for the acetyl group in
HDAC-catalyzed reaction, a spectrophotometric HDAC assay could potentially be
developed via quantifying thioacetate released from the HDAC-catalyzed
dethioacetylation reaction.
H3C H3C O S HN HN
H H OH OH H2N H2N O O ε ε N -acetyl-lysine N -thioacetyl-lysine
Figure 4. Structural comparison of Nε-acetyl lysine and Nε-thioacetyl lysine.
15 Results and Discussion
To test our hypothesis, three peptides shown in Figure 5, i.e. peptides 1a, 1b, and
1c were prepared. The peptide template was derived from the C-terminal region of the
human tumor suppressor p53 protein (amino acid residues 372-389) with the in vivo acetylation site (382) being Lys (peptide 1c), Nε-acetyl lysine (peptide 1b), or Nε- thioacetyl lysine (peptide 1a).
Figure 5. Analytical RP-HPLC profiles for the purified peptides used in this study. Peptide sequences are H2N-KKGQSTSRHKXLMFKTEG-COOH with X = Lys (peptide 1c), Nε-acetyl lysine (peptide 1b), and Nε-thioacetyl lysine (peptide 1a).
All the peptides were synthesized by employing Fmoc-chemistry based solid phase
peptide synthesis (SPPS)56, and were purified by preparative reversed phase high
16 performance liquid chromatography (RP-HPLC). Their masses were confirmed by either
matrix assisted laser desorption ionization-time of flight (MALDI-TOF) or electrospray
ionization (ESI) mass spectrometic analysis. Peptide 1c was used as the synthetic
authentic de(thio)acetylation peptide product, whereas peptide 1b was evaluated side-by- side with peptide 1a. For incorporating Nε-thioacetyl lysine into peptide 1a, the required
Nα-Fmoc- Nε thioacetyl lysine was synthesized as shown in Scheme 1.
In addition, for the biochemical analysis, human HDAC8 was initially chosen as a
representative member from the Zn2+-dependent family of protein deacetylase enzymes.
Peptides 1a and 1b were first evaluated as potential substrates for human HDAC8
in a RP-HPLC-based assay. Both peptides 1a and 1b formed the de(thio)acetylated
peptide product (i.e. peptide 1c), after incubation with HDAC8 at 37 °C for 1 hour (h).
Figure 6 shows two representative RP-HPLC assay chromatograms under the above
HDAC8 assay condition.
H3C S H2N HN
O H Ethyl Dithioacetate O H OH OH O N Ethanol: 5 % (w/v) aq Na CO O N H 2 3 H O (1:1 (v/v)). O
Scheme 1. Chemical synthesis of Nα-Fmoc- Nε thioacetyl lysine.
Two other experiments further confirmed the genuine enzymatic
de(thio)acetylation as shown in Figure 6. First, a non-enzymatic reaction was done in
17 which no peptide product formed above the HPLC reliable detection limit of 1µM.
Second, another p53 C-terminal peptide (amino acid residue 372-389) was made with
Lys381 being Nε-thioacetylated. This peptide was subjected to the same HDAC8 assay and produced no peptide product. The interesting discovery from our assay was that
peptides 1a and 1b were comparably de(thio)acetylated by human HDAC8 to form
-1 -1 peptide 1c with estimated kobs being 0.38 min and 0.41 min .
A
Peptide 1a UV(214 nm) / mAU
B
Peptide 1b UV(214 nm) / mAU / nm) UV(214
Figure 6. Representative RP-HPLC chromatograms from HDAC8-catalyzed de(thio)acetylation of peptides 1a & 1b, parts A and B and non-enzymatic reactions of peptides 1a & 1b, parts C and D.
This suggests that, when placed within an appropriate amino acid sequence, the thioacetyl group can serve as a functional mimic for the acetyl group for the enzymatic deacetylation reactions catalyzed by HDAC8. Based on this finding, we set out to test the feasibility of developing a spectrophotometric HDAC8 assay by quantifying the 18 thioacetate released from the enzymatic reaction. The thioacetate would then react with
Ellman’s reagent, 5,5’-dithiobis(2-nitrobenzoate) (DTNB) and produce 2-nitro-5-
thiobenzoate (TNB), which is monitored at 412 nm (Figure 7).
H3C S H N HN 3 HDAC8 O
H SCH3 H + HN thioacetate HN O O peptide 1c peptide 1a
NO2 COO NO O NO2 2 COO COO SCH3 S S + thioacetate + S S S O OOC 2-nitro-5-thiobenzoate H C NO (TNB) monitored 3 2 at 412 nm 5,5'-dithiobis(2-nitrobenzoate) (DTNB)
Figure 7. Reaction scheme for the DTNB-based spectrophotometric assay developed in this study. Peptide sequence: H2N-KKGQSTSRHKXLMFKTEG-COOH with X = Lys (peptide 1c), Nε-thioacetyl-lysine (peptide 1a).
19 DTNB-based spectrophotometric assay condition establishment
DTNB has been used previously to selectively react and quantify thiol-containing
compounds such as free thiols and thioacids. The reaction product, TNB has a maximum
absorbance at 412 nm.57 The reaction between DTNB and free thiols has been known to
occur much faster than with thioacids (very fast vs. ~50-60 minutes needed for achieving
maximum absorbance at 412 nm.57 Thus DTNB is used primarily to quantify free thiols.
However, DTNB may still be used to selectively react with thioacids and quantify thioacids. To demonstrate the ability of DTNB to quantify a thioacid released from an
enzymatic reaction, a detailed kinetic study was performed using DTNB and the
commercially available thioacetic acid (Aldrich, Milwaukee, WI, USA). This study was
needed to make sure a complete conversion of the thioacid to TNB occurred in the
presence of an excess amount of DTNB. Following the procedure detailed in
“Experimental Section”, the obtained A412 values were plotted against time for each
individual thioacetic acid concentration. As indicated in Figure 8, a maximum A412 was
obtained or closely approached starting at 60 min following DTNB addition for all the
thioacetic acid concentrations examined. We thus used 60 min as the fixed post-DTNB
time point for the actual enzymatic assays.
Assay with recombinant HDAC8
Using peptide 1a as an in vitro substrate for HDAC8, the HPLC-based and the
DTNB-based assays were both performed to evaluate the HDAC8-catalyzed
dethioacetylation reaction, following the procedure detailed in “Experimental Section”.
For the DTNB-based assay, the extinction coefficient (ε) value for TNB reported in
20 literature (13,700 M–1 cm–1)57 was used for our calculations even though a very close average value (13,505 M–1 cm–1) was also obtained from the standard curve (Figure 9) for
thioacetic acid that was generated according to the DTNB-based assay procedure
0.4
0.35
0.3
0.25
, A U 0.2 412 A
0.15
0.1
0.05
0 0 102030405060708090100 Time, minutes
Figure 8. Kinetic analysis of DTNB reacted with the commercially available thioacetic acid. Each data point corresponded to an average of the duplicate measurements that agreed with each other within 10%. Concentrations of thioacetic acid used are: ○, 10 μM; ●, 25 μM; ■, 50 μM; ♦, 75 μM; ▲, 100 μM.
21 established in the current study. Figure 10 shows the DTNB-based and the HPLC-based assay results, it is clear that these two assay formats gave rise to mutually agreeable measurements (within 8%) for HDAC8 activity when peptide 1a was employed as a substrate, even though the well-established HPLC-based assay measured the peptide product whereas our newly developed DTNB-based assay measured another product, i.e. thioacetate.
1.6 y = 13505x + 0.032 R2 = 0.9976 1.4
1.2
1
AU 0.8 412, A
0.6
0.4
0.2
0 0.00E+00 2.00E-05 4.00E-05 6.00E-05 8.00E-05 1.00E-04 1.20E-04 [thioacetic acid], M
Figure 9. Standard curve generated with the commercially available thioacetic acid. Each data point corresponded to an average of the duplicate measurements that agreed with each other within 20%.
Assay with HeLa nuclear extracts
In order to examine the selectivity of our newly developed DTNB-based
spectrophotometric assay among different classical HDAC enzymes, we performed both
22 the HPLC-based and the DTNB-based assays with the HeLa nuclear extracts enriched in
HDAC1 and 2. The procedures for the HDAC8 assay were followed. It is apparent from
Figure 10 that no detectable dethioacetylation of peptide 1a was observed with both the
HPLC-based and the DTNB-based assay formats, when HDAC8 was replaced with the
HeLa nuclear extracts. That only the intact peptide 1a was recovered from the assay mixture as revealed by the HPLC-based assay (Figure 11) argued against the possibility that the degradation of peptide 1a under assay conditions contributed to the lack of detectable dethioacetylation.
Figure 10. Comparative analyses of enzymatic dethioacetylation of peptide 1a to form peptide 1c (product). HPLC: HPLC-based assay quantifying the released peptide 1c; UV- Vis: DTNB-based spectrophotometric assay quantifying the released thioacetate. 23 250
200
AcNH-RH(ThAcK)(ThAcK)-CONH2 150
100
UV (214 nm) / mAU nm) UV (214 50
0 20 25 30 35 40 Rt / min
400
peptide 1a 300
200
100 impurity UV (214 nm) / mAU / nm) (214 UV
0 20 25 30 35 40 Rt / min
Figure 11. HPLC chromatograms showing only intact peptide 1a & AcNH- RH(ThAcK)(ThAcK)-CONH2 were recovered from HPLC-based assay with HeLa nuclear extracts. All assays were performed in duplicates and identical results were obtained. A minor impurity in the purified peptide 1a sample was present in the chromatogram at tR~29.5 min, rather than the dethioacetylated product, peptide 1c.
24 However, under the same assay conditions, peptide 1b was able to be deacetylated
significantly by the HeLa nuclear extracts, in that 2.3% and 6.5% of substrate turnover
were already observed at 30 min when 4 μL and 12 μL of the HeLa nuclear extracts (9
mg of protein per mL) were respectively used, with kobs = 0.23 ± 0.01 μM / (min • 4 μL of
the nuclear extracts), as judged by HPLC analysis. This same phenomenon was also
observed when AcNH-RH(AcK)-(AcK)-CONH2 and AcNH-RH(ThAcK)(ThAcK)-
58 CONH2 (analogs of H2N-RH(AcK)(AcK)-COOH ) were used, with the former being deacetylated significantly (kobs = 0.29 ± 0.02 μM / (min • 4 μL of the nuclear extracts) at
30 min), but no detectable dethioacetylation was observed for the latter. Again, only intact AcNH-RH(ThAcK)(ThAcK)-CONH2 was recovered from the assay mixture as
revealed by the HPLC-based assay (Figure 11). Taken together, these results suggested
that our newly developed spectrophotometric assay is selective for HDAC8 versus
HDAC1/2. Based on the currently available theoretical and experimental studies, 5,26,58-64 while the catalytic domain is highly conserved among the classical HDAC enzymes, the acetyl-lysine binding pocket in HDAC8 seems to be more malleable as compared to those in other classical HDACs. This could explain our observed capability and incapability respectively for HDAC8 and HDACs present in HeLa nuclear extracts to catalyze dethioacetylation reaction, due to the larger Van der Waals radius of S versus O.
Conclusion
We have demonstrated that Nε-thioacetyl-lysine, when placed within an
appropriate amino acid sequence, could serve as a functional mimic for Nε-acetyl-lysine
in a HDAC8 catalyzed reaction. Using this novel unnatural amino acid we were able to 25 develop a new spectrophotometric HDAC8 assay. In our assay, the thioacetate product
released from the enzymatic dethioacetylation of peptide 1a was reacted with DTNB to produce a quantifiable chromophore, TNB at 412 nm. We determined this assay was selective for HDAC8 versus HDAC1/2 and other classical HDAC enzymes. The application of this new spectrophotometric assay offers a few advantages over the current
available assays. First, this assay format is fast and environmentally friendly compared
to the existing HDAC8 assays which involve either a radioactive assay or HPLC
assay.51-53 Also, as compared to all three currently existing HDAC assay formats, our assay is the most cost-effective format because a widely available UV-Vis
spectrophotometer was used and the substrate was easily made via simple organic and
peptide synthesis. The further application of this new spectrophotometric assay will lead
to high-throughput screening of HDAC8-selective inhibitors and for selective activity
reporting of HDAC8 activity under (patho)physiological conditions.
Experimental Section
This part outlines all of the materials involved in the experimental section.
a) For the synthesis of Nα-Fmoc-Nε-thioacetyl-lysine: Nα-Fmoc-lysine was
purchased from Novabiochem (La Jolla, CA, USA). Ethanol and ethyl dithioacetate were
purchased from Sigma (St. Louis, MO, USA). Sodium carbonate was purchased from
Fisher (Pittsburgh, PA, USA). Silica gel (70-230 mesh, 60 Å) was purchased from Sigma
(St. Louis, MO, USA). Thin layer chromatography (TLC) was performed on TLC plates
from EMD Chemicals (San Diego, CA, USA) (thickness: 0.2 mm, with aluminum
backing).
26 b) For peptide synthesis and purification: All Fmoc-protected amino acids (except
Nα-Fmoc-Nε-thioacetyl-lysine), resins, the coupling reagent 2-(1H-benzotriazole-1-yl)-
1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU), and the additive N-
hydroxybenzotriazole (HOBt) were purchased from Novabiochem. Nα-Fmoc-Nε- thioacetyl-lysine was used to incorporate Nε-thioacetyl-lysine into peptides.
Trifluoroacetic acid (TFA), N,N-dimethylformamide (DMF), and acetonitrile were
purchased from EMD biosciences (San Diego, CA, USA). Anhydrous diethyl ether was
purchased from Fisher. 4-methylmorpholine (NMM), piperidine, phenol, thioanisole, and
ethanedithiol were purchased from Aldrich (Milwaukee, WI, USA).
c) For assays: DTNB, Trizma, guanidinium chloride, ethylenediaminetetracetic
acid (EDTA) disodium salt, and an 1M solution of MgCl2 (molecular biology grade) were
purchased from Sigma. Thioacetic acid, 96% (Cat #T30805) was purchased from
Aldrich. The bovine serum albumin (BSA) with reduced fatty acid content was also
purchased from SIGMA (Cat. #A3803), and was used for all the assays. NaCl, KCl, and
NaH2PO4 were purchased from Fisher. The human recombinant HDAC8 and the HeLa
nuclear extracts (enriched in HDAC1 and 2) were purchased from BIOMOL
International, L.P. (Plymouth Meeting, PA, USA) (Cat. #SE145-0100 and KI140-0100, respectively).
Synthesis of Nα-Fmoc-Nε−thioacetyl-lysine
To a stirred suspension of Nα-Fmoc-lysine (368 mg, 1 mmole) in ethanol (2.12 mL) was added dropwise at 0°C a 5 % (w/v) aqueous solution of sodium carbonate
(Na2CO3) (2.12 mL). Next, ethyl dithioacetate (126 µL, 1.1 mmole) was added dropwise 27 at 0°C. The reaction mixture was stirred at RT for 5 h. Then a 50 % (v/v) solution of
ethanol in double deionized water (ddH2O) (3 mL) was added, and the ethanol was
removed from the mixture by reduced pressure and the remainder of the aqueous solution
was acidified with 6 N HCl to pH ~1-2. The product was then extracted with
dichloromethane (DCM). The combined organics were washed with brine, dried over
anhydrous sodium sulfate (Na2SO4), filtered, and concentrated under reduced pressure.
The resulting oily residue was subjected to silica gel chromatography, affording an off
1 white solid (302 mg, 71 % yield): H NMR (300 MHz, CDCl3): δ 9.33 (br, 1H,
C(=S)NH), 7.97-7.30 (m, 8H, Harom), 5.73 (brd, 1H, J=6.6 Hz, OC(=O)NH), 4.50-4.05
(m, 4H, Fluorenyl H9, CH2O, and Halpha), 3.59 (br, 2H, CH2NH), 2.49 (s, 3H, CH3), 1.89-
13 1.28 (m, 6H, CH2CH2CH2); C NMR (75 MHz, CDCl3): δ 200.9 (C(=S)NH), 176.2
(COOH), 156.7 (NHC(=O)O), 143.5 (Carom), 141.3 (Carom), 128.0 (Carom), 127.3 (Carom),
125.1 (Carom), 120.2 (Carom), 67.4 (CH2O), 53.5 (Calpha), 47.1 (Fluorenyl C9), 46.1
(CH2NH), 33.9 (CH2), 32.0 (CH2), 27.1 (CH2), 22.8 (CH3); HRMS (ESI) calcd. for
+ C23H26N2NaO4S ([M + Na] ) 449.15055; found: 449.14955.
Peptide synthesis and purification
The following peptides used in the current study were all synthesized based on the
Fmoc chemistry strategy56 on a PS3 peptide synthesizer (Protein Technologies Inc.,
Tucson, AZ, USA): i) H2N-KKGQSTSRHK(K)LMFKTEG-COOH (peptide 1c shown in
ε Figure 5); ii) H2N-KKGQSTSRHK(ThAcK)LMFKTEG-COOH (ThAcK = N -
thioacetyl-lysine, peptide 1a shown in Figure 5); iii) H2N-
KKGQSTSRHK(AcK)LMFKTEG-COOH (AcK = Nε-acetyl-lysine, peptide 1b shown in
28 Figure 5); iv) AcNH-RH(AcK)(AcK)-CONH2; and v) AcNH-RH(ThAcK)(ThAcK)-
CONH2. Whereas the first three peptides were all synthesized from the Wang resin
preloaded with Fmoc-Gly, the last two peptides were synthesized from the Rink amide resin.
For each peptide coupling reaction, 4 equivalents of a Fmoc-protected amino acid,
3.8-4.0 equivalents of the coupling reagent HBTU and the additive HOBt were used in the presence of 0.4 M NMM/DMF, and the coupling reaction was allowed to proceed at
RT for 1 h. A 20% (v/v) piperidine/DMF solution was used for Fmoc removal. All the
peptides were cleaved from the resins by reagent K (83.6% (v/v) trifluoroacetic acid,
5.9% (v/v) phenol, 4.2% (v/v) ddH2O, 4.2% (v/v) thioanisole, 2.1% (v/v) ethanedithiol),
precipitated in cold diethyl ether, and purified by RP-HPLC on a preparative C18 column
(100 Å, 2.14 x 25 cm). The column was eluted with a gradient of ddH2O containing
0.05% (v/v) of TFA and acetonitrile containing 0.05% (v/v) of TFA at 10 mL/min and monitored at 214 nm. The pooled HPLC fractions were stripped of acetonitrile and lyophilized to give all peptides as puffy white solids. Peptide purity (>95%) was verified by RP-HPLC on an analytical C18 column (100 Å, 0.46 x 25 cm). The column was eluted with a gradient of ddH2O containing 0.05% (v/v) of TFA and acetonitrile
containing 0.05% (v/v) of TFA from 0-50 % acetonitrile containing 0.05 % (v/v) in 1h at
1 mL/min and monitored at 214 nm. The molecular weights of all purified peptides were confirmed by either MALDI-TOF or ESI mass spectrometric analysis. The first peptide:
MS (MALDI-TOF) m/e 2091 [M+H]+; the second peptide: MS (MALDI-TOF) m/e 2149
[M+H]+; the third peptide: MS (MALDI-TOF) m/e 2133 [M+H]+; the fourth peptide: MS
(ESI) m/e 693 [M+H]+; the fifth peptide: MS (ESI) m/e 725 [M+H]+.
29 Kinetic analysis of the reaction between DTNB and the commercially available thioacetic
acid
A HDAC8 assay solution (300 μL) (without HDAC8 and its substrate but with
added thioacetic acid (0, 10, 25, 50, 75, or 100 μM, final concentrations)) that contained
25 mM Tris•HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 1 mg/mL
BSA was diluted 3-fold by the addition of 600 μL of the following quench buffer: 3.2 M
guanidinium chloride in 100 mM of sodium phosphate (pH 6.8). To each of the above
solutions with different final thioacetic acid concentrations was added 100 μL of a DTNB
solution (400 mM sodium phosphate (pH 6.8), 20 mM DTNB, and 100 mM EDTA), and each of the resulting solutions was further incubated at RT for 0, 5, 10, 15, 20, 30, 60, or
90 min, and A412 was recorded at each time point with a Cary 100 UV-Vis
spectrophotometer (Varian, Inc., Walnut Creek, CA, USA). The results are depicted in
Figure 10.
HPLC-based enzymatic assay
The HDAC8-catalyzed dethioacetylation reaction was performed in the same
HDAC8 assay solution as described above, except that no exogenous thioacetic acid was
added but human recombinant HDAC8 and its substrate (i.e. peptide 1a shown in Figure
5) were added to final concentrations of 375 nM (or 1.5 μM) and 0.3 mM, respectively.
An enzymatic reaction was initiated by the addition of HDAC8 at 37 ºC or RT and was
allowed to incubate at 37 ºC or RT until quenched at different times with the following
stop solution: 1.0 M HCl and 0.16M acetic acid or 3.2 M guanidinium chloride in 100
mM of sodium phosphate (pH 6.8). One portion (40 μL) of the HDAC8 assay mixture 30 was quenched with 80 μL of the above stop solution, and was analyzed by RP-HPLC
with a C18 analytical column (100 Å, 0.46 x 25 cm), eluting with the following gradient
of ddH2O containing 0.05% (v/v) TFA (mobile phase A) and acetonitrile containing
0.05% (v/v) TFA (mobile phase B): linear increase from 0% B to 35% B from 0–40 min
(1 mL/min), and ultraviolet (UV) monitoring at 214 nm. This HPLC gradient gave a
good separation of peptides 1a, 1b, and 1c. The enzymatically formed dethioacetylated
product (i.e. peptide 1c) was confirmed by its comigration with the chemically
synthesized authentic sample and by mass spectrometric analysis with MALDI-TOF, and
was quantified by HPLC peak integration and comparison with that of synthetic authentic
sample. The nearly same assay protocol was used when the HeLa nuclear extracts were
used instead of human HDAC8, except that the following quench buffer was used only:
3.2 M guanidinium chloride in 100 mM of sodium phosphate (pH 6.8).
DTNB-based enzymatic assay
A second portion (300 μL) of the above HDAC8 assay mixture was quenched with 600 μL of the stop solution: 3.2 M guanidinium chloride in 100 mM of sodium phosphate (pH 6.8), and was analyzed by the DTNB-based assay procedure established in the current study (see “Results and Discussion”), i.e. 60 min after the quench and DTNB
(100 μL) addition, A412 was recorded for each sample derived at different time points of
the same assay reaction. The same assay protocol was used when the HeLa nuclear
extracts were used instead of human HDAC8.
31 CHAPTER II
EMPLOYING Nε-THIOACETYL-LYSINE TO DEVELOP POTENT AND/OR
SELECTIVE INHIBITORS FOR HUMAN CLASS III PROTEIN DEACETYLASES
Overview of class III protein deacetylases
Class III protein deacetylases, or sirtuins, are NAD+-dependent protein
deacetylases that are highly conserved from bacteria to humans. Sirtuins have a complex
proposed catalytic mechanism where the acetylated substrate and NAD+ bind and yield
nicotinamide, the deacetylated product, and O-acetyl-ADP-ribose.65-69 Sirtuins are
involved in the regulation of many cellular processes such as apoptosis, cell cycle
progression, gene silencing, and longevity. Several small molecules have been designed
and shown to inhibit sirtuin activity. However, we are still far from having a clear picture
of how sirtuins function. This introduction will give a review of sirtuins focusing on the
structure, function, regulation, and inhibitors discovered for the following three enzymes,
SIRT1, 2, and 3.
Structural aspects of sirtuins
The structural aspects of the yeast silent information regulator-2 (Sir2) protein
(the founding member of the class III protein deacetylases) and other Sir2 homologues will be used to discuss sirtuin protein structures. Sir2 proteins contain a highly
32 conserved catalytic core domain composed of ~270 residues. There are additional N- and
C-terminal extensions present which are involved in protein-specific functions.70 To understand the catalytic domain structure and other various parts of Sir2, crystal structures of several Sir2 enzymes have been solved.
The crystal structures of Sir2 enzymes from bacteria to humans have been determined and give a general understanding of Sir2’s structure.62,71-78 Two subdomains,
a larger and smaller one are present in the catalytic domain. An inverted Rossman fold is
formed by six β-strands enclosed by α-helices that are present on each side of the larger
domain. The smaller domain is composed of three antiparallel β-strands with two α-
helices. Also in the smaller domain are two pairs of cysteine residues which hold a
structural zinc(II) ion. A cleft is formed between the two domains where the two
substrates, NAD+ and the acetylated peptide are found.62,71-78 A brief understanding of
the structure of Sir2 permits an analysis of the various functions of sirtuins.
Biological functions of sirtuins
Sirtuins are involved in the control of gene transcription, cytoskeletal
organization, metabolic control, and HIV infection.3,4,6,34,35,59,79,80-88 SIRT1 is able to remove an acetyl group from the p53 protein C-terminal region33,89 at position K382.89-91
SIRT1 and p53 interact in the nucleus where initiation of DNA damage increases the interaction of SIRT1 and p53.90,91 p53 target genes are activated once acetylation of p53 occurs. This action leads to cell cycle arrest and apoptosis. When p53 is deacetylated by
SIRT1, p53-mediated transcriptional activation is reduced.89-91 This results in repression
of apoptosis stemming from DNA damage or oxidative stress. SIRT2 is found within the
33 microtubule network and recognizes alpha-tubulin as a substrate with the deacetylation
site occurring at K40.35 SIRT2 prefers the alpha-tubulin peptide as a substrate over other
histone peptides. This evidence suggested that SIRT2 could have evolved to perform the deacetylation of alpha-tubulin.35 SIRT3 is involved in metabolic control. The conversion
of acetate to acetyl-coenzyme A (acetyl-CoA) is catalyzed in vivo by the enzyme known as acetyl-coenzyme A synthetase (AceCS). Both AceCS1 and AceCS2 are able to catalyze the conversion of acetate to acetyl-CoA in vivo.84,85 While AceCS1 is a
cytoplasmic enzyme, AceCS2 is localized within the mitochondrial matrix. In humans,
SIRT3 has been shown to deacetylate acetylated AceCS2 at position 642, thus activating
its catalytic activity.92 In mice, murine SIRT3 has been shown to deacetylate acetylated
AceCS2 at position 635,93 and murine SIRT1 has been shown to deacetylate acetylated
AceCS1 at position 661,93 thus activating them. The acetyl-CoA generated within the mitochondrial matrix enters the citric acid cycle where ATP and NADH production increases.85 The acetyl-CoA produced by AceCS1 within cytoplasm can be used for
fatty acid synthesis, decreasing NADPH concentrations.85 SIRT1 has also been shown to
mediate Tat deacetylation in vitro and in vivo at position 50, thus activating its
transcriptional activity.88 The human immunodeficiency virus (HIV) promoter is
activated transcriptionally by the HIV Tat protein. Thus SIRT1 is also important in the
control of HIV infection.88
Regulation of sirtuins
Various sirtuins are subjected to different regulatory mechanisms. Our focus is on the examination of SIRT1, 2, and 3 and how their functions are regulated.
34 The seven human sirtuins are found in various parts of the cell. The subcellular localization of SIRT1 is mainly in the nucleus and is the most conserved mammalian sirtuin.89,90,94-98 However, SIRT1 is also found in the cytoplasm. It was discovered that
SIRT1 can be regulated by nucleocytoplasmic shuttling.99 SIRT6 and 7 are also found in the nucleus, while SIRT2 is localized in the cytoplasmic compartment associated with the microtubule network.35,100,101 SIRT2 is also found in the nucleus. SIRT2 and chromatin can interact during the eukaryotic cell cycle. SIRT2 is involved in the regeneration of condensed chromatin. The formation of condensed chromatin may be regulated by the deacetylation of histone H4 at Lys16.86
SIRT3 is present in the mitochondria and can be transported from the nucleus to the mitochondria.102-104 SIRT4 is mainly present in the mitochondrial matrix. Also, the localization of SIRT5 occurs in the mitochondria.98 It is important to understand where different sirtuins are found inside the cell so we can have a better understanding of their physiological functions mediated through distinct endogenous substrates.
To date, physiological substrates have been identified for three of the four human sirtuins that possess bona fide deacetylase activity, i.e. SIRT1, 2, and 3. Even though
SIRT5 has also been shown to possess histone deacetylase activity in vitro, its physiological substrate(s) is still currently unknown.105 SIRT1 is able to deacetylate Nε- acetylated transcription factors including p53, FOXO3, BCL6, NF-κB, MyoD, the apoptosis regulator Ku70, and murine AceCS1 (mediated by murine SIRT1). Two substrates, alpha-tubulin and lysine-16 Nε-acetylated histone H4 protein are deacetylated by SIRT2. The physiological substrate of SIRT3 was identified recently. SIRT3 can deacetylate AceCS2.34, 65,83,85-87,89,92
35 Existing inhibition strategies and inhibitors for human class III protein
deacetylases
The development of sirtuin inhibitors has been slow and few inhibitors have been reported for members within the sirtuin family, the exception is SIRT1. Also many
reported inhibitors are weak and/or non-selective. Known inhibitors act on multiple
sirtuin members and other enzymes outside of the family. Therefore, the summary of
current inhibitors focuses on a few important ones along with the inhibition strategies
used to find and develop sirtuin inhibitors.
To date, only one potent and selective inhibitor has been reported. An indole
inhibitor was very potent against SIRT1 with an IC50 ~ 98 nM. It was 200-fold less
potent and 500-fold less potent for SIRT2 and SIRT3.106 Other small molecule inhibitors
are sirtinol, M15, and splitomicin. These molecules are analogs of
α−substituted β−naphthol. Sirtinol and M15 inhibited human SIRT2 and yeast Sir2 in
vitro, while splitomicin showed selectivity against different yeast sirtuins.107,108 The
cellular functions of sirtuins present in plant and mammalian cells were discovered using
these α−substituted β−naphthol analogs.109-111 One example of an inhibitor that is not very selective is nicotinamide. Nicotinamide is a byproduct of sirtuin deacetylase reactions. Nicotinamide is able to inhibit SIRT1, 2, 3, and other yeast sirtuins.34,90,102
This compound works by capturing the ADP-ribosyl-enzyme-acetyl peptide intermediate and then regenerating NAD+.112 As new structural and mechanistic information becomes
available, more inhibitors will be discovered. The various strategies employed to
discover inhibitors will enable more new compounds to be found in the future.
36 Developing new and using previous inhibition strategies will aid in the finding of
sirtuin inhibitors. To date, the most conventional strategies involve employing
radioactive assays, fluorescent assays, forward chemical genetics, and phenotypic assays to screen chemical libraries, as well as virtual screening and in silico methodologies to identify sirtuin inhibitors. These different techniques can be used in combination or alone to screen for inhibitors.
The traditional way of measuring enzyme activity involves using a radioactive
assay where the substrates are radioactively labeled and the product is quantified by a
liquid scintillation counter.113 This technique is very time consuming and dangerous to
the environment and user. A more convenient assay involves using a fluorescent
substrate where fluorescence changes over time can be measured to identify
inhibitors.106,114,115 Other approaches to screen large libraries of chemicals involve using
chemical genetics. In this approach, small organic molecules are examined based on
their ability to increase or decrease a known phenotype. When a preferred outcome is
obtained, the compounds are then screened in vitro to determine if they target the desired
protein. This technique was used to discover the inhibitors, M15 and sirtinol.116 A very different method involves using an in silico methodology where an in silico intestinal absorption test along with compounds showing encouraging binding to a conserved hydrophobic pocket in the NAD+ binding site are conducted. This method identified
inhibitors for SIRT2.116 While, some of these techniques allow for high-throughput
screening to be conducted, the assays themselves could improve. As inhibition strategies
and techniques improve, the search for and discovery of sirtuin inhibitors will advance
and increase.
37 Statement of the Problems and Objectives
The objective of my research described in this chapter involves inhibitory studies of class III protein deacetylases. To date, only a few inhibitors of sirtuins have been identified for these enzymes except SIRT1. Developing an inhibitor for one or more of these enzymes will help in obtaining a better understanding of sirtuins and possibly the catalytic mechanisms involved for each enzyme. Besides discovering inhibitors for mechanistic purposes, sirtuin inhibitors hold the potential for therapeutic benefits.
Sirtuin inhibition/activation may hold the potentials for treating metabolic and
age-related diseases and cancer. Sirtuin activity and ageing may be intertwined due to
two pieces of evidence. First, Sir2 activation in yeast is required for both resveratrol and
calorie restriction to extend the lifespan of yeast. Second, calorie restriction has shown
positive outcomes on age-associated morbidity in primates.117 Sirtuin activity is related
to human ageing. Researchers believe compounds that effect calorie restriction may
show signs of benefiting other metabolic diseases such as metabolic syndrome.118
Sirtuins also play a role in treating cancer. Various sirtuin inhibitors possess anticancer activity.119 Silencing SIRT1 in epithelial cancer cells can cause them to die.120 Sirtuin
modulators may hold therapeutic potentials in managing neurodegenerative diseases
including Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. In a
Huntington’s disease mouse model, sirtuins may play a role in protecting neurons
associated with Alzheimer’s disease and Huntington’s disease.121 Recently, a SIRT2
inhibitor guarded against dopaminergic cell death in a model of Parkinson’s disease in
vitro and in vivo.122 These examples demonstrate the potential sirtuin
inhibition/activation can have in age and metabolic diseases and cancer.
38 Design Rationale
We have developed a chemical tool, Nε-thioacetyl-lysine that is able to give us a better understanding of classical protein deacetylases. However, we were also interested in examining its possible application(s) for studying and/or modulating human sirtuins, which involve a different catalytic mechanism than the classical protein deacetylases.
The key features for the sirtuin catalytic mechanism involves the formation of a high- energy O-alkyl intermediate after nicotinamide cleavage from NAD+, followed by the formation and collapse of a bicyclic intermediate as shown in Figure 12.65-69,116 Together with this information and our observation that peptide 1a was processed 7-fold slower than 1b during the SIRT1-catalyzed nicotinamide formation step, but ~400-fold slower for dethioacetylation of 1a as compared to deacetylation of 1b (see below) lead us to believe peptide 1a is able to be processed by SIRT1, but forms a catalytically less efficient longer-lived intermediate along the reaction coordinate after the nicotinamide cleavage and before the collapse of the bicyclic intermediate which yields the dethioacetylated peptide product (i.e. peptide 1c), when compared to the normal processing of peptide 1b by SIRT1. Also sirtuins follow an ordered sequential kinetic mechanism where lysine Nε-acetylated binds first followed by NAD+ binding. This ternary complex is then competent so the chemical cleavage step can occur.123 Peptide 1a can thus be regarded as an authentic mechanism-based SIRT1 inhibitor due to the fact the longer-lived intermediate produced during the SIRT1-catalyzed processing of peptide 1a acted as an effective bisubstrate analog inhibitor. The catalytic mechanism of sirtuins should be well conserved because the catalytic domains are highly conserved among the various sirtuins.5,7,65 We therefore wanted to apply this mechanism-based SIRT1
39 inhibition by peptide 1a to other sirtuin members to serve as a general and efficient replacement of sirtuin substrates to generate sirtuin inhibitors.
+ NAD + Lysine Nε-acetylated substrate
N O Nicotinamide
NH2
H3C O H2N N H O O O O O O N N O P P NH N O O O H N O-alkyl amidate HO OH O O B: H H intermediate
H2N N O O O O P O N O P O CH3 O N O O O H N N O NH H HO OH HO Bicyclic intermediate
2'-O-acetyl-ADP ribose + Deacetylated product (& 3'-O-acetyl-ADP ribose)
Figure 12. A brief representation of the proposed sirtuin chemical mechanism. (Modified from reference 65)
Results and Discussion
The possible application(s) of the chemical tool, Nε-thioacetyl-lysine that we developed for studying and/or modulating human sirtuins, three peptides, i.e. peptides 1a,
1b, and 1c were used. The peptide template of these three peptides is derived from the C- terminal region of the human tumor suppressor p53 protein (amino acid residues 372-
389). The peptide sequences are H2N-KKGQSTSRHKXLMFKTEG-COOH with X =
Nε-thioacetyl lysine (peptide 1a), Nε-acetyl lysine (peptide 1b), and Lys (peptide 1c). As
40 described in Chapter I, all the peptides were synthesized by employing Fmoc-chemistry based SPPS56, and were purified by RP-HPLC. Their masses were confirmed by either
MALDI-TOF or ESI mass spectrometric analysis. Peptide 1c was used as the synthetic authentic de(thio)acetylation peptide product, whereas peptide 1b was evaluated side-by- side with peptide 1a. For incorporating Nε-thioacetyl lysine into peptide 1a, the required
Nα-Fmoc-Nε thioacetyl lysine was synthesized as previously shown in Scheme 1.
Peptides 1a and 1b were first evaluated as potential substrates for human SIRT1.
Peptides 1a and 1b were evaluated side-by-side in the enzymatic reactions with peptide
1b serving as the positive control. The enzymatic reaction occurred for 10 min at 37°C.
Peptide 1c was not processed like peptide 1b. Under this assay condition, peptide 1b was
able to form the deacetylated peptide product, but no dethioacetylated product was
produced from peptide 1a. Representative HPLC chromatograms are shown in Figure 13.
The kobs for the SIRT1-catalyzed deacetylation of peptide 1b was estimated to be 9.29
min-1. Through a more extensive time course analysis of SIRT1-catalyzed
-1 dethioacetylation of peptide 1a, the kobs was estimated to be 0.023 min . Peptide 1a is
thus processed ~400-fold less efficiently by SIRT1 as compared to peptide 1b.
To understand why peptides 1a and 1b were processed differently by SIRT1 a
further in-depth analysis examining nicotinamide production was conducted. The
proposed catalytic mechanism is based on structural and biochemical
evidence.6,65,68,77,123,124 Most believe the first step in the reaction mechanism involves the cleavage of nicotinamide from coenzyme NAD+, which forms a high-energy O-alkyl
amidate intermediate.
41
Figure 13. Representative HPLC assay chromatograms for SIRT1 assay with peptides 1a & 1b.
We believe two factors may have influenced the dramatic difference between the two
rates of the dethioacetylated and deacetylated catalyzed reactions for SIRT1. First,
replacing acetyl with a thioacetyl group may have altered the nicotinamide cleavage
reaction. Second, the catalytic formation of peptide 1c after nicotinamide cleavage is
perturbed when the acetyl group is replaced with thioacetyl. Two examine these two possibilities, an HPLC assay was employed to evaluate the time-dependent enzymatic production of nicotinamide from peptides 1a and 1b. The same assay conditions as previously used for examining SIRT1-catalyzed de(thio)acetylation reactions were used for examining nicotinamide production. The kobs for the SIRT1-catalzyed nicotinamide
production was around 5.60 min-1 for peptide 1b and approximately 0.46 min-1 for
peptide 1a. Based on the 7.4-fold difference for the rate constants for peptides 1a and 1b,
42 we believe the second factor above may influence more profoundly the observed
differences in the SIRT1-catalyzed deacetylation and dethioacetylation reactions. It thus appeared that, due to the close structural similarity between acetyl and thioacetyl
moieties, peptide 1a is still be able to be processed by SIRT1, but forms a catalytically
less competent longer-lived intermediate after the nicotinamide cleavage step, as compared to SIRT1 processing of peptide 1b. Therefore, the Nε-thioacetyl-lysine-
containing peptides (e.g. peptide 1a) could be very useful biochemical/ biophysical probes for dissecting the intermediate events for sirtuin-catalyzed deacetylation reaction.
Peptide 1a is currently being evaluated at Johns Hopkins University School of Medicine.
The research findings will be released in the near future.
Peptide 1a was further examined as a potential inhibitor for the SIRT1-catalyzed
deacetylation reaction. Peptide 1a was ~260 fold stronger as an inhibitor than a known
inhibitor, nicotinamide as shown in Table 2.125,126 Encouraged by this result, two
extremely truncated peptide 1a analogs, Nα-Fmoc-Nε-thioacetyl-lysine and Nα-acetyl-Nε-
thioacetyl-lysine whose chemical syntheses were shown in Schemes 1 and 2 were tested
to determine their inhibition potency against SIRT1. Both of these small compounds behaved as much weaker inhibitors compared to peptide 1a as shown in Table 2. The
amino acid residues around Nε-thioacetyl-lysine in peptide 1a appear to be necessary for
a strong binding interaction with SIRT1 and influence the inhibition potency greatly.
However, a more conservatively truncated peptide 1a analog, the pentapeptide (H2N-
HKK(ThAcK)LM-COOH, peptide 1d) was found to be only a ~6-fold weaker SIRT1 inhibitor than peptide 1a. This result appeared to be consistent with the previous X-ray structural analysis of a sirtuin enzyme with peptide 1b, where the Nε-acetyl-lysine and the
43 two amino acid residues on each side of Nε-acetyl-lysine were shown to be the peptide residues that make predominant binding interactions with the sirtuin enzyme used.
Table 2. Inhibition of SIRT1a
Compound IC50 (μM) Nicotinamide 520 Peptide 1a 1.7 + 0.4
H2N-HKK(thioacetyl)LM-COOH 10.4 + 0.3 α ε N -Fmoc-N -thioacetyl-lysine 2,000 (IC25) Nα-acetyl-Nε-thioacetyl-lysine No inhibition @ 2mM
a Substrate concentrations used, 0.5 mM β-NAD+, 0.3 mM peptide 1b.
Our findings for SIRT1-catalyzed reaction with Nε-thioacetyl-lysine containing peptides lead us to evaluate the Nε-thioacetyl-lysine moiety in the peptide templates derived from the physiological substrates for SIRT2 and SIRT3. Currently, among human sirtuins that possess bona fide protein deacetylase activity, physiological substrates have been identified only for SIRT1, 2, and 3. We hypothesized that the thioacetyl group can be utilized as a general and effective inhibition strategy for all sirtuins because sirtuins possess well conserved catalytic domains.5,7,65
H3C S H2N HN
O H Ethyl Dithioacetate O H OH OH H3C N Ethanol: 5 % (w/v) aq Na CO H C N H 2 3 3 O (1:1 (v/v)). H O
Scheme 2. Chemical synthesis of Nα-Acetyl-Nε thioacetyl lysine.
44 To test our hypothesis, more peptides were synthesized based on a template
derived from the SIRT2 substrate human α-tubulin (peptide 2) and derived from the
SIRT3 substrate human AceCS2 (peptide 3a-c) as shown in Figure 14. All peptides were
synthesized employing Fmoc-chemistry based solid phase peptide synthesis strategies56 using Nα-Fmoc-Nε-thioacetyl-lysine to incorporate Nε-thioacetyl-lysine into peptides.
As mentioned above, peptides 1b and 1c were used as the substrate and synthetic
authentic deacetylation peptide product for the SIRT1 assay, peptides 3b and 3c were
used as the substrate and the synthetic authentic deacetylation peptide product for SIRT3
assay. Also peptide 1b was utilized as an in vitro substrate for the SIRT2 assay because
we discovered this peptide can be processed by SIRT2 but about 8-fold less efficiently
than by SIRT1. However, peptide 1b still formed product that could be analyzed reliably
on the HPLC system when longer reaction times were used.
Peptide 1a: H N-KKGQSTSRHK(ThAcK)LMFKTEG-COOH 2 H3C H3C O S Peptide 1b: H2N-KKGQSTSRHK(AcK)LMFKTEG-COOH H2N HN HN Peptide 1c: H2N-KKGQSTSRHK(K)LMFKTEG-COOH Peptide 1d: H2N-HK(ThAcK)LM-COOH Peptide 2: H N-MPSD(ThAcK)TIGG-COOH H H H 2 OH OH OH Peptide 3a: H N-KRLPKTRSG(ThAcK)VMRRLLRKII-COOH H2N H2N H2N 2 O O O ε Peptide 3b: H2N-KRLPKTRSG(AcK)VMRRLLRKII-COOH Lysine N -acetyl-lysine Nε-thioacetyl-lysine (K) (AcK) (ThAcK) Peptide 3c: H2N-KRLPKTRSG(K)VMRRLLRKII-COOH
Figure 14. Peptides used in this study. The following peptide templates were used in this study: Peptide1a-c, SIRT1 substrate human p53 tumor suppressor protein (372-389); Peptide 1d, p53 (380-384); Peptide 2, SIRT2 substrate humanα -tubulin (36-44); Peptide 3a-c, SIRT3 substrate human Acetyl-coenzyme A synthetase 2 (AceCS2) (633-652).
45 We discovered peptides 2 and 3a to be potent inhibitors for SIRT2 (IC50 ~ 11 μM) and SIRT3 (IC50 ~ 5 μM) as shown in Table 3. Peptide 3a was the very first potent
SIRT3 inhibitor ever discovered. Our hypothesis was supported by these findings, where peptide sirtuin substrates were successfully converted into potent peptide sirtuin inhibitors by replacing Nε-acetyl-lysine with Nε-thioacetyl-lysine. Next, peptides 1a, 2, and 3a were further examined for their potential selective inhibition of SIRT1, 2, and 3.
A few conclusions can be deduced from the data in Table 3. First, peptide 1a was about equally effective as an inhibitor of either SIRT1 or SIRT2, but was a 35-fold weaker inhibitor for SIRT3. Second, peptide 2 inhibited SIRT1 and SIRT3 but was 10-fold and
40-fold less potent as compared to SIRT2. Third, peptide 3a was about equally effective as an inhibitor of either SIRT2 or SIRT3, and a ~5 fold stronger inhibitor for SIRT1. The observation of selective inhibition provided further evidence for the different substrate specificity for different sirtuins. However, a better understanding of how individual sirtuins are able to recognize their substrates could not be deduced by this study. In the current study, a few findings were quite surprising: the strong inhibition of peptide 1a against SIRT2 and peptide 3a against SIRT1 and SIRT2 because peptides 1a and 3a were based on peptide templates originated from the SIRT1 and SIRT3 physiological substrates. It might be possible under certain in vivo conditions for SIRT2 to accept human p53 protein and AceCS2 as its substrates and SIRT1 might be able to accept
AceCS2 as its substrate.
As mentioned in chapter I, we showed that peptides 1a and 1b were comparably de(thio)acetylated by human HDAC8. This liability of peptide 1a toward HDAC8 is likely to decrease its significance as a chemical biological research tool or a prospective
46 therapeutic agent. Thus, we next sought to determine if peptide 2 and 3a could also be dethioacetylated by HDAC8. The same HPLC-based HDAC8 assay was utilized as previously described.
Table 3. Sirtuin inhibitor evaluationa
b Compound IC50 (μM) HDAC8 SIRT1 SIRT2 SIRT3 1a 1.7 ± 0.4c 1.8 ± 0.3 67.3 ± 2.4 +d 1d 10.4 ± 0.3 NDe ND ND 2 116.8 ± 12.0 11.4 ± 1.1 449.4 ± 18.4 –f 3a 0.9 ± 0.2 4.3 ± 0.3 4.5 ± 2.0 – a Substrate concentrations used in an inhibition assay: 0.5 mM β-NAD+, 0.3 mM peptide substrate. b Mean ± standard deviation of duplicate measurements. c Also see Ref. 8. d Sensitive to HDAC8. e Not Determined. f Resistant to HDAC8.
Peptides 1b, 2, and 3a were incubated for 2 h at RT in the HDAC8 assay buffer. ~10 % substrate turnover from peptide 1b was seen. However, no detectable formation of the dethioacetylated peptide products was determined from both peptides 2 and 3a (Figure
15). Thus peptides 2 and 3a were not dethioacetylated by HDAC8 under the same experimental conditions unlike peptides 1a and 1b.
In Chapter I of this thesis, a novel HDAC8-selective spectrophotometric assay was described. In these experiments, we observed the inability of HDAC1 and 2 from
HeLa cell nuclear extract to process peptide 1a and another unrelated Nε-thioacetyl- containing peptide. The positive control containing the Nε-acetyl-lysine containing peptide was significantly deacetylated by HeLa cell nuclear extract. We concluded that the replacement of Nε-thioacetyl-lysine for Nε-acetyl-lysine precluded deacetylation by
47 HDACs present in HeLa nuclear extract. We did not test HeLa nuclear extract with
peptides 2 and 3a because it was very likely the same results would be obtained.
300
2h @ RT 200
Peptide 1b 100
Peptide 1c UV(214nm) / mAU
0
20 25 30 35 40
tR / min
300
2 h @ RT Peptide 2 200 Impurity
100
UV(214nm) / mAU 0
20 25 30 35 40
tR / min
300
2 h @ RT 200 Peptide 3a
100
UV(214nm) / mAU
0 20 25 30 35 40
tR / min
Figure 15. Representative HPLC chromatograms from HDAC8 assays with peptide 1b, 2, and 3a. All assays were performed in duplicate and essentially the same HPLC chromatograms were obtained for duplicates. The small peak with tR~27 min in the second chromatogram was from a minor impurity in the purified peptide 2 sample, rather than the dethioacetylated product.
Conclusion
We have developed a simple and efficient strategy for developing potent and/or selective, and intracellular protein deacetylase-resistant sirtuin inhibitors. Our simple
48 strategy involves replacing the Nε-acetyl-lysine with Nε-thioacetyl-lysine in peptide
sirtuin substrates. A potent inhibitor for SIRT1, a potent and selective SIRT2 inhibitor
and a potent pan-sirtuin (among SIRT1, 2, and 3) inhibitor peptide have been
documented in the current study with hopes of employing each peptide as a chemical
biological research tool for understanding sirtuin biology. Also peptides 1a, 2, and 3a
will serve as valuable lead compounds for discovering more sirtuin inhibitors that are
potent, selective, metabolically stable, and permeable to the cellular membrane. As
described previously, a truncated pentapeptide of 1a was made and found to be only a 6-
fold weaker inhibitor of SIRT1 than peptide 1a as shown in Table 3. Sirtuins are
evolutionarily conserved enzymes from prokaryotes to higher eukaryotes. Also the
catalytic domains are highly conserved between the different sirtuins.5,7,65 Other
pentapeptides derived from peptides 2 and 3a may also serve as more structurally manageable lead compounds if the key binding interactions for SIRT2 and SIRT3 are preserved. Further studies will be performed to confirm our prediction with hopes of finding new lead compounds.
Experimental Section
This section outlines the various experimental procedures used to study protein
deacetylases in Chapter II.
Peptide synthesis and purification
All peptides were synthesized using Fmoc chemistry based SPPS56 on a PS3
peptide synthesizer (Protein Technologies Inc., Tucson, AZ, USA), analyzed, and
49 purified by RP-HPLC. Essentially the same experimental procedures as detailed in
Chapter I were followed. The molecular weights of new purified peptides (i.e. peptides
1d, 2, and 3a-c) (>95% pure based on analytic RP-HPLC analysis) were also confirmed
by either MALDI-TOF or ESI mass spectrometric analysis. Peptide 1d: MS (ESI) m/e
714 [M+H]+; Peptide 2: MS (MALDI-TOF) m/e 964 [M+H]+; Peptide 3a: MS (MALDI-
TOF) m/e 2508 [M+H]+; Peptide 3b: MS (MALDI-TOF) m/e 2492 [M+H]+; Peptide 3c:
MS (MALDI-TOF) m/e 2450 [M+H]+.
Synthesis of Nα-acetyl-Nε-thioacetyl-lysine
The same synthetic procedure for the synthesis of Nα-Fmoc-Nε-thioacetyl lysine
was followed for the synthesis of Nα-acetyl-Nε-thioacetyl lysine from ethyl dithioacetate
and Nα-Acetyl-lysine (purchased from Bachem, King of Prussia, PA). The product was
1 obtained as an off-white solid: H NMR (300 MHz, DMSO-d6): δ 10.08 (br, 1H,
C(=S)NH), 7.64 (brd, 1H, J=7.2 Hz, C(=O)NH), 3.98 (br, 1H, Halpha), 3.41 (br, 2H,
CH2NH), 2.36 (s, 3H, CH3C(=S)), 1.82 (s, 3H, CH3(C=O)), 1.66-1.27 (m, 6H,
13 CH2CH2CH2); C NMR (75 MHz, DMSO-d6): δ 198.7 (C(=S)NH), 174.9 (COOH),
168.6 (C(=O)NH), 53.3 (Calpha), 45.6 (CH2NH), 32.8 (CH2), 32.0 (CH2), 27.1 (CH2), 23.0
+ (CH3), 22.9 (CH3); MS(ESI): m/z 247 [M + H] .
Human sirtuin inhibition assays
The SIRT1 assay data included in this report came from the assays utilizing GST-
SIRT1. However, we have discovered the activities of SIRT1 and GST-SIRT1 toward
both peptides 1a and 1b are equivalent. GST-SIRT1 (whose plasmid is a kind gift from 50 Prof. Tony Kouzarides) was expressed and purified from e. coli as described
previously.87,91,127 Human GST-free SIRT1 was purchased from BIOMOL International,
L.P. (Plymouth Meeting, PA, USA).
Initial SIRT1 assay
A typical time course assay solution contained the following components: 25 mM
+ Tris•HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.5 mM β-NAD , 0.3 mM peptide 1a or 1b, 1.4 μM human GST-SIRT1 (or GST-free SIRT1). The addition of
enzyme started the enzymatic reaction at 37°C until quenched at various time points
using a stop solution composed of 100 mM HCl and 0.16 M acetic acid. The stopped assay solutions were examined by RP-HPLC with a C18 analytical column, as with the
HDAC8 assays described in Chapter I. Using the same assay conditions, no peptide 1c formation occurred above the HPLC reliable detection limit of ~1 μM for the non- enzymatic reaction. SIRT1 inhibition assay solutions had the following components:
+ 25 mM Tris•HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.5 mM β-NAD ,
0.3 mM peptide 1b, an inhibitor with varied concentrations including 0, and 1.4 μM
human GST-SIRT1. An enzymatic reaction was started by adding enzyme at 37°C and
was incubated at 37 °C for 10 min until quenched using a stop solution composed of 100
mM HCl and 0.16 M acetic acid. The peptide 1c formation was noted and quantified
with analytical RP-HPLC. Stock solutions of peptide 1a and nicotinamide were prepared
α ε α ε in ddH2O. Stock solutions of N -Fmoc-N -thioacetyl-lysine and N -acetyl-N -thioacetyl-
lysine were prepared in DMSO, so that a final DMSO concentration of 4% (v/v) in an
51 assay solution was acquired. This final DMSO concentration had no effect on
deacetylase activity. Inhibition potency was expressed as an IC50 value. The IC50 was estimated as the concentration of inhibitor which allowed 50 % as much peptide 1c formation in 10 minutes as compared to an assay which lacked the inhibitor.
SIRT2 & SIRT3 assays
For SIRT2 and SIRT3 inhibition assays, we purchased and used the tag-free human SIRT2 (Cat. # SE251-0500) and the tag-free human SIRT3 (Cat. #SE270-0500)
from BIOMOL International L.P. (Plymouth Meeting, PA, USA). The inhibition assays
with human SIRT1, 2, and 3 enzymes basically involved the same procedures and were
performed as described above for the HPLC-based SIRT1 assay. To summarize, a
typical sirtuin inhibition assay solution had the following components: 25 mM (or 50 mM
for SIRT2 assay) Tris•HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/mL BSA (SIGMA Cat. #A3803 with reduced fatty acid content, for SIRT2 assay only), 0.5 mM β-NAD+, 0.3 mM substrate (peptide 1b for SIRT1 and SIRT2 assays;
peptide 3b for SIRT3 assay), an inhibitor (peptide 1a, 1d, 2, or 3a) with varied
concentrations including 0, and an enzyme (150 nM GST-SIRT1; 0.3 μM SIRT2; or 2.0
μM SIRT3). An enzymatic reaction was started by adding the enzyme at 37 °C and was
incubated at 37 °C for 10 min (for SIRT1 assay) or 60 min (for SIRT2 and SIRT3 assays)
until quenched using the following stop solution composed of 100 mM HCl and 0.16 M
acetic acid. Turnover of the limiting substrate was maintained at ≤12%. The quenched
assay solutions were analyzed by RP-HPLC with a C18 analytical column (100 Å, 0.46 x
25 cm), eluting with the following gradients of ddH2O containing 0.05% (v/v) TFA
52 (mobile phase A) and acetonitrile containing 0.05% (v/v) TFA (mobile phase B): linear
increase from 0% B to 35% B (for SIRT1 assay) or 40% B (for SIRT2 and SIRT3 assays)
from 0–40 min (1 mL/min), and UV monitoring at 214 nm. The sirtuin-catalyzed
deacetylation products (peptide 1c in SIRT1 and SIRT2 assays; peptide 3c in SIRT3
assay) were confirmed by their comigration with the chemically synthesized authentic
samples and by MALDI-TOF mass spectrometric analysis, and were quantified by HPLC
peak integration and comparison with those of synthetic authentic samples. Under the same assay conditions, no detectable formation of the deacetylation products (i.e. peptides 1c and 3c) was observed for non-enzymatic reactions. Also using the same
assay conditions, peptides 1a and 3a did not give any detectable formation of peptides 1c
and 3c, respectively, via dethioacetylation. No detectable formation of the corresponding
dethioacetylated peptides from peptides 1d and 2 was discovered using the same assay
conditions. All peptide stock solutions were prepared in ddH2O. Inhibition potency was
estimated based on IC50 values defined as before.
Human HDAC8 assay
The human recombinant HDAC8 (Cat. #SE145-0100) was purchased from
BIOMOL International L.P. (Plymouth Meeting, PA, USA). The HPLC-based HDAC8
assay was performed as above described. To summarize, a HDAC8 assay solution had the following components: 25 mM Tris•HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg/mL BSA (SIGMA Cat. #A3803 with reduced fatty acid content), 0.3
mM peptide 1b, 2, or 3a, and 1.5 μM HDAC8. An enzymatic reaction was started by
adding HDAC8 at RT and was incubated at RT for 2 h before quenched using the
53 following stop solution composed of 1.0 M HCl and 0.16 M acetic acid. The stopped
assay solutions were examined by RP-HPLC with a C18 analytical column (100 Å, 0.46 x 25 cm), eluting with the following gradients of ddH2O containing 0.05% (v/v) TFA
(mobile phase A) and acetonitrile containing 0.05% (v/v) TFA (mobile phase B): linear
increase from 0% B to 35% B (for the assay with peptide 2 or 1b) or 40% B (for the assay
with peptide 3a or 1b) from 0–40 min (1 mL/min), and UV monitoring at 214 nm. The
deacetylated peptide (i.e. peptide 1c) formed from peptide 1b was seen as described in
Chapter I. However, no detectable formation of the corresponding dethioacetylated
peptides from peptides 2 and 3a was observed. The enzymatically formed peptide 1c was
confirmed by its comigration with the chemically synthesized authentic sample and by
MALDI-TOF mass spectrometric analysis, and was quantified by HPLC peak integration
and comparison with that of synthetic authentic sample. Using the same assay
conditions, no detectable formation of peptides 1c was discovered for the non-enzymatic
reactions.
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66 END NOTES
Portions of both chapters I and II were adopted and reprinted from Fatkins, D.G.;
Monnot, A.D.; Zheng, W., Nε-thioacetyl-lysine: a multi-facet functional probe for
enzymatic protein lysine Nε-deacetylation, Bioorganic and Medicinal Chemistry Letters,
16, 3651-6, Copyright Elsevier, 2006 and Fatkins, D.G.; Zheng W., A
Spectrophotometric Assay for HDAC8, Analytical Biochemistry, in press, Copyright
Elsevier, 2007 with permission.
67