Acetyltransferase in Neuronal Mitochondria and Is Hydrolyzed by Aspartoacylase in Oligodendrocytes

Home , Amidase, Aspartoacylase

A Dissertation

entitled

Developing Approaches to Treat Canavan Disease

Qinzhe Wang

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in Chemistry

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Dr. Ronald E. Viola, Committee Chair

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Dr. Donald Ronning, Committee Member

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Dr. Dragan Isailovic, Committee Member

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Dr. Robert M. Blumenthal, Committee Member

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Dr. Amanda Bryant-Friedrich, Dean

College of Graduate Studies

The University of Toledo

May 2017

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Developing Approaches to Treat Canavan Disease

Qinzhe Wang

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry

The University of Toledo

May 2017

Canavan disease is a progressive, fatal neurological disorder that is caused by defects in the human ASPA gene, which leads to an interruption in the metabolism of N- acetylaspartic acid (NAA). NAA is the second most abundant amino acid in the human brain. It is synthesized by aspartate N-acetyltransferase in neuronal mitochondria and is hydrolyzed by aspartoacylase in oligodendrocytes. Multiple therapies are currently under investigation to identify a treatment for Canavan disease.

Aspartoacylase has previously been successfully expressed, kinetically characterized, and structurally characterized. To provide further insights about this enzyme, a series of mass spectrometry-based protein analysis studies have been performed. The amino acid sequence of aspartoacylase was confirmed by high-performance liquid chromatography/matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry. No post-translational modifications, in particular asparagine-linked glycosylation that were previously implicated, were identified.

Aspartate N-acetyltransferase (ANAT) has been shown to be a membrane associated enzyme with a putative membrane region of about 30 amino acid residues. While

iii detergent extraction and several protein engineering approaches in the putative membrane domain failed to produce a soluble and active form of the enzyme, a tandem affinity purification approach using maltose-binding protein as fusion tag was able to produce an active and soluble enzyme form suitable for inhibitor development. Aspartate N- acetyltransferase was found to have high substrate specificity. Only three compounds, β- methylaspartate, 2,3-diaminosuccinate, and L-glutamate, were identified as alternative substrates from 160 different amino acid analogs that were examined. Several factors that could affect ANAT activity were also tested. Triton X-100 and Tween 20 were identified to have the least impact on ANAT activity after a screening of a detergent library. The effect of variations in pH identified a pK value of 6.8, which could reflect an active site histidine residue functioning as a general base.

As an initial effort to develop selective inhibitors for ANAT as a novel substrate reduction therapy for Canavan disease, several weak to moderate ANAT inhibitors were identified through focused library screening of amino acid analogs and small molecule metabolites. A surprisingly potent hit with carboxybenzyl-aspartic acid led to the exploration of a series of di-carboxylic acids. Optimizing the chain lengths, and the nature of the side chain linkage resulted in inhibitors with low micromolar inhibition constants. This was followed by targeted inhibitor optimizations starting with a benzylaminoethylphthalate core structure, which has led to several sub-micromolar inhibitors against this important human enzyme. These sets of newly synthesized ANAT inhibitors are now being modified to improve their cellular uptake properties in order to start efficacy studies in an animal model of Canavan disease.

To my family, thank you for everything!

Acknowledgements

First and foremost I am deeply indebted to my Ph.D. advisor, Dr. Ronald E. Viola, for all his excellent guidance, patience and encouragement during the past five years of research. His continuing support has enabled me to undertake challenging research problems and to keep learning by trying new ideas and new experimental procedures.

I would also like to thank my committee members, Dr. Donald Ronning, Dr. Dragan

Isailovic, and Dr. Robert M. Blumenthal for their knowledge and support. Their prompt feedback and valuable suggestions greatly enhanced the quality of the research. I am also thankful for the friendship and help provided by the past and current members of the

Viola group, especially, Dr. Alexander G. Pavlovsky, Dr. Mojun Zhao, Dr. Stephen P.

Zano, Dr. Nitesh Poddar, Dr. Pravin Bhansali, Dr. Yasanandana S. Wijayasinghe, Dr.

Bharani Thangavelu, Vinay Mutthamsetty, Gwenn G. Parungao, Gopal Dahal, and

Muhammad Hussain. I thank them for their help in various aspects of my research.

I thank the Department of Chemistry and Biochemistry, The University of Toledo, for providing me with financial support and the grants from NIH, Jacob’s Cure foundation and Turing Pharmaceuticals LLC for Canavan disease research.

Finally, I would like to express my greatest thanks to my family members: my wife,

Dr. Danyang Zhu, my parents, Hongli Wang and Juan Zhang, and my brother Guanqiang

Wang for their understanding, words of encouragement, support, sacrifices of time and love to assure the completion of the dissertation.

vi Table of Contents

Abstract ...... iii

Acknowledgements ...... vi

Table of Contents ...... vii

List of Tables ...... xi

List of Figures ...... xii

List of Abbreviations ...... xv

Chapter 1: Introduction ...... 1

1.1 Canavan disease ...... 1

1.2 N-acetyl-L-aspartate metabolism in brain and proposed N-acetyl-L-aspartic acid

metabolic pathway ...... 4

1.3 Aspartoacylase ...... 7

1.4 Aspartate N-acetyltransferase ...... 13

1.5 Current treatment therapies for Canavan disease ...... 15

1.5.1 Metabolite Therapy ...... 15

1.5.2 Gene Replacement Therapy ...... 16

1.5.3 Enzyme Replacement Therapy ...... 16

1.5.4 Enzyme Enhancement Therapy ...... 17

1.5.5 Substrate Reduction Therapy ...... 17

vii 1.6 Chapter outline and organization ...... 18

Chapter 2: Characterization of human Aspartoacylase ...... 19

2.1 Introduction ...... 19

2.2 Potential N-glycosylation site ...... 22

2.3 Expression and purification of human aspartoacylase from Pichia pastoris ...... 25

2.4 Comparative enzyme activity analysis for ASPA glycosylation ...... 28

2.5 Gel mobility shift analysis for post-translational modification ...... 30

2.6 Molecular peak determination by MALDI-TOF mass spectrometry ...... 33

2.7 Solid phase extraction of glycopeptides ...... 35

2.8 Mass spectrometry based protein sequencing ...... 38

2.9 Codon optimization of human aspartoacylase gene for E. coli expression system ...... 41

2.10 Expression and Purification of wild type Aspartoacylase enzyme from E. coli ...... 42

2.11 Summary for post-translational modification analysis for human aspartoacylase expressed from Pichia ...... 44

2.12 Developing treatment therapies targeting aspartoacylase for Canavan disease ...... 44

2.12.1 Progress on Enzyme Replacement Therapy ...... 44

2.12.2 Progress on Enzyme Enhancement Therapy ...... 46

Chapter 3: Purification and characterization of Aspartate-N-acetyltransferase ...... 50

3.1 Introduction ...... 50

3.2 Detergent extraction of native aspartate-N-acetyltransferase ...... 52

3.3 Membrane anchor removal ...... 55

3.4 Previous attempts at purification of Aspartate-N-acetyltransferase ...... 59

viii 3.5 Codon optimization of human aspartate N-acetyltransferase gene for E. coli expression

...... 61

3.6 Dual Affinity Purification system ...... 65

3.7 Purification of MBP-ANAT-H6 fusion protein ...... 66

3.8 Confirmation of the expression of ANAT by mass spectrometry ...... 69

3.9 Enzyme Activity of Aspartate N-acetyltransferase ...... 71

3.9.1 The PDH based activity assay ...... 71

3.9.2 The DTNB based activity assay ...... 72

3.10 Detergents affect the enzyme activity of Aspartate N-acetyltransferase ...... 76

3.11 Other factors affecting ANAT activity ...... 78

3.12 pH activity profiles of ANAT ...... 80

3.13 Crystallization trials of MBP-ANAT-his fusion ...... 82

3.14 Summary and Future Directions ...... 84

Chapter 4 Inhibitor development for Aspartate-N-acetyltransferase ...... 85

4.1 Introduction ...... 85

4.2 Assay development for inhibitor screening ...... 87

4.3 Initial inhibitors identified by Library screening ...... 89

4.4 Inhibitor optimization starting from di-carboxylic acid analogue core structures ...... 94

4.5 Inhibitor optimization starting from the phthalic acid core structure ...... 96

4.6 Summary and Future Directions ...... 99

References ...... 105

Appendices ...... 119

ix Appendix A1 ...... 119

Appendix A2 ...... 120

Appendix A3 ...... 121

Appendix A4 ...... 122

Appendix B1 ...... 123

Appendix C1 ...... 124

Appendix C2 ...... 125

x List of Tables

1.1 Comparison of constructs for ASPA wild type enzyme 13

2.1 Predicted glycosylation sites by NetNGlyc 1.0 server 24

2.2 Blind docking study of ASPA stabilizers 49

3.1 Detergent Extraction of Native Aspartate N-acetyltransferase 54

3.2 Substrate Kinetic Parameters for Aspartate N-acetyltransferase 76

4.1 Summary of Compound Library Screening 92

4.2 Compounds with a carboxybenzyl group and their inhibition constants 94

A.1 Typical components in de-glycosylation reaction 119

xi List of Figures

1-1 N-acetyl-L-aspartic acid metabolic pathway 6

1-2 Crystal structure of ASPA wild type enzyme 9

1-3 Clinical mutants of ASPA in Canavan disease 11

2-1 Sequence alignments of aspartoacylase homologs from different species 21

2-2 Asparagine residues of human aspartoacylase. 22

2-3 Additional putative glycosylation sites identified by NetNGlyc server 24

2-4 Anion exchange chromatogram of Pichia expressed human aspartoacylase 27

2-5 Aspartoacylase assay with aspartase as coupling enzyme 28

2-6 Kinetic assay of human aspartoacylase during PNGase F digestion 30

2-7 Kinetic assay of the mock “PNGase F treatment” reaction 30

2-8 Gel mobility assay for glycan detection 32

2-9 MALDI-MS of intact aspartoacylase. 34

2-10 Hydrazine chemistry basic principle 36

2-11 All three N-glycosylation sites were confirmed from fetuin 37

2-12 A representive chromatogram shown on nano-HPLC seperation. 39

2-13 Sequencing aspartoacylase by mass spectrometry 40

2-14 Sequencing of N117 containing peptide of aspartoacylase. 40

xii 2-15 Expression of codon optimized aspartoacylase wild type enzyme in E. coli.

2-16 Surface lysine modification of aspartoacylase 46

2-17 ASPA Dimer interface as a preferred binding site. 48

3-1 Sequence alignment between ANAT homologs 56

3-2 Illustration of membrane anchor removal 57

3-3 Design of Membrane anchor removal constructs 58

3-4 Different fusion constructs of human aspartate N-acetyltransferase 61

3-5 Number of deposited DNA sequences in GenBank 64

3-6 Chromatograms of two affinity purification steps for MBP-ANAT-his fusion protein 67

3-7 SDS-PAGE of MBP-ANAT-his during two-step affinity purification 68

3-8 PMF analysis for ANAT 70

3-9 Conformation of ANAT expression by peptide sequencing 71

3-10 ANAT activity assays 74

3-11 Reciprocal plot for MBP-ANAT-his fusion enzyme 75

3-12 Effects of detergents on ANAT activity 77

3-13 Effect of organic solvent on ANAT activity 79

3-14 V/K pH profile of MBP-ANAT-his fusion enzyme 81

3-15 pH and buffer screen for crystallization 83

4-1 Structures of moderate inhibitors identified from the metabolite library 93

xiii 4-2 Di-carboxylic acid core 95

4-3 Initial ANAT inhibitors identified from screening a 4-aminomethylphthalate derivatives 101

4-4 Benzyl substituents: methyl and bromo functional groups 102

4-5 Trifluoromethyl effect and the additional influence of a hydrophilic functional group 103

4-6 Substitutions on benzylaminoethylphthalates 104

A-1 Codon optimized nucleotide sequence for human aspartoacylase 120

A-2 hASPA E. coli cloning construct synthesized by Life Technologies. 121

A-3 N-terminal PEGylation reaction of ASPA 122

B-1 Hydrodynamic radius of MBP-ANAT-His fusion protein 123

C-1 Positive and negative controls for DTNB assay 124

C-2 Representative kinetic absorbance curve for ANAT inhibitor 125

xiv List of Abbreviations

AceCS1………………... Acetyl-Coenzyme A synthase1 aCSF…………………....Artificial cerebrospinal fluid ANAT ………………….Aspartate N-acetyltransferase ASADH…………………Aspartate semialdehyde dehydrogenase ASPA……………………Aspartoacylase AspNAT ………………..Aspartate N-acetyltransferase Acy2…………………….Aspartoacylase gene

BBB……………………..Blood brain barrier

Cbz………………………N-[(benzyloxy)carbonyl] CD…….…………………Canavan disease CHAPS………………….Cholamido propane sulfonate CMC…………………….Critical micelle concentration CNS…….………………. Central Nervous System CV…………………….....Column volume Cymal5………………..…5-Cyclohexyl-1-Pentyl-β-D-maltoside C8E4…………………….Octyl tetraglycol C8E5…………………….Octyl pentaglycol C12E8………………...…Dodecyl octaglycol

DDM………………….…Dodecylmaltoside DLS……………...... Dynamic Light Scattering DM……………………....Decylmaltoside DMSO …………...... Dimethyl Sulfoxide DTNB……………………5-(3-Carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid DTT……………………...Dithiothreitol

EDTA…………………....Ethylenediaminetetraacetic acid

GCP…………...………....Glutamate carboxypeptidase GST……………………...Glutathione sulfur transferase GTA…………………...…Glycerol triacetate

HEK-293T………………. Human embryonic kidney cells 293 HGNC………………..…. HUGO Gene Nomenclature Committee

IMAC……………………. Immobilized Metal Affinity Chromatography IPTG……………………... Isopropyl β-D-1-thiogalactopyranoside

LB……………………...…Luria-Bertani LDAO…………………… lauryldimethylamine-N-oxide mAAT………………...…. Mitochondrial aspartate aminotransferase MALDI………………..… Matrix Assisted Laser Desorption/Ionization MBP…………………...….Maltose binding protein Mega-9………………...….Nonanoyl-N-methylglucoside mGluR3…….……………. Metabotropic glutamate receptor type 3 MGM…………………..... Minimal Glycerol Medium MM……………………….Minimal Methanol Medium MS………………………..Mass spectrometry MS/MS…………………...Tandem mass spectrometry

NAA…….………………. N-acetyl-L-aspartate NAAG…………………… N-acetylaspartylglutamate NaDC3…..………………. Na+ dependent dicarboxylate transporter 3 NCBI…………………….. National Center for Biotechnology Information NG…………………….…..Nonylglucoside Ni-NTA………………….. Ni-Nitrilotriacetate NIH………………………. National Institutes of Health NLM……………………... National Library of Medicine

OG…………………….…..octylglucoside OMIM……..…………….. Online Mendelian Inheritance in Man

PaiA………………………Polyamine N-acetyltransferase PCR……………………… Polymerase Chain Reaction PDB……………………… Protein Data Bank PDH…………………...…. pyruvate dehydrogenase PEG………………………..Polyethylene glycol PMF……………………….Peptide mass fingerprinting PNGase F……………...…. Peptide:N-Glycosidase F PTM……………………….post-translational modification

SDS………………………. Sodium Dodecyl Sulfate SDS-PAGE…………..Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis SPE………………………..Solid phase extraction

xvi SPEG……………………...Solid Phase Extraction of Glycopeptides

TAP………………………..Tandem affinity purification TEV………………………..Tobacco etch virus TFA………………………. Trifluoroacetic acid TMAO……………………..Trimethylamine N-oxide TRX…………………...….. Thioredoxin TOF…………………….….Time of Flight

YPDS…………………...... Yeast extract-Peptone-Dextrose-Sorbitol

xvii

Chapter 1

Introduction

1.1 Canavan disease

Canavan Disease (CD), also called Canavan-van Bogaert-Bertrand disease, was first described by Myrtelle Canavan in 1931 1. A disease with similar symptoms was later reported by van Bogaert and Bertrand in 1949 2, 3. Canavan disease is an autosomal recessive disorder (OMIM #271900) caused by mutations in a single gene. The child will have disease symptoms only if two copies of the mutated genes are present, which means that if the husband and wife both carry one copy of the Canavan disease gene, then there is a 25% chance that their child will become a CD patient. CD patients are mostly identified from the Ashkenazi Jewish population, but many cases have also been found in a wide range of ethnic populations.

The early disease symptoms typically show up at ages between 3 and 9 months, when

Canavan children may develop hypotonia and lack of head control as an early signature.

Progressive spongy degeneration of brain white matters was confirmed in a follow up study using ultrasonic imaging4. Other clinical symptoms of Canavan disease includes ataxia, optic atrophy, poor sucking ability, mental retardation, and epileptic seizures 5, 6.

Macrocephaly is a common symptom for Canavan disease while microcephaly has also

1 been observed from a CD patient 7. A mouse model suggested that CD can also cause hearing impairment 8. CD typically progresses rapidly and this disease is without any efficient clinical treatments.

Canavan disease can be definitively diagnosed by high N-acetylaspartate (NAA) accumulation in both plasma and urine, with an NAA signal seen by proton magnetic resonance spectroscopy 9 and finally confirmed by DNA sequencing of the defective

Canavan disease gene for mutations. Depending on the clinical outcome, Canavan disease can be categorized into three different forms: an extremely severe form (neonatal form) that is recognized immediately after birth, a severe form (infantile form) and a milder form (juvenile form). Although most Canavan disease patients suffer from severe disease symptoms that lead to early death 10, juvenile forms of CD have also been reported. For example, Janson et al 11, described two sisters with developmental delay but without macrocephaly, hypotonia, spasticity, or seizures. In infantile Canavan disease, the NAA concentration in urine is 60 times higher than that in normal people, and 12 times higher than that observed in the mild type of CD.

Canavan disease is a genetic disorder caused by defects in a single gene, which codes for aspartoacylase (ASPA) 12. More than 70 mutations has been reported and are included in the Human Gene Mutation Database at the Institute of Medical Genetics at Cardiff

(www.hgmd.cf.ac.uk). Missense mutations, gene splicing, small deletions, small insertions, small indels, gross deletions, and gene truncations are all present as genetic defects in Canavan disease.

There has been a long standing debate about the underlying cause of Canavan disease.

The first hypothesis focuses on the building blocks of lipid synthesis. Aspartoacylase is

2 the enzyme responsible for producing free acetate from N-acetyl-L-aspartate. The released acetic acid molecule participates in myelin synthesis 13. In Canavan disease, the impaired aspartoacylase cannot produce enough acetate, which leads to the inability to maintain myelin sheath synthesis 14. For example, Mehta et al discovered that the 14C labeled acetyl moiety of N-acetylaspartate is incorporated into lipids in both brain and liver tissues 15. Further study identified that the biosynthesis of six classes of myelin- associated lipids were affected after inactivating the ASPA gene in a mouse model of

Canavan disease 16. A detailed review of the evidence for defective myelin lipid synthesis caused by lack of acetate from NAA can be found in Namboodiri et al. 17

The alternative hypothesis suggests that it is the toxic levels of accumulation of N- acetyl-L-aspartate (NAA), instead of a lack of acetate in the brain, that causes the cerebral myelin damage 12. NAA is synthesized in neuronal mitochondria and is hydrolyzed in the cytoplasm of oligodendrocytes. Even back in the 1930s to 1950s, mitochondrial defects and spongiformation were observed in CD patients. Since NAA is an osmolyte, disruption of the migration of NAA from neurons to oligodendrocytes may lead to osmotic dysregulation 18. NAA accumulation was also shown to induce seizures

19, 20. The tremor rat is a aspartoacylase gene deletion mutant, with high NAA concentration in the brain, identified in the Kyoto:Wistar colony, and exhibiting absence- like seizures and spongiform degeneration 20. Also, direct injection of NAA into normal rat cerebroventricle induces seizures. It is now known that NAA is a substrate for N- acetylaspartylglutamate (NAAG) biosynthesis, a dipeptide neurotransmitter 21. Thus, toxic accumulation of NAA may lead to neuron overexcitation. A metabolomic study confirmed NAA as a serum biomarker for schizophrenia 22. NAAG is higher in brain

3 white matter (1.5 – 2.7 mM) and lower in brain gray matter (0.6 – 1.5 mM) 23, and

NAAG synthetase has been identified and characterized recently 24.

1.2 N-acetyl-L-aspartate metabolism in brain and proposed N-acetyl-L-aspartic acid metabolic pathway

In 1956, Tallan et al discovered N-acetyl-L-aspartate (NAA) in the brain of cats 25.

They reported the detection of a “bound form” of aspartic acid. Acid hydrolysis of the brain tissue powder released 80 mg of aspartic acid per 100 grams of tissue and the precursor was identified as NAA. Later, NAA was also identified in various other species

26. N-acetyl-L-aspartate is the second most abundant amino acid, after glutamate, in the human brain. NAA has been shown to be distributed in the brain at about 8-10 mM based on an earlier NMR spectroscopy analysis of human brains 23. Later, specific antibodies against NAA demonstrated that NAA is present in neuronal cell bodies as well as in oligodendrocytes/myelin in adult rodent brain 27. In 1959, Goldstein identified an aspartate acylating activity from rat brain slices, suggested an enzyme-catalyzed reaction based on heat inactivation, and determined the optimal pH range to be from pH 6.0 to pH

6.8 28. However, the gene responsible for this aspartate N-acetyltransferase (ANAT or

Asp-NAT) activity wasn’t determined until 51 years later. In 2010 Wiame et al 29 tested

NAT8L and NAT14 in human embryonic kidney-293 cells expressing the large T-antigen of SV40 (HEK-293T) cell line, and confirmed that NAT8L is responsible for NAA synthesis from L-aspartate and acetyl-CoA.

To emphasize the significance of NAA metabolism in brain development and to distinguish from other amino acid metabolic pathways, the brain NAA metabolism

4 among several cell types has been designated as the N-acetyl-L-aspartic acid metabolic pathway. The citric acid cycle and the malate-aspartate shuttle provides oxaloacetate through malate dehydrogenase in the mitochondria. Aspartate is then generated from glutamate and oxaloacetate by mitochondrial aspartate aminotransferase (mAAT) (EC

2.6.1.1). Aspartate is then converted to N-acetylaspartate by aspartate N-acetyltransferase using acetyl-Coenzyme A as the acetyl donor. NAAG is synthesized in neuronal cells and is released by a depolarization-induced, calcium-dependent fashion 21. NAAG is then hydrolyzed by two glial peptidases, glutamate carboxypeptidase-II (GCPII) or glutamate carboxypeptidase-III (GCPIII) 30, 31. GCPII is expressed exclusively in glial astrocytes based on a study in adult rat nervous system 32. Released NAA is then transported into the circulatory system or to oligodendrocytes by the sodium-coupled high-affinity

33 carboxylate transporter NaDC3 and is hydrolyzed by ASPA into L-aspartate and acetate

(Figure 1-1). The released acetate has three possible fates: the first is to participate in myelin synthesis through cytosolic acetyl-Coenzyme A synthase1 (AceCS1). The second is to provide energy through the TCA cycle, and the last route is to participate in histone acetylation and gene regulation. The synthesis of NAA parallels with the development of the infant brain34.

It seems that although the N-acetyl-L-aspartic acid metabolic pathway is highly dynamic, the NAA concentration must be maintained at certain levels for normal brain function. Too high an NAA concentration leads to seizures and spongiform degeneration

20, while an NAA concentration that is too low leads to brain damage 35, hypoacetylaspartia in patient 36, and even sudden death in a mouse model 37. Abnormally high levels of NAA were also discovered in Sickle Cell disease 38, while NAA levels

5 were found to be significantly lowered in Alzheimer’s disease 39 and in multiple sclerosis40.

Figure 1-1: N-acetyl-L-aspartic acid metabolic pathway. Five important enzyme steps are shown: mitochondrial aspartate aminotransferase (mAAT), aspartate N-acetyltransferase

(AspNAT), NAAG synthetase, glutamate carboxypeptidase (GCP), and aspartoacylase.

The NAA transporter sodium dicarboxylate co-transporter (NaDC3) is responsible for

NAA transport across the cell membrane. The intercellular transport of NAA is shown by a dashed line.

6 1.3 Aspartoacylase

In 1988, Matalon et al discovered the correlation between Canavan disease and aspartoacylase activity 12 and, 5 years later, the same research group cloned the human

ASPA cDNA and confirmed that the isolated protein had aspartoacylase activity in bacteria 41. It is now known that the ASPA gene (HGNC_ID:756) (or acy2 gene) is located at chromosome 17 (17p13.2) from chr17:3,377,403 to 3,402,699. The ASPA gene has 5 introns and 6 exons, which can be translated into a 313-amino acid enzyme with a molecular mass of 36 kDa. Exons 1 through 4 express the amino terminal domain of aspartoacylase and Exons 5 and 6 express the carboxyl terminal domain. Recombinantly expressed aspartoacylase is a homo-dimer, based on dynamic light scattering and native gel electrophoresis experiments 42, 43. Aspartoacylase is a metalloenzyme requiring one zinc per subunit for catalysis. Aspartoacylase, also called aminoacylase II, belongs to the aminoacylase family which contains aminoacylase I, II, and III. Compared to aminoacylase I and III, the human ASPA wild type enzyme has a stricter requirement for its substrate, recognizing only NAA versus many other N-acetyl amino acids 44-46.

However the N-formyl-, N-chloroacetyl-, N-dichloroacetyl- and N-trifluoroacetyl-derived

L-aspartates have also been found to be substrates for aspartoacylase 47. Aspartoacylase shows sigmoidal behavior if the NAA concentration is lower than 0.6 mM, while it shows substrate inhibition if the NAA concentration is higher than 2 mM 42. A large scale human yeast two-hybrid experiment showed that ASPA does not interact with ~8000 human proteins tested 48, and no other protein partners have been identified to date.

Structures of wild type aspartoacylase from rat and human have been reported by Bitto et al 49 in the apo-enzyme form, and the complex structure of human aspartoacylase with

7 an intermediate analogue, N-phosphonomethyl-L-aspartate, has been reported by Le Coq et al 43. To further understand the possible structural perturbations caused by clinical mutations, structures of aspartoacylase from four mutant enzyme forms, K213E, Y231C,

F295S, and E285A have been solved by Wijayasinghe et al 50. Since the crystal structures of aspartoacylase wild type as well as several clinical mutants have been solved 43, 49, 50, an enhanced knowledge about the functioning of this essential human enzyme has been obtained. Based on the crystal structure, aspartoacylase is a homo-dimer with N-terminal and C-terminal domains. The N-terminal domain belongs to the alpha/beta fold with six parallel beta-sheets in the middle and eight alpha helixes surround them. The C-terminal domain contains four beta sheets. Two long antiparallel beta sheets wrap around N- terminal domain. As shown in Figure 1-2, the electron density is poorly defined around the first 8-10 amino acid residues at the N- terminal and the final 3 amino acid residues at

C- terminal of ASPA in the crystal structure of the wild type enzyme 43, 49 as well as in the mutant structures 50. The termini are close to each other (6.4Å and 6.7Å between E9 and C310 in 2O53; 4.8 and 5.0Å between E9 to C310 in 2I3C; 5.9 and 11Å between E9 and C310 in 2Q51; 5.8 and 7.0Å between H10 and C310 in 2O4H). The terminal residues do not directly participate in catalysis and, because they are 30 Å away from active site, they are not likely to control access to the catalytic site. It is interesting that a clinical mutation with low catalytic activity, K213E, is found in this region. The K213E mutation lowers the unfolding temperature of this mutant by 2 oC compared to wild type enzyme51.

Also 750 mM urea is sufficient to lower the mutant enzyme activity by half 51, a significantly lower concentration than is needed for native aspartoacylase.

Figure 1-2: Crystal structure of ASPA wild type enzyme. PDB ID: 2O4H. Dimer structure of aspartoacylase is shown in both side-view and top-view. The monomer on the left is color coded according to the peptides expressed by each exon from N-terminal to

C-terminal. The monomer on the right is color coded according to domain organization.

The position of the catalytic zinc atom and an intermediate analog is shown.

9 More than 70 clinical mutants of aspartoacylase have been identified that lead to impaired enzyme activity, including 49 missense or nonsense mutations, 5 splicing mutation, 12 small deletions, 2 small insertions, 8 gross deletions 52. The three most common mutations in patients are E285A, Y231X (a truncation due to stop codon), and

A305E. The widely distributed mutation sites on the aspartoacylase protein sequence are shown in Figure 1-3. Patients who have lost any portion of the aspartoacylase gene due to large deletions 10 show severe symptoms at birth, compared to the normal delayed onset at 3-4 month. Although most of the mutants were reported as “loss of activity” in the original reports, the activity of 16 clinical mutants were found to be measurable by using a more sensitive coupled assay51.

Figure 1-3: Clinical mutants of ASPA in Canavan disease. The distribution of point mutations on the ASPA primary sequence and several gene truncation mutants are shown. The most common clinical mutants are color coded in red.

11 It is interesting that the clinical mutants of ASPA are distributed throughout the primary sequence of aspartoacylase. The majority of these mutations are located far away from the enzyme active site. Two hypotheses are presented here that could explain the loss of activity from these remote mutations. First, the low aspartoacylase activity may come from a lower stability of these enzyme forms in vivo. Indeed, it has been shown that two clinical point mutations, K213E and E214X are within 8 Å to residues H10 and

C310, which are the N-and C-terminals shown in the crystal structure. One patient with a

K213E/G274R double mutation had a mild clinical phenotype 53 and four patients with the G274R mutant in one family had moderate to severe symptoms 54. Transiently expressing aspartoacylase mutants in COS-7 cells (monkey kidney fibroblasts) showed that K213E expressed at similar levels as the wild type enzyme and had the same cellular activity as the wild type enzyme 55. Although the G274R mutation had similar mRNA levels to wild type, significant less protein accumulated55. The double mutant further lowered the protein levels 55. The E214X truncation mutation completely loses the C- terminal domain, thus leading to an altered protein that tends to degrade very quickly in cells 55. A stop codon mutant, X314W, adds an additional 45 amino acid residues to the

C-terminal, causing an early disease progression 56. These studies suggest changes in protein stability with different mutations.

Second, although a mutation may be located far away from enzyme active site, it may affect the enzyme activity if the enzyme kinetics is limited by conformational dynamics.

Kots et al modeled the catalytic cycle of aspartoacylase and suggested that the enzyme’s regeneration into its native conformation is the rate-limiting step of the aspartoacylase catalyzed reaction 57. A comparison of the enzyme activity from three different

12 aspartoacylase constructs (two made in our lab and one made by Herga et al 58) possess

different stability and activity as shown in Table 1.1. All three constructs were expressed

in E. coli strains, which would exclude complications from potential post-translational

modifications.

Table 1.1: Comparison of constructs for ASPA wild type enzyme.

Molecular Theoretical Activity Amino acid sequence weight (number Stability Reference (U/mg) of Amino acids) pI

o N’-MGSSHHHHHH Unstable at 4 C 47

37761.2 (332) 6.36 0.083 SSGLVPRGS-ASPA overnight

o N’-GF-ASPA without initial stable at 4 C for 58

35808.1 (314) 6.06 14.2* Methionine residue several days

stable at 4 oC for 42 and this ASPA-HHHHH-C’ 36420.8 (318) 6.25 5-10 several days work 59

*activity measured by a different assay compared to the other two constructs.

In the amino acid sequence column, the underlined ASPA stands for the protein

sequence of aspartoacylase. N’ stands for N-terminal and C’ stands for C-terminal. Other

amino acid residues are represented in single letter notation.

1.4 Aspartate N-acetyltransferase

After identification of NAA in the brain 25, 60, additional studies had been focusing on

identifying the enzymes responsible for NAA synthesis. In 1959, Goldstein reported that

NAA is formed by an enzyme-catalyzed reaction 28. Although some reaction conditions

were recorded, there was very limited knowledge of the NAA synthetic enzyme itself.

13 From that point, although significant progress had been made, it was not until 2010 when

Wiame et al. discovered the gene coding for aspartate N-acetyltransferase 29.

It is now known that the NAT8L gene (HGNC_ID:26742) (or FLJ37478, Hcml3,

"Shati" gene) is located at chromosome 4 (4p16.3) from chr4:2,059,512 to 2,069,089.

The NAT8L gene has 2 introns and 3 exons, which can be translated into a 302-amino acid enzyme with a molecular mass of 32.8 kDa. Exon 1 expresses the amino acid residues 1 to 125. Exon 2 expresses the amino acid residues 126 to 180. Exon 3 expresses the amino acid residues 181 to 302. Currently, there is only one documented case in which a genetic defect in ANAT led to hypoacetylaspartia 36, 61. That patient has a homozygous 19-bp deletion in exon 1 and the mutation leads to an expression frame shift, which eliminated ANAT activity 29.

The NAT8L gene encoded protein, Aspartate N-acetyltransferase (EC 2.3.1.17), ANAT or Asp-NAT, is an acyltransferase that transferring acetyl groups from acetyl-Coenzyme

A to L-aspartic acid. This enzyme is also named N-acetylaspartate synthase based on its biological function. ANAT, has a conserved sequence that is in the GCN5 or NAT superfamily at C-terminal region.

Amino acid sequence analysis of the ANAT protein also identified a hydrophobic region of 30 amino acid residues, which suggests that ANAT could be a membrane bound protein. Indeed, back in 1967, Knizley discovered that the NAA synthetic activity was found in an acetone powder of cat brain, rather than in the soluble fraction, which raised the question of a possible membrane associated enzyme for the first time 62. In

1985, after almost two decades, Truckenmiller et al discovered that ANAT was membrane-associated and that the ANAT activity could be solubilized and partially

14 recovered by treatment with detergents such as Triton X-100 and CHAPS 63. It is now known that ANAT is a membrane bound protein localized in the neuronal mitochondria and that ANAT activity is sensitive to detergents. However, inefficient solubilization and detergent sensitivity are two factors that have limited further biochemical characterization of ANAT.

1.5 Current treatment therapies for Canavan disease

Canavan disease is a genetic disorder because it has defined gene mutations. Canavan disease is also a metabolic disease because the symptoms are due to the disruption of the

N-acetyl aspartate metabolic pathway in the brain. Unlike a traditional single gene genetic disease where mutations cause a simple phenotype, such as the E6V mutation on the beta globin gene causing aggregation of hemoglobin S molecules under low oxygen condition in Sickle-cell disease 64, genetic defects in an enzyme that induce a metabolic disorder can lead to multiple symptoms. Since the disease symptoms are complex, multiple therapeutic approaches could be applied. Five different therapeutic approaches are undergoing extensive research for the possible treatment of Canavan disease, including one at the gene level and four at the protein or metabolic level.

1.5.1 Metabolite Therapy

Triacetin-based acetate supplementation therapy uses glycerol triacetate (GTA or triacetin) to deliver acetic acid into the brain. GTA has been proven to be safe for human consumption and has been used in many different areas 65. The rationale behind acetate supplementation therapy is to address the consequences causes by the lack of

15 aspartoacylase activity in patients: lower levels of acetate production. Recently, Clanton et al supported this approach by suggesting that it is possible to reduce seizures by altering the flux through brain metabolic pathways 66.

1.5.2 Gene Replacement Therapy

The Gene Replacement Therapy approach transfers a copy of the wild type ASPA gene into the target species by using an adenovirus vector in order to express fully functional wild type aspartoacylase67. Since the target cells are in the brain, recombinant adeno-associated virus is commonly used to cross the blood brain barrier. In 2013,

Ahmed et al rescued the early death of CD mice model by using single dose of recombinant adeno-associated viruses, but they still observed some abnormalities in myelin lipids that will need to be addressed 67.

1.5.3 Enzyme Replacement Therapy

Enzyme replacement therapy is a medical treatment that aims to replace the defective enzyme by supplying external produced functional enzyme. Polyethylene glycol is one type of large polymer that shows good biocompatibility. Modification of enzyme surface lysine residues using polyethylene glycol (PEGylation) reduces the immunogenicity of the exogenous enzyme, and the PEGylated ASPA is able to pass the blood brain barrier (BBB) 68. Enzyme replacement therapy has been developed and successfully used for certain lysosomal storage diseases 69, but the need to bypass the

BBB to deliver the functional enzyme for the treatment of CD is an additional challenge.

16 1.5.4 Enzyme Enhancement Therapy

The aim of Enzyme enhancement therapy is to identify small molecule pharmacological chaperones that will stabilize as well as increase the activity of aspartoacylase mutants in vivo. A comparison between the mRNA level and protein expression level in COS-7 cells showed that several mutants have low protein expression levels compared to wild type, even at the same mRNA expression level 55. This suggests two possibilities: protein folding errors due to the present of mutations or protein stability issues which lead to fast degradation in vivo. Small molecule chaperones has been developed for the treatment of GM1 gangliosidosis 70, and initial studies suggest that a similar approach could be used in the treatment of CD.

1.5.5 Substrate Reduction Therapy

Substrate reduction therapy aims to reduce the NAA levels in the brain by using either small molecules such as lithium citrate 71 or by inhibiting the N-acetylaspartate synthetase through the development of potent inhibitors. Baslow et al compared the effect of small molecules on the NAA level in tremor rats. Among the compounds examined, including ethanol, 4-methylpyrazole, 4-pyrazole carboxylic acid, pyrazole-3,5- dicarboxylic acid, 1-H Pyrazole-1-carboxamidine, lithium chloride and sodium valproate, only lithium chloride was found to reduce the rat brain NAA levels by more than 10% 72.

Lithium may be functioning by inhibiting the release of NAA from neurons 73.

Since NAA is produced by aspartate N-acetyltransferase, developing potent enzyme inhibitors has the potential to selectively reduce the NAA concentration without other side effects as may happen in lithium therapy. This idea is supported by a report from

17 Guo et al showing that a mouse model without both the ASPA and NAT8L genes was found to have normal CNS myelination 74.

1.6 Chapter outline and organization

Despite more than eighty years of research on Canavan disease and related enzymes, there are still many unanswered questions.

The first area concerns the post-translational modification hypothesis of human aspartoacylase. Aspartoacylase was proposed to be a post-translationally modified enzyme, especially suggestions of glycosylation and phosphorylation. In Chapter 2 of this dissertation, we have explored the possible factors that could affect aspartoacylase expression, activity and stability. We performed mass spectrometry based protein sequencing studies to explore potential modifications on aspartoacylase. No additional post-translational modifications were observed.

Second, human aspartate N-acetyltransferase has been shown to be a membrane associated enzyme, and expression and purification of this enzyme to high purity represented a major challenge for the application of Substrate Reduction Therapy for the treatment of Canavan disease. In Chapter 3 of this dissertation, we demonstrate the successful expression and purification of human aspartate N-acetyltransferase in a prokaryotic host and its subsequent characterization.

Third, for our initial effort towards the Substrate Reduction Therapy for Canavan disease, we have identified preliminary ANAT inhibitors through focused library screening, and carried out further optimization of these identified core structures

(Chapter 4) to produce reasonably potent ANAT inhibitors.

Chapter 2

Characterization of Human Aspartoacylase

2.1 Introduction

Human aspartoacylase is an essential enzyme in the human brain, participating in the

N-acetylaspartic acid metabolic pathway. Before this study, the molecular identity41, the metal dependency42, the structure of wild type enzyme43, 49, and the effects of clinical mutations on enzyme activity and stability51 were all established. However, there is one remaining question in literature, which is about the potential post-translational modifications (PTMs), such as glycosylation, of this enzyme.

On the one hand, there are multiple lines of evidence that suggest aspartoacylase functions as a glycoprotein. First, sequence alignment showed a consensus glycosylation motif, Asn-Xxx-Thr/Ser (Xxx can be any amino acid except for proline) at N117, and the motif is conserved among multiple homologs of aspartoacylase from eukaryotic species.

Second, a significant difference in aspartoacylase activity was observed from different expression hosts. Aspartoacylase expressed from Pichia pastoris had a 100-fold higher activity compared to the enzyme expressed from E. coli 42, 47. A conserved mutation

(N117Q) of the potential glycosylation site on yeast expressed enzyme led to an enzyme

19 with decreased activity and stability similar to E. coli expressed aspartoacylase42. Third, glycosidase treatment and glycan enrichment study using Pichia expressed aspartoacylase resulted in one major peak and two small peaks observed by MALDI- mass spectrometry that corresponded to complex-type oligosaccharides42. The effects of endoglycosidases treatment on aspartoacylase activity were also studied. Peptide:N-

Glycosidase F (PNGase F) treatment reduced the enzyme dramatically, while

Endoglycosidase H (Endo H), an amidase that cannot cleave complex-type oligosaccharides 75, didn’t affect the enzyme activity.

On the other hand, the evidence suggesting aspartoacylase is not a glycoprotein, or at least not the N-glycosylation is required for enzyme activity came from the crystal structure. No additional electron density was observed at the potential glycosylation site

N117 from either apo- or complex form of human aspartoacylase 43, 49. The same result was observed for the rat homolog 76. In each case the N117 side chain was completely buried. The human aspartoacylase from dissolved crystals has same activity, thus excluding the possibility that the crystallized form was an inactive form of human aspartoacylase. GST-fusion aspartoacylase was expressed from E. coli and the thrombin- released aspartoacylase has been reported to have comparable activity to the Pichia expressed enzyme58.

To provide a more consistent picture for these discrepancies, a detailed study was conducted focusing on possible PTMs. Five different experiments will be presented in this chapter with the aim of testing the glycosylation hypothesis, including a reexamination of the de-glycosylation experiments on aspartoacylase enzyme activity, a gel mobility based assay for glycan removal, a MALDI experiment for molecular weight

20 determination, and a tandem mass spectrometry based protein sequencing study. In addition, a new construct for E. coli expression led to a fully active enzyme. These newly obtained data suggest that aspartoacylase is not glycosylated when expressed in Pichia and glycosylation is not required for aspartoacylase activity.

Figure 2-1: Sequence alignments of aspartoacylase homologs from different species.

Uniprot database contains machine annotated protein sequences with or without human annotation. The human annotated sequences (A and C) are more likely to be real aspartoacylase homologs from different species. Despite the conserved amino acid sequence patterns that were observed from the machine annotated sequences (C and D), experimental validations are still needed.

Figure 2-2: Asparagine residues of human aspartoacylase.

Aspartoacylase is shown from three orientations. While multiple asparagine residues

(colored in blue) are on the surface of human aspartoacylase, Asn117 site (colored in red) is completely buried inside.

2.2 Potential N-glycosylation site

Glycosylation is one of the most common types of protein post-translational modifications. It is estimated that about 20% of proteins from all species recorded in the

Swiss-Prot database are glycosylated based on updated statistics in 201177. Glycosylation enhances the hydrophilic properties of targeted proteins and also has an effect on the conformation of modified proteins. According to the nature of the glycosidic bond, biological glycosylations can be categorized into asparagine-linked (N-linked, or N-

22 glycosylation), serine/threonine-linked (O-linked, O-glycosylation) and other less common types of glycosylations. N-glycosylation has been very well studied. The pre- synthesized oligosaccharides are transferred to the nascent polypeptide by oligosaccharyltransferases in the lumen of the ER membrane, in a peptide sequence- dependent manner. The consensus sequence, named sequon, for this type of glycosylation is Asn-Xxx-Ser/Thr, where Xxx can be any amino acid residue except proline. An Asn-

Xxx-Cys sequon has also been identified in rare instances 76. Based on this consensus sequence human aspartoacylase has only one potential N-glycosylation site at Asn117.

During our characterization of this enzyme it was important to investigate whether this glycosylation site is occupied or not, and how its occupancy could potentially affect the enzyme property of aspartoacylase.

Although it is unlikely that an N-glycosylation event could happen on an asparagine residue that is not part of the glycosylation sequon, non-classical N-glycosylation events have been observed according to comprehensive glycoproteomic studies78.

Aspartoacylase contains a total of 15 asparagine residues. To predict non-classical N- glycosylation sites, NetNGlyc server from SIB Bioinformatics Resource Portal has been used. The NetNGlyc server is widely used to predict N-glycosylation sites for human proteins based on amino acid sequence information. It uses artificial neural networks for the surrounding sequences of a sequon, and has an overall prediction accuracy of 76%.

Based on this analysis four out of the fifteen asparagines have a somewhat higher potential for N-glycosylation (Table 2.1). Three of these asparagines with higher scores are also solvent exposed: Asn37, Asn77 and Asn134 (Figure 2-3).

Table 2.1: Predicted glycosylation sites by NetNGlyc 1.0 server.

(Threshold=0.5) ------SeqName Position Potential Jury N-Glyc ------Sequence 23 NELT 0.5958 (7/9) + Sequence 37 NGAE 0.7680 (9/9) +++ Sequence 54 NPRA 0.6852 (8/9) + WARNING: PRO-X1. Sequence 70 NRIF 0.5009 (3/9) + Sequence 77 NLGK 0.6890 (9/9) ++ Sequence 97 NHLF 0.4014 (7/9) - Sequence 117 NTTS 0.6867 (9/9) ++ SEQUON NXS/T. Sequence 121 NMGC 0.6162 (6/9) + Sequence 133 NNFL 0.5000 (4/9) + Sequence 134 NFLI 0.6803 (9/9) ++ Sequence 210 NEGK 0.5972 (7/9) + Sequence 236 NGEI 0.6321 (7/9) + Sequence 246 NLQD 0.6631 (8/9) + Sequence 284 NEAA 0.4950 (5/9) - Sequence 304 NAKS 0.6248 (6/9) +

Besides the N117 site in a NTT sequon, three other asparagine residues N37, N77 and

N134 were predicted to be potential glycosylation sites if the sequon constraints were removed. The N54 site is unlikely to be a glycosylation site due to the presence of a proline residue following the asparagine.

24 Figure 2-3: Additional putative glycosylation sites identified by NetNGlyc server. The potential N-glycosylation sites are those above the threshold (0.5), with the highest peaks indicating the greatest potential for glycosylation. The distribution of potential N- glycosylation sites is shown along the primary sequence, with the solvent exposed sites that have the highest potential labeled.

2.3 Expression and purification of human aspartoacylase from Pichia pastoris

Pichia pastoris is one of the most commonly used eukaryotic systems for heterologous protein expression. In contrast to bacterial cells, Pichia can support different protein modifications, including N-linked post-translational glycosylation. The aspartoacylase wild type gene was cloned into pPICZ A vector using EcoRI and Xho I restriction sites. A hexa-histidine tag was placed at the carboxyl-terminus to facilitate enzyme purification. The resulting plasmid was amplified in XL10 competent E. coli cells using the low salt formula of Luria-Bertani (LB) broth with 50 µg/ml Zeocin as the selecting antibiotic. The restriction enzyme Sac I was used to linearize the plasmid in order to check the integrity of the construct and to perform homologous recombination of human aspartoacylase into the yeast chromosome. Three different Pichia stains, X-33,

GS115, and KM71H were examined, and KM71H was chosen based on the optimal expression levels of the enzyme42. X-33 is a wild-type Pichia strain and GS115 strain

(his4) has a mutation in the histidine dehydrogenase gene, thus is depended on exogenous histidine from the media. While both X-33 and GS-115 strains grow fast in methanol, the

KM71H strain (arg4 aox1::ARG4) grows slower in methanol due to loss of the AOX1 gene.

25 Cells containing human aspartoacylase gene were selected on Yeast extract-Peptone-

Dextrose-Sorbitol (YPDS) plates after growth for 3 days at 28 oC. Colonies were picked and cultured in 10 ml Minimal Glycerol Medium (MGM) for ~22 h and cells were then transferred to 1L Minimal Glycerol Medium once the OD600 reached 10, followed by incubation for ~36 hours under the same conditions to increase biomass. The expected

OD600 after 36 hours was between 10-15. The cells were then collected by centrifuge at

5000 rpm at 25 oC for 5 min. Cells were transferred to 500 ml of Minimal Methanol

Medium (MM). The growth media was supplemented with 1% methanol after 24 hours to compensate for evaporation. Cells were harvested after another 24 h by centrifuging at

8000 rpm for 15 min at 4 oC. Cell paste was kept at -80 oC until further usage. Typically,

15 g of cell paste were obtained per liter of cell culture.

Pichia expressed human aspartoacylase (hASPA) was purified by Immobilized-Metal

Affinity Chromatography (IMAC) using home-packed columns with Ni Sepharose High

Performance resin (GE Healthcare) and further polished by ion exchange chromatography on Source 15Q resin (GE Healthcare). Briefly, 5 g of cell paste was suspended in 50 ml Ni-NTA Buffer A containing 20 mM potassium-phosphate, pH 7.4,

20 mM imidazole, 500 mM NaCl, 5 mM β-mercaptoethanol, and 5% glycerol. To prevent protein digestion in vitro, 100 µl of protease inhibitor cocktail (Sigma, P8340) was added per 50 ml buffer. Pichia cells were mechanically disrupted by glass beads on a beads beater for 10 cycles, 1 min on and 5 min off for each cycle. After centrifuging at 10000 rpm for 30 min, the supernatant was filtered using 0.8-micron syringe filters. The soluble lysate was then loaded onto a Ni-NTA affinity column equilibrated with Buffer A using either an AKTA Explorer 100 or AKTA FPLC chromatography system (GE Healthcare).

26 The enzyme was eluted by a linear gradient of the concentration of imidazole from 20 mM to 400 mM in 10 column volumes (CV). Kinetic assay and SDS-PAGE were performed on the collected fractions and the active fractions were pooled and dialyzed against ion-exchange buffer A, containing 50 mM Hepes and 1.0 mM DTT at pH 7.5 overnight. The second purification was done by eluting the enzyme with a linear gradient of sodium chloride from 0-500 mM in 35 column volumes on a Source15 Q anion exchange resin. The active fractions were combined and dialyzed into 50 mM Hepes at pH 7.4, containing 100 mM NaCl and 1 mM DTT. After concentration to less than 2 ml of total volume using an Amicon Ultra 15 concentrator (10 kDa norminal molecular weight cutoff, Millipore), the protein concentration was measured by using a NanoDrop

2000 spectrophotometer. Concentrated aspartoacylase was stored at -20 oC until further usage. The protein yield for Pichia expressed aspartoacylase is about 5 mg from 15 g of cells.

20130215ASPA15Q(1361062156)001:10_UV1_280nm 20130215ASPA15Q(1361062156)001:10_UV3_260nm 20130215ASPA15Q(1361062156)001:10_Conc 20130215ASPA15Q(1361062156)001:10_Fractions 20130215ASPA15Q(1361062156)001:10_Logbook

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 3839 0 50 100 150 200 250 300 350 ml

27 Figure 2-4: Anion exchange chromatogram of Pichia expressed human aspartoacylase.

Two peaks usually showed up in the chromatograph of Source 15Q column. The second peak (fractions 15-19) corresponds to aspartoacylase. The blue curve corrresponds to the

UV absorption at 280 nm and the magenta curve corresponds to UV absorption at 260 nm.

Both UV curves are shown on the same scale.

2.4 Comparative enzyme activity analysis for ASPA glycosylation

The catalytic activity of aspartoacylase was measured by a coupled assay that uses L- aspartase as the coupling enzyme and N-acetylaspartate (NAA) as the substrate 47. NAA is converted into L-aspartate, which was then converted into fumarate by aspartase.

Increasing absorbance at 240 nm was monitored on an UV plate reader (SpectraMax) followed the formation of fumarate (2.53 cm-1mM-1). Typical assay buffer contains 25 mM Hepes, 1 mM Mg(OAc)2, 0.1 mM 2-mercaptoethanol at pH 7-7.2. The assay was performed by adding 940 µl buffer, 10 µl L-aspartase (~20 µg), 10 µl NAA to achieve a

0.75 mM final concentration, and 40 µl of aspartoacylase.

Figure 2-5: Aspartoacylase assay with aspartase as coupling enzyme. Adapted from

Moore et al47.

28 Previous experiments from our laboratory have suggested that glycosylation has significant roles in maintaining the activity and stability of human aspartoacylase expressed from Pichia pastoris. The activity of the wild type human enzyme is around 10 units/mg, while the E. coli expressed murine aspartoacylase, which would not be glycosylated in this bacterial expression system, has a much lower activity (0.08 units/mg)42. Comparative enzyme activity assays were conducted on human aspartoacylase with and without glycosidase treatment to confirm the effect of de- glycosylation on enzyme activity. Human ASPA was digested in 50 mM Hepes, 100 mM

NaCl, pH 7.4 in native conditions at 37 oC for 1 hour. An example of the reaction mixture is listed in Appendix A1. One hour PNGase F treatment at 37 oC has a significant effect on aspartoacylase activity as shown in Figure 2-6. While this result may be interpreted as supportive evidence that an N-glycan has an effect on aspartoacylase activity, a control study led to a different explanation. The storage buffer for PNGase F from New England

Biolabs contains 20 mM Tris-HCl, 50 mM NaCl, 5 mM EDTA and 50% Glycerol. To our surprise, the storage buffer itself, without PNGase F, has the same effect on aspartoacylase activity (Figure 2-7). Further study demonstrated that the decrease in aspartoacylase activity was the result of the presence of EDTA in the enzyme storage buffer. During the de-glycosylation reaction, the final concentration of EDTA was 0.83 mM, which could either have removed the essential catalytic metal, or behaved as an inhibitor. De-glycosylation experiments using other endoglycosidases, such as Endo H, didn’t have any effect on the catalytic activity of aspartoacylase.

Figure 2-6: Kinetic assay of human aspartoacylase during PNGase F digestion in native condition. Each reaction was conducted in duplicate.

Figure 2-7: Kinetic assay of the mock “PNGase F treatment” reaction using the same buffer storage conditions but leaving out the PNGase enzyme. The extended lag in

“PNGase F” treated sample suggests the inhibition of aspartoacylase activity.

2.5 Gel mobility shift analysis for post-translational modification

On SDS-PAGE, proteins are primarily separated based on differences in molecular weight, due to the denaturing effect of SDS. Gel mobility shift analysis following endoglycosidase digestion provides a good measurement of changes in molecular weight due to changes in glycosylation. Aspartoacylase expressed from Pichia pastoris strains don’t

30 have the enzymes capable of the complex-type N-glycan synthesis that could be found on human enzymes. The resulting protein, if it is glycosylated, should contain simpler high mannose type N-glycans79.

Chicken ovalbumin was selected as a positive control for a deglycosylation study because it has only one well-characterized N-glycan site. The de-glycosylation experiment was conducted as described above, and the migration patterns of aspartoacylase with and without PNGase F treatment were compared by SDS-PAGE. The gel bands for aspartoacylase appear at the same position before and after treatment, consistent with the predicted molecular weight of about 36 kDa (Figure 2-8). The control glycoprotein, ovalbumin, shows the expected measurable decrease of molecular weight on after deglycosylation. This result suggests that, contrary to the previous interpretation of the kinetic studies 42, the yeast-expressed aspartoacylase doesn’t have an N-glycan attached to it.

Figure 2-8: Gel mobility assay for glycan detection. SDS-PAGE of PNGase F-treated (+) and untreated (-) aspartoacylase (ASPA) (lanes 2-3) and treated (+) and untreated (-) ovalbumin (lanes 4-5). While the cleavage of a single N-glycan is observable for ovalbumin on SDS-PAGE, no mobility shift was observed for ASPA.

32 2.6 Molecular peak determination by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) is a soft ionization technique that is particularly suitable to analyze large biomolecules. MALDI coupled to a time-of-flight

(TOF) analysis is commonly used to determine the molecular weights of proteins. In this study, two criteria are used to determine if aspartoacylase is a glycoprotein. First, glycoproteins have a different preference for certain MALDI matrices. While common non-glycoproteins prefer sinapinic acid, glycoproteins are easier to ionize in 2,5- dihydroxybenzoic acid or a combination of different matrices. Also, MALDI signals for glycoproteins are generally weaker than non-glycoprotein because of the heterogeneous nature of N-glycans. Second, a group of molecular ions with mass differences expected for the integral number of mono sugar units should be detected, which is different from the molecular ion peak commonly seen in non-glycoproteins. Human aspartoacylase, after Source 15Q purification, was dialyzed into 10 mM ammonium acetate buffer at pH

5.5. Ammonium acetate buffer is a volatile buffer which is compatible with MALDI analysis. It is worth noting that Hepes buffer will inhibit the MALDI signal at concentrations as low as 50 mM. The protein and matrix are mixed at 1:1 ratio (v:v) and dried under reduced pressure. MALDI spectra were recorded on a Bruker’s

Ultraflextreme MALDI TOF/TOF instrument that was used in linear positive ion mode.

The calculated molecular weight for wild type human aspartoacylase is 36,421 Da. As shown in Figure 2-11, +2 and +1 molecular ions are observed at 36,327 m/z and 18,150 m/z. The difference between these measured values and the calculated molecular weight is less than 0.26%. Therefore, MALDI-TOF data suggest that aspartoacylase is not glycosylated, consistent with the gel mobility shift experiments described above.

Figure 2-9: MALDI-MS of intact aspartoacylase. The +1 and +2 charges of intact aspartoacylase are shown. No sign of glycan was observed.

34 2.7 Solid-phase extraction of glycopeptides

Gel mobility analysis and MALDI molecular weight measurement for human aspartoacylase both give consistent results, suggesting that this protein is not glycosylated.

But the two experiments mentioned above cannot rule out the possibility that only a small portion of the Pichia expressed aspartoacylase is indeed glycosylated, and that the glycosylated aspartoacylase has significantly higher activity. More than 90% of the yeast- expressed aspartoacylase could be non-glycosylated, and, given the differences in ionizability, the non-glycosylated version could have been selected exclusively in the

MALDI molecular weight measurements. To rule out that possibility, a glycan specific enrichment method was employed.

Various methods have been used to enrich glycopeptides based either on the physical or chemical properties of carbohydrates. For example, glycopeptides can be enriched based on their hydrophilic nature by using Sepharose CL-4B resin 80, 81. Glycopeptides can also be isolated by affinity purification using lectins82, which are selective carbohydrate binding proteins. The most robust method is Solid Phase Extraction of

Glycopeptides (SPEG) based on a reversible hydrazone formation mechanism83, 84. This approach relies on the chemical oxidation of the hydroxyl groups of sugars and the subsequent coupling reaction to a solid phase support modified with hydrazide functional groups (Figure 2-10).

Figure 2-10. Hydrazine chemistry basic principle, modified from Zhang et al83.

Tryptic peptides from Pichia expressed human aspartoacylase and a control glycoprotein fetuin were further purified using C18 SPE columns (Waters). The column was first washed with 5 ml acetonitrile, 2 ml 50% acetonitrile/0.1% Trifluoroacetic acid

(TFA) and equilibrated with 3 ml 0.1% TFA in water. Each sample was loaded onto the column and washed three times with 3 ml of 0.1% TFA. Peptides were eluted with 400 µl

50% acetonitrile/0.1% TFA. To oxidize the glycans, 50 µl of 100 mM sodium (meta)- periodate was added to make a final concentration of 10 mM periodate ions. Peptides were oxidized at room temperature for 1 h in the dark. After 10 times dilution using 1%

TFA to lower the acetonitrile concentration, the excess of sodium periodate was removed by the C18 SPE column. The oxidized glycopeptides were immobilized to a hydrazide resin at room temperature overnight with shaking. To accelerate the reaction, 1 mM aniline was added as a catalyst. Non-glycopeptides were removed after extensive washing steps with 50% acetonitrile/0.1% TFA, 1.5 M NaCl, water, and 50 mM ammonium bicarbonate (pH 7.5), 3 times each for a total of 12 times. Then, the formally

36 glycopeptides were released by PNGase F at 37 oC for 16 hours. The released peptides were purified by a Zip-Tip C18 or Sep-Pak C18 SPE column and subjected to MALDI-

TOF/TOF sequencing using the positive ion mode. α-Cyano-4-hydroxycinnamic acid was used as the matrix. Three glycosylation sites, corresponding to the correct number of known sites, were successfully identified form control glycoprotein fetuin (Figure 2-11), but no glycosylation sites were identified from human aspartoacylase.

Figure 2-11: All three N-glycosylation sites were confirmed from fetuin using the SPEG protocol. The difference between the calculated peptide masses and detected masses are smaller than 0.2 Da.

37 2.8 Mass spectrometry-based protein sequencing

The glycan-targeted method confirms that Pichia expressed human aspartoacylase is not a glycoprotein. However, it is still possible that other types of post-translational modifications are present. Aspartoacylase was subjected to tandem mass spectrometry- based protein sequencing. Aspartoacylase was digested with either trypsin or Glu-C overnight. The digested peptide mixture was first detected by MALDI-MS for mass fingerprinting analysis and were then separated on an UltiMate 3000 Nano LC System with an Acclaim PepMap 100 C18 Protein and Peptide Column (Figure 2-12). Mobile phase A was HPLC grade water plus 0.05% TFA and mobile phase B was 90% HPLC grade acetonitrile/water plus 0.05% TFA. A 75 min linear gradient from 3% B to 60% B, followed by a linear gradient from 60% B to 100% B in 10 min, were used at a flow rate of 300 nl/min to separate the peptides (Figure 2-13) The fractions from this column were mixed with the DHB matrix and loaded onto a MALDI target (AnchorChip, Bruker) by a

PROTEINEER fc II system (Bruker). The spotting interval was 15 s, each spot contained

75 nl of sample, and more than 300 spots were typically collected for each nano-HPLC separation. MS/MS data were collected using LIFT mode. As shown in Figure 2-11, 242 out of 313 (77.3%) amino acid residues were covered either by peptide mass fingerprinting or by tandem sequencing. Peptides covering 137 amino acid residues

(43.7%) were sequence confirmed by tandem sequencing. 11 out of 15 asparagine residues were covered and no evidence of any post-translational modifications were obtained. Further sequencing effort identified the N117 containing peptide and the sequencing result showed no modification at the N117 site (Figure 2-14).

Figure 2-12: A representive chromatogram of nano-HPLC separation.

Tryptic peptides of purified aspartoacylase were separated on an UltiMate 3000 Nano LC

System with an Acclaim PepMap 100 C18 Protein and Peptide Column. The fractions from this column were mixed with MALDI matrix and spotted on MALDI target for further analysis. The black curve corrresponds to absorption at 214 nm and the red curve corresponds to absorption at 280 nm.

Figure 2-13: Sequencing of aspartoacylase by mass spectrometry

The peptides that are not covered by any method are in red.

Figure 2-14: Sequencing of N117 containing peptide of aspartoacylase.

40 A representative spectrum for peptide containing N117 site. The b and y ion series are shown.

2.9 Codon optimization of human aspartoacylase gene for E. coli expression system

Codon bias has been shown to be one of the major factors affecting heterologous protein expression85. For example, the rare codons used by certain human proteins may cause translation errors, such as translation stalling, early termination, and amino acid substitution86. To solve this problem, several genetic modified E. coli strains with an additional plasmid that contains genes expressing the tRNAs coding for rare codons supplemented have been available on the market. An advantage of using codon- supplemented strains is that no modification of human genes is necessary. But maintaining an additional plasmid requires a second antibiotic. And the expression level of target protein is affected by the expression levels of multiple supplemented tRNA genes.

Codon optimization has been shown as one powerful technique to enhance the expression of human proteins in E. coli by leading to optimized mRNA stability and enhanced tRNA availability87. To test if codon optimization could help with the expression of human aspartoacylase in E. coli, the human ASPA gene was codon optimized using GeneOptimizer® software and synthesized by GeneArt® Gene Synthesis service (Life Technologies) (Appendix A2 and A3). Changes in the endonuclease cutting sites of Nde I and Xho I were avoided during the codon optimization. Also, the oligonucleotide for a hexahistidine tag was attached at the 3’ terminus of the synthetic gene. The codon-optimized gene was cloned into a pET41a vector as a fusion protein

41 using Nde I and Xho I restriction sites. The calculated molecular weight for the expected protein is 36.4 kDa.

2.10 Expression and Purification of wild-type Aspartoacylase enzyme from E. coli

The codon optimized human aspartoacylase wild type was successfully expressed and purified from an E. coli BL21(DE3) strain. Briefly, BL21(DE3) cells were transformed using the expression construct, with 37 oC chosen as the initial growth temperature for E. coli before induction without optimization. Later, 28 oC was identified as the best inducing temperature, determined by varying the induction temperature from 16 oC to 37 o C. IPTG was added into cell culture at a final concentration of 1 mM once the OD600 reached at least 0.5. Cells were harvested after 5 h by centrifugation at 10,000g for 10 min, and were kept at -80 oC for further usage.

The wild type aspartoacylase was purified by Ni-affinity chromatography, followed by anion exchange chromatography to homogeneity, similar to the procedure used for the yeast-expressed enzyme. Briefly, 5 g of cell paste was dissolved in 50 ml NiNTA buffer

A containing 50 mM HEPES, 0.5 M sodium chloride, 20 mM imidazole, 5% glycerol and

5 mM β-mercaptoethanol at pH 7.4 for 30 min. EDTA-free Protease inhibitor cocktail

(P8340, Sigma) was added to prevent sample degradation. After homogenization using a hand homogenizer for 30 times, the cells were broken by sonication (pulse on 30 s then off for 2 min, total pulse on for 10 min). After centrifuge at 15,000 x g at 4 oC for 30 min, the sample was loaded on the Ni-affinity column. After washing out the unbound proteins, aspartoacylase was eluted by a linear imidazole gradient from 20-400 mM. The expression fractions were checked by kinetic assay and SDS-PAGE (Figure 2-15).

Figure 2-15: Expression of codon optimized aspartoacylase wild type enzyme in E. coli.

Codon optimized ASPA gene was successfully amplified (A) and ligated into pET41a vector using NdeI and XhoI restriction enzymes. The purified aspartoacylase showed up as single 36 kDa band on SDS-PAGE as expected (B). The activity of E. coli expressed

ASPA was calculated to be 6.7 U/mg, which is comparable to the activity of Pichia expressed enzyme. The lag shown in ASPA reaction (C) reflects the slower activity of aspartoacylase in the coupled assay. No lag is observed in control reaction where only aspartase and L-aspartic acid are present.

43 2.11 Summary for post-translational modification analysis for human aspartoacylase expressed from Pichia

Human protein structures can be more complicated than bacterial proteins due to a range of types of post-translational modifications. Post-translational modifications will alter protein surface property and stability, and activity if the protein of interest is an enzyme. Aspartoacylase is an essential human enzyme participating in brain metabolism and mutations of aspartoacylase lead to a fatal human disease called Canavan disease.

Initial sequence analysis of human cDNA suggested the possibility of post-translational modifications such as glycosylation or phosphorylation. Our initial studies gave contradictory results about whether aspartoacylase is a glycoprotein or not. The additional studies described here have conclusively demonstrated that yeast-expressed aspartoacylase is not a glycoprotein. And no evidence has been found for any other post- translational modifications.

2.12 Developing treatment therapies targeting aspartoacylase for Canavan disease

Two different treatment therapies are being developing in our laboratory, enzyme replacement therapy and enzyme enhancement therapy, with aspartoacylase as the target for each of these therapeutic approaches.

2.12.1 Progress on Enzyme Replacement Therapy

Enzyme Replacement Therapy uses polyethylene glycol-modified human aspartoacylase as the enzyme form for this approach. It has been demonstrated that global modification of aspartoacylase through surface lysine residues retained more than 80% of

44 its enzymatic activity in vitro51 and was able to effectively lower the elevated substrate levels observed in Canavan mice68, 88. Since the mechanism for PEGylated aspartoacylase to traverse blood-brain barrier has not been fully characterized, multiple experiments were designed to probe this mechanism. One potential issue was noticed immediately.

Aspartoacylase has 23 lysine residues per monomer, and all of these lysines are exposed to solvent to some extent. Global modification of lysine residues results in a heterogeneous mixture, with different degrees of modification as well as modification site heterogeneity (Figure 2-16). Both cation and anion chromatography resins were used in an attempt to separate these PEGylated enzyme mixtures, but with only limited success.

The un-PEGylated enzyme could be separated from PEGylated enzyme, but the individual modified enzyme forms could not be completed separated from each other. To reduce the heterogeneity problem, N-terminal PEGylation experiments were conducted at a lower pH (5.5 vs. 8.3). While most of aspartoacylase remained unmodified, about 20% of aspartoacylase were modified and subsequently showed only one band on SDS-PAGE

(Appendix A4). The next step would be to optimize the yield of modified enzyme and then to remove the unmodified aspartoacylase using HPLC as described89 to yield a homogeneously modified enzyme for animal studies.

Figure 2-16: Surface lysine modification of aspartoacylase. Methoxy-PEG-

CO(CH2)2COO-NHS, Mw 5,000 was used to modify human aspartoacylase. Although all enzymes were PEGylated based on SDS-PAGE, sample was heterogeneous.

2.12.2 Progress on Enzyme Enhancement Therapy

Osmolyte buffers, such as trimethylamine N-oxide (TMAO), have the ability to assist certain protein mutants to fold properly 90. Since Canavan disease is caused by mutations in the aspartoacylase enzyme and most aspartoacylase mutants are much less stable 51, we

46 hypothesized that compounds that could preferably bind to an allosteric site on aspartoacylase and stabilize it may ether increase the enzyme activity in vitro, or increase the stability of the enzyme in vivo. Previously, the ligand binding hot spots on aspartoacylase were probed using FTMap91, which is a computational mapping server that performs blind docking calculations for multiple small organic molecules for submitted biomacromolecules and then performs clustering to identify “druggable” sites.

Experimentally, high-throughput thermofluor assays were conducted for ASPA mutants and multiple compounds showed a dose-dependent stabilization92. To understand the interaction sites between the identified compounds and aspartoacylase mutants, a blind docking study was performed using Autodock Vina93. The dimer structures of wild type

ASPA as well as E285A mutant were used. The docking grid was enlarged to cover the whole enzyme dimer, which enabled the global energy evaluation between protein and small molecule ligands without human intervention. The lowest binding energy and binding position for each compound on E285A mutant is shown in Figure 2-17. Most of the compounds were proposed to bind to the ASPA dimer interface as shown in Figure 2-

17. It was noticed that N-acetyl-glutamate preferentially interacted with the active site residues in the E285A mutant enzyme, but not in the WT ASPA, suggesting an enlarged substrate binding pocket in the mutant form. The inhibition patterns of N-acetyl-L- glutamate against WT ASPA as well as E285A mutant were tested using our kinetic assay. While N-acetyl-L-glutamate didn’t inhibit WT ASPA at the highest concentration of 10 mM, it was found to be a weak inhibitor for E285A mutant (Ki = 4.34 ± 0.82 mM).

47 E285A

diprotin B

NAAG

ellagic acid patulin glutathione

captopril

Figure 2-17: ASPA Dimer interface as a preferred binding site.

Each monomer of ASPA E285A mutant is colored differently. The dimer interface of

ASPA E285A with docked compounds is shown. While most of the compounds binds to the dimer interface, patulin binds preferably to the enzyme active site.

48 Table 2.2: Blind docking study of ASPA stabilizers.

WT E285A Difference in (kcal/mol) Binding site (kcal/mol) Binding site binding energy (kcal/mol) Acetyl-Glu -4.8 Dimer interface -5.7 Active site -0.9

Patulin -5.2 Active site -6.0 Active site -0.8

Glutathione -5.5 Dimer interface -6.3 Dimer interface -0.8

captopril -4.9 Dimer interface -5.2 Dimer interface -0.3

diprotin B -6.2 Dimer interface -6.3 Dimer interface -0.1

Ellagic acid -7.9 Dimer interface -7.8 Dimer interface 0.1

NAAG -5.9 Dimer interface -5.5 Dimer interface 0.4

Chapter 3

Purification and Characterization of Aspartate-N- acetyltransferase

3.1 Introduction

Aspartate N-acetyltransferase is the enzyme responsible for the synthesis of N-acetyl-

L-aspartic acid in the neuronal mitochondria. Although this is not the defective enzyme in

Canavan disease, increasing evidence suggests that the activity of this enzyme is directly related to CD symptoms. In 2000, Matalon et al developed a mouse model in which a 10 bp deletion was introduced in exon four of the ASPA gene. The gene knockout mice display many of the CD symptoms, such as neurological impairment, macrocephaly, and a high concentration of NAA in the brain and urine 94. While most of the attention had been focused on aspartoacylase, the defective enzyme, a recent study reported decreased

CD symptoms by introducing a second gene knock-out in the NAT8L gene, which codes for aspartate N-acetyltransferase 74. The double knock-out mice appear to have normal myelination. These new results highlights the importance of ANAT in CD symptoms.

Unfortunately, expression and purification of ANAT is still challenging due to the

50 inherent properties of the enzyme, in particular the fact that ANAT is a membrane- associated protein 95.

Aspartate N-Acetyltransferase has not been easy to solubilize. Several different detergents have been used in an attempt to solubilize this enzyme. In 1985, Truckenmiller et al discovered that about 60% of the ANAT activity could be recovered from rat brain homogenates after treatment using 1% Triton X-100 63. It was also reported that 3-((3- cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS) was less efficient in

ANAT solubilization. Treatment with 0.1 mM, 1 mM and 10 mM CHAPS resulted in

0%, 15% and 35% recovery of ANAT activity, respectively 63. Higher activity recovery, about 50%, was observed by increasing the solubilization time from 1 h to overnight 96.

Lu et al reported that 0.5% Triton CF-54 also increased ANAT solubilization by 10 to

25% 97. Using a higher concentration, above 2%, caused a significant increase in soluble

ANAT activity. In 2010, Wiame et al discovered that octylglucoside (OG) is also capable of solubilizing ANAT, although the efficiency with this detergent is less than CHAPS at low concentration 29.

From these studies it was determined that ANAT activity is sensitive to the presence of detergents. For example, while 1% Triton X-100 is good for ANAT solubilization, further increases in detergent concentration up to 10% completely destroyed the enzyme activity 63. For CHAPS, solubilization of ANAT increased with increasing CHAPS concentrations from 0.1 mM up to 10 mM, while further increases destroyed ANAT activity irreversibly 96. At least 60% of ANAT activity is lost at 12 mM CHAPS concentration and 90% of ANAT activity is lost at 20 mM CHAPS concentration.

51 Compared to CHAPS, octylglucoside (OG) is a milder detergent for ANAT solubilization according to Wiame et al 29. 5 mM of OG caused loss of about half of the ANAT activity.

At this point, four different detergents had been applied for ANAT solubilization, with different degrees of success. In order to get a more complete picture of the detergent effects it is necessary to expand the list of detergents and include different types of detergents to test for better ANAT solubilization. Since inefficient solubilization and detergent sensitivity may ultimately limit our ability to apply the traditional approaches for membrane protein analysis, even when using optimized detergents, alternative approaches have also been evaluated.

Three different approaches to obtain a soluble and active form of ANAT are presented in this Chapter. First, a panel of detergents was explored to extract a native ANAT form with only a carboxyl terminal hexahistidine tag added. Second, a protein engineering study was conducted with the aim of altering or removing the putative membrane anchor domain. Third, chimeric protein constructs were prepared with different solubilizing protein fusion partners.

3.2 Detergent extraction of native aspartate-N-acetyltransferase

Detergent extractions of native aspartate-N-acetyltransferase was performed in our laboratory by Dr. Mojun Zhao and Gwenn G. Parungao as a collaborative project, and those results are briefly described here 98.

The ANAT fusion enzyme used for detergent extraction, containing only a carboxyl terminal hexa-histidine tag, was constructed by Dr. Stephen Zano and is referred to as

“native ANAT” 99. To make this construct the human NAT8L gene was cloned into the

52 pETDEST42 plasmid, including a linker with 31 amino acid residues and a carboxyl terminal hexahistidine tag. E. coli BL21(DE3) cells were selected as the expression host.

The protein was expressed at 28 °C for 5 h in Luria-Bertani (LB) medium containing 100

µg/mL ampicillin and 1 mM isopropyl α-D-thiogalactopyranoside (IPTG). The cells were lysed by sonication in a buffer containing 50 mM HEPES, 500 mM NaCl and 10% glycerol at pH 7.4. The membrane fractions were collected by ultracentrifugation

(193,000 x g) of the supernatant obtained from a lower speed centrifugation (15,560 x g).

Solubilization experiments were conducted with different detergents at different detergent concentrations and temperatures. The extraction efficiency for each detergent was calculated according to the density of the bands from western blot analysis with an anti-Histag antibody.

A portion of the results of these detergent extraction experiments are summarized in

Table 3.1, adapted from Wang, et al 98. At 1.5 times the critical micelle concentration

(CMC) level and 4 oC, the ionic detergent sodium dodecanoyl sarcosine and the zwitterionic detergent lauryldimethylamine-N-oxide (LDAO) were the most effective at solubilizing ANAT. It was quite surprising that even buffer alone was able to solubilize a low amount of ANAT.

53 Table 3.1: Detergent Extraction of Native Aspartate N-acetyltransferase (adapted from Wang, et al. 98)

Extracted enzyme relative to control 1.5 x 1.5 x 8.7 x Detergents CMC CMC CMC at 4 °C at RT at RT buffer control 1.00 1.00 1.00 Non-ionic detergents Triton X-100 1.27c --a --b Tween-20 0.84 --a 1.15 octylglucoside (OG) --b 0.34 --b nonylglucoside (NG) 0.28 1.27c 1.47c nonanoyl-N-methylglucoside (Mega-9) 1.04 0.81 --b decylmaltoside (DM) 1.43c 0.46 1.30c dodecylmaltoside (DDM) 0.11 1.01 1.88c cyclohexylmaltoside (cymal5) 0.63 1.06 1.73c a c octyl tetraglycol (C8E4) 0.93 -- 1.37 c octyl pentaglycol (C8E5) 0.77 1.42 0.44 c b dodecyl octaglycol (C12E8) 0.80 1.90 -- Ionic detergents sodium dodecylsulfate (SDS) 0.08 0.83 3.27c sodium dodecanoyl sarcosine 1.45c 1.37c 1.25c Zwitterionic detergents decyl-N,N-dimethylamine-N-oxide 0.55 1.03 0.54 cholamido propane sulfonate (CHAPS) 0.84 0.72 --b lauryldimethylamine-N-oxide (LDAO) 1.55c 0.53 --b a not tested b no significant extraction observed c conditions with significantly enhanced protein extraction are shown in BOLD

54 3.3 Membrane anchor removal

The second approach tested to produce a soluble and active form of ANAT is based on a model proposed by Tahay et al 100, in which ANAT is predicted to have a 30-amino acid hydrophobic helix-turn-helix region, according to a sequence alignment between human ANAT and a homologous bacterial enzyme, polyamine N-acetyltransferase (PaiA) from B. subtilis.100. Tahay et al discovered that deletion of the first seven amino acid residues at the amino terminus seems to have a positive effect on ANAT activity.

However, deletion of the first 37 amino acid residues causes a decrease in ANAT activity. It is interesting that the first 67 amino acid residues are not needed for this enzyme to be active because a truncation starting at Met68 still has about half of the enzymatic activity compared to wild type enzyme 100. Site-directed mutagenesis along the

ANAT sequence suggested that not only the predicted NAT domain at carboxyl terminus, but also the amino acid residues before the putative membrane domain, are needed for

ANAT to be active. Based on the published structure of spermidine acetyltransferase from Bacillus subtilis 100, a model for ANAT was proposed. In this model, the putative

30-amino acid hydrophobic region is assigned as a membrane insertion domain and doesn’t directly participate in catalytic center formation. If this model is correct then there is a possibility to replace this anchor domain with a smaller linker without eliminating ANAT activity.

To locate the boundaries between this membrane region and regions in the cytosol, the amino acid sequences of five closely related ANATs from mouse (NAT8L_MOUSE), rat

(NAT8L_RAT), western clawed frog (NAT8L_XENTR), and zebra fish

55 (NAT8L_DANRE), and the bacterial spermidine acetyltransferase (PAIA_BACSU) from

Bacillus subtilis were aligned (Figure 3-1).

Figure 3-1: Sequence alignment between ANAT homologs and a polyamine N- acetyltransferase (PaiA) from B. subtilis. The proposed membrane anchor region in the

56 mammalian enzymes is highlighted in yellow, the turn region in the soluble bacterial enzyme is shown in red.

Protein engineering

Figure 3-2: Illustration of membrane anchor removal. The upper panel adapted from

Tahay et al. 100 The bottom panel is from PDB ID 1TIQ. The turn region is in yellow.

Figure 3-3: Design of Membrane anchor removal constructs.

The hydrophobic membrane domain of ANAT was replaced by two short peptides,

“KEQN” or “NDTFKEQN”, corresponding to the turn region that is present in the soluble PaiA enzyme structure. The underlined sequence displays the predicted membrane region for ANAT.

The general idea of membrane anchor removal is shown in Figure 3-2. Two loop engineering constructs were designed to test this approach. The first construct was made exactly according to the sequence alignment in Figure 3-1. Unfortunately, the expressed protein was still found primarily in the insoluble fraction. To exclude the possibility that this failure to achieve a soluble protein was due to incomplete loop replacement, the second construct was made by removing four more amino acids in human ANAT.

Unfortunately, the expressed protein with this modification was still not soluble. A

58 preliminary Ni-IMAC purification was conducted to increase the purity of the expressed enzymes, but didn’t yield any detectable ANAT activity from either construct.

3.4 Previous attempts at purification of Aspartate-N-acetyltransferase

Beside these detergent extraction and protein engineering approaches, the additional of fusion protein partners can also been used to increase the solubility of certain proteins.

During the past few years, several previous graduate students and postdocs in our laboratory have made contributions towards this approach and have made significant progress. More than ten different constructs have been produced during the past years with the aim of producing a soluble form of ANAT.

The first problem to be addressed was the unreliable expression of ANAT. Initially, a

ANAT-His construct in pDEST41 was produced by Dr. Stephen Zano 99. Although he was able to identify one protein band that is labeled by the anti-histag antibody in western blot, he discovered that most of proteins are still in the flow-through during metal affinity chromatography, and also that this enzyme form is not stable and significant activity loss was observed after dialysis. In an attempt to increase the binding of ANAT to the Ni-

NTA column, Dr. Yasanandana S. Wijayasinghe produced a His-ANAT-His construct in a pET28 vector. But, no expression was observed during the pilot expression with this construct 92.

The expression problem was finally solved by introducing large fusion protein partners such as a glutathione sulfur transferase tag (GST), a thioredoxin tag (TRX) or a maltose binding protein tag (MBP). Because the ANAT gene in pDEST41 was codon- optimized for mammalian cell culture, it is possible that the low protein yield that was observed is due to the need for rare codon usage. To increase the expression of ANAT, a

59 GST-His-ANAT-His construct in pET41a was made by Dr. Mojun Zhao using an E. coli codon optimized ANAT gene. However, she noticed that there was a background signal present using the established pyruvate dehydrogenase based ANAT activity assay. Also, smaller bands were observed in western blot analysis using anti-His tag antibody, bands that suggested the presence of protein truncations. Dr. Yasanandana S. Wijayasinghe produced a Trx-His-ANAT-His construct in a pDR32 vector, which is a modified version of pET32 (gift from Dr. Donald Ronning). A His-MBP-ANAT-His construct was produced by Gwenn G. Parungao from a modified pET28a vector (also from Dr.

Ronning). The different fusion constructs are shown in Figure 3-4. The lengths of linkers of the different fusion proteins vary from 21 to 68-amino acid residues. All fusion constructs contain polyhistidine tags for IMAC based purification. Despite having these different fusion partners, the various ANAT fusion proteins are still not fully solubilized.

The second problem that was encountered was the presence of western blot active truncations. It was observed that the addition of an EDTA-free protease inhibitor cocktail

(P8340, Sigma) still did not reduce the presence of these protein truncations. Several of these constructs, such as Trx-His-ANAT-His, showed more extensive truncations even during the pilot expression experiments. Multiple attempts were made to separate the full-length fusion proteins from the truncated version without significant progress. The

GST-his-ANAT-his and his-MBP-ANAT-his constructs each have two different affinity tags, thus could be purified by using two affinity columns in tandem. Unfortunately, the truncated proteins still could not be separated due to the existence of two histidine tags.

Regardless of which affinity column was applied first, small bands were still found by

SDS-PAGE as well as by western blot.

Figure 3-4. Different fusion constructs of human aspartate N-acetyltransferase (ANAT)98.

The relative size of each fusion protein is displayed. The NAT8L gene is shown in green, the poly-histidine tag in yellow, and the linker region in purple. The thioredoxin (TRX) fusion partner is shown in red, the glutathione S-transferase (GST) in orange and the maltose binding protein (MBP) is in blue.

3.5 Codon optimization of human aspartate N-acetyltransferase gene for E. coli expression

The E. coli codon-optimized human aspartate N-acetyltransferase gene, named

NAT8L_HUMAN_Ecoli, was designed and synthesized by Genscript (Piscataway, NJ).

The codon-optimized DNA sequence has been deposited in GenBank under accession number KU321638 (GI: 973447314), and the translated protein sequence has an accession number of ALX35610.1.

GenBank is a comprehensive public database maintained by the National Center for

Biotechnology Information (NCBI) of the National Library of Medicine (NLM), National

Institutes of Health (NIH) 101. It is the primary database of DNA and protein amino acid sequences for biological sciences. Traditionally, most of the DNA sequences found in

61 GenBank have been naturally existed genetic information. Recently, there has been a significant increase in the number of artificial DNA sequences due to the increased need for heterologous gene expression and codon optimization. The submitted sequences are categorized into various divisions based either on the source taxonomy or the sequencing techniques. The division of synthetic and chimeric sequences (SYN) and DNA sequences isolated from environment (ENV) are focused on here. The number of deposited DNA sequences from the SYN division, the ENV division and the total number of deposited

DNA sequences in GenBank from 2009 to 2016 are plotted in Figure 3-5 101-108.

Compared to the exponential growth of total recorded DNA sequences in GenBank, the synthetic genes and genes isolated from environment are still small (Panel A, Figure 3-5).

But the number of sequences from SYN and ENV divisions are growing steadily over time (Panel B, Figure 3-5). In 2012, there were 928 million synthetic DNA sequences in

GenBank, an annual increase 494.2%. In 2016, the number was over 1 billion 108. Due to the decreased cost of gene synthesis and the availability of codon optimization software, it is likely that the number of artificial DNA sequences will be increasing at an even greater rate.

Despite having the same translated amino acid sequences, the codon-optimized synthetic genes can be quite different from the original gene. Take a mammalian protein coding gene for example. First, the introns are removed for bacterial expression. Next, the codons are optimized for the target host and can be very different from the original sequence. Cis-regulating sequences such as specific promoters are also removed. At this point it is really necessary to treat synthetic genes as new genes just like organic chemists doing with newly synthesized organic molecules.

62 To organize the increasing number of codon optimized genes, a new naming scheme is proposed here. The naming scheme is an extension of the standard protein naming system from UniProt Knowledgebase (UniProtKB), which is the main database on functional information on proteins with manual annotation 109. The name of a synthetic gene contains three parts. The first part is the gene name, compliant with existing nomenclature guidelines 110. The second part is the native organism where the gene expressed in and the last part is the target organism where protein expression will take place. All parts are connected by underscores. And last, italic type is used to differentiate between gene and protein. The final expressed protein shouldn’t have any significant difference compared to the native version of the same protein, as long as no posttranslational modifications or folding issues are involved. Thus, a special naming system for heterologous expressed protein is not necessary. For example, the E. coli codon-optimized human aspartate N-acetyltransferase gene is named

NAT8L_HUMAN_Ecoli. The expressed protein will be still named as human aspartate N- acetyltransferase.

63 A)

2.5E+12

2E+12

1.5E+12 SYN

1E+12 ENV

TOTAL 5E+11

Number of deposited DNA sequences 0 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 Year

6E+09

5E+09

4E+09

3E+09 SYN

2E+09 ENV

1E+09

Number of deposited DNA sequences 0 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 Year

Figure 3-5: Number of deposited DNA sequences in GenBank.

The numbers of deposited DNA sequences from SYN division, ENV division and the total number of deposited DNA sequences in GenBank from 2009 to 2016 are shown. A)

Data points for SYN sequences are not seen due to scaling. B) Comparison of DNA sequences from SYN and ENV division in GenBank.

64 3.6 Dual Affinity Purification system

Tandem affinity purification of proteins (TAP) is an effective and highly specific technique to purify a target protein without requiring prior knowledge about the properties of the target protein. The original version of this technique was developed in the 1990s 111, 112 using calmodulin binding peptide and Protein A attached at the carboxyl terminus of a target protein. After on-column cleavage of Protein A by tobacco etch virus

(TEV) protease, the target protein binds to the second affinity column which contains calmodulin beads and was then eluted by native elution using ethylene glycol-bis(β- aminoethyl ether)-N,N,N',N'-tetraacetic acid (EDTA). High purity protein was obtained from this TAP scheme.

There is, however, a disadvantage with the tandem affinity purification method described above, especially when applied to membrane protein purification. The two affinity probes are combined as a “TAP tag”, which is then positioned either at the amino or carboxyl terminus of the target protein. However, if any protein degradation occurs during the protein expression or purification step, then truncated versions of these proteins will also co-elute during the purification procedure.

To solve that problem, it is possible to utilize a different affinity purification approach in which one affinity probe (maltose binding protein) was positioned at the amino terminus of the target protein while a hexa-histidine tag was placed at the carboxyl terminus. In theory only the full-length recombinant protein will go through both steps of the two-step purification. For ANAT purification, the MBP tag was selected as the N- terminal affinity tag and the hexahistidine tag was selected as the C-terminal affinity tag.

65 The MBP-ANAT-his construct (Figure 3-2) was produced from the his-MBP-ANAT- his construct by site-directed mutagenesis using Q5® Site-Directed Mutagenesis Kit from

New England Biolabs. The forward and reverse primers are:

5’-AAAACTGAAGAAGGTAAACTGG-3’ and 5’-GCCCATGGTATATCTCCTTC-

3’, respectively.

3.7 Purification of MBP-ANAT-H6 fusion protein

The MBP-ANAT-His construct was produced from the his-MBP-ANAT-His construct by mutagenesis. NiCo21(DE3) competent E. coli cells containing the MBP-ANAT-His construct were selected on LB plates with 30 µg/ml kanamycin at 37 °C for 16 h.

Colonies from these plates were used to inoculate starter cultures containing 10 ml of LB media. After 16 h growth at 37 °C, each starter culture was diluted 100-fold into 1 L of

LB media, and cell growth was continued for about 2 h until A600 reached 0.6. IPTG was then added to a final concentration of 0.5 mM, and protein expression was induced at 16

°C for 20 h. The dual affinity tagged human ANAT was initially purified by immobilized metal-affinity chromatography (IMAC). The column was washed with Buffer A, containing 20 mM potassium phosphate, pH 7.4, 300 mM sodium chloride, 10% glycerol and 20 mM imidazole, and then eluted with a linear gradient of Buffer B (buffer A containing 400 mM imidazole). After the IMAC column, the active fractions were pooled and loaded onto a 30 ml Dextrin Sepharose column and highly purified ANAT fusion was then obtained by elution with a 0-10 mM linear maltose gradient. Protein concentration was measured by NanoDrop 2000 UV-Vis Spectrophotometer (Thermo

Scientific). The purity of ANAT fusion was confirmed by SDS-PAGE and the activity

66 was assayed by a thiol-exchange reaction using 5-(3-Carboxy-4-nitrophenyl)disulfanyl-2- nitrobenzoic acid (DTNB). It was observed that the purified MBP-ANAT-His protein eluted as a sharp peak during the second affinity purification step (Figure 3-6). The calculated molecular mass for MBP-ANAT-His is 76.8 kDa. Although several other protein bands showed up in the first affinity purification step, almost all of the smaller bands were removed in the second purification step (Figure 3-7).

2015103101 NiNTA20151024 production001:10_UV1_280nm 2015103101 NiNTA20151024 production001:10_Conc 2015103101 NiNTA20151024 production001:10_Fractions

mAU

3000 280 nm

2015102601 dextrin HP 30ml 20151023 production001:10_UV1_280nm 2015102601 dextrin HP 30ml 20151023 production001:10_Conc 2015102601 dextrin HP 30ml 20151023 production001:10_Fractions

mAU B) Dextrin step 200

2500 A) Ni-NTA step

150

2000

100 1500

50 1000

500 0

F3 Waste 3 4 5 6 7 8 9 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 0 100 200 300 400 500 ml

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 0 50 100 150 200 ml

Figure 3-6. Chromatograms of two affinity purification steps for MBP-ANAT-His fusion protein. MBP-ANAT-His protein was eluted from Ni-NTA column (panel A) during a 20 to 400 mM imidazole gradient. Active fractions were pooled and loaded onto a dextrin

67 column. Purified MBP-ANAT-His protein were eluted as a sharp peak during a maltose gradient (0 to 10 mM) (panel B).

Figure 3-7. SDS-PAGE of MBP-ANAT-his during two-step affinity purification.

Active fractions containing MBP-ANAT-his protein from Ni-NTA column and dextrin column were loaded onto SDS-PAGE. The first lane on the left is molecular weight marker. Lane 1 contains pooled active fractions after Ni-NTA purification, and Lane 2 contains pooled active fractions after the Dextrin column.

68 3.8 Confirmation of the expression of ANAT by mass spectrometry

In order to confirm the expression of ANAT enzyme, peptide mass fingerprinting

(PMF) analysis was carried out as previously described 59, 113 with minor modifications.

Briefly, the affinity-tag purified ANAT fusion enzyme was separated on a precast gradient gel (Invitrogen, NuPAGE, 4-12%) and stained by Coomassie Brilliant Blue R-

250 dye dissolved in a methanol/acetic acid mixture. Protein bands corresponding to the correct molecular weight of ANAT were excised, destained with neat acetonitrile and

50% acetonitrile, reduced with 10 mM dithiothreitol at 56 oC for 30 minutes and then alkylated with 55 mM iodoacetamide in the dark. Trypsin (1.3 mg in 10 mM ammonium bicarbonate buffer) was added to each sample and the tryptic digestion was performed at

37 ºC overnight. Trifluoroacetic acid (TFA) was added to 1% final concentration to terminate the digestion. 1 µl of the sample was mixed with 1 µl of a saturated solution of

α-cyano-4-hydroxycinnamic acid in 33% acetonitrile/water with 0.067% TFA and then 1

µl of the mixture was spotted onto the MALDI target. MALDI-MS spectra were recorded by accumulating 4000-5000 laser shots per spectrum in positive ion mode with 500 –

4000 m/z as the detection range in a Bruker’s Ultraflextreme MALDI-TOF/TOF mass spectrometer. Proteins were identified from the SwissProt database with Homo sapiens as the taxonomy filter using the Mascot search algorithm (Matrix Science). ANAT was identified by peptide mass finger-printing analysis with a protein score of 71 (p < 0.05).

Seven peaks matches the trypsin digested peptides from ANAT protein as shown in

Figure 3-8. Because the sequence coverage is low (18%), it was necessary to perform peptide sequencing by tandem mass spectrometry in order to confirm the sequence of the peptide. Several peaks were selected for tandem sequencing and a peptide,

69 IVAAEDHEALPGAK, has been successfully sequenced on Bruker Ultraflextreme

MALDI-TOF/TOF mass spectrometer operated in LIFT mode (Figure 3-9).

Figure 3-8: PMF analysis for ANAT.

ANAT was identified by peptide mass finger printing analysis. The top panel shows the sequence coverage for ANAT and the bottom panel shows that seven peaks matches the trypsin digested peptides from ANAT protein.

Figure 3-9. Conformation of ANAT expression by peptide sequencing.

A peptide of ANAT has been sequenced by tandem mass spectrometry. The peptide sequence is shown on the top with fragmented b and y ions labeled.

3.9 Enzyme Activity of Aspartate N-acetyltransferase

Two different activity assays have been used in this study to measure ANAT activity.

The first is a pyruvate dehydrogenase (PDH) based coupling assay, where acetyl- coenzyme A is recycled. The other assay is based on a thiol exchange reaction using 5-(3- carboxy-4-nitrophenyl)disulfanyl-2-nitrobenzoic acid (DTNB). The PDH based assay was initially used to test the activity of the Trx-his-ANAT-his fusion protein, later switching to the DTNB based assay since the PDH based assay cannot be used to determine the kinetic constant for acetyl-coenzyme A.

3.9.1 The PDH based activity assay. ANAT activity was assayed in 40 mM sodium phosphate buffer, pH 7.2, 100 mM NaCl, 5% glycerol, 1 mM CHAPS, 1 mM DTT. The generation of coenzyme A was coupled to the activity of pyruvate dehydrogenase. Each assay used 0.05 U of pyruvate dehydrogenase from porcine heart. The details of this

ANAT activity assay is shown in Figure 3-10, Panel A.

71 3.9.2 The DTNB based activity assay. ANAT activity was also assayed by monitoring the production of coenzyme A using DTNB. The DTNB based activity assay contains 20 mM HEPES, pH 7.4, 150 mM NaCl, 5% glycerol, 40 µM DTNB, 40 µM acetyl-CoA, 2 mM L-aspartate, 25 µg of enzyme and, when tested, various amounts of inhibitors in a total volume of 200 µl. The increasing absorption at 412 nm (ε = 14.15 mM−1cm−1) was monitored for up to 10 min during the reaction. To optimize the salt components in this ANAT assay, and to mimic the in vivo conditions for ANAT, an artificial cerebrospinal fluid (aCSF) was prepared according to Toriumi et al 114 and used as buffer to assay ANAT activity. The aCSF contains 147 mM NaCl, 4 mM KCl, 2.3 mM

CaCl2 at pH 7.4. However, no changes were observed in the ANAT activity upon inclusion of aCSF.

The kinetic properties of the MBP-ANAT-his fusion protein was determined by varying the substrate concentrations of L-aspartate from 0.01-2 mM and acetyl-CoA from

2.5-50 µM. Each measurement was performed in duplicate. The resulting reciprocal plot suggests an ordered or random sequential enzyme mechanism (Figure 3-11). No signal was observed in the assay if either of the substrates, acetyl-coenzyme A or L-aspartic acid, was omitted, which also supports the proposed sequential mechanism. The obtained kinetic data were fitted to the equation for a sequential enzyme mechanism (Eq. 1) to determine the kinetic parameters.

�� = !"#$ ! [!] (Eq 1) !" ! !!" ! ! ! ! !!"#!$

72 where Ka and Kb are the Michaelis constants, and Kia is the binding constant for substrate A.

The maximum velocity (Vmax) of expressed MBP-ANAT-his fusion was determined to be 71 mU/mg and the Km for acetyl-CoA is 3.1 µM. The Km for the native amino acid substrate L-aspartic acid is 160 µM (Table 3.2).

Next, the substrate specificity of ANAT was tested. An in-house library of 160 amino acid analogs was constructed and tested for possible alternative substrates of ANAT.

Only three compounds from this library, β-methylaspartate, 2,3-diaminosuccinate, and L- glutamate, were identified as alternative substrates. These results suggest that ANAT is a very specific enzyme for L-aspartate (Table 3.2).

Figure 3-10: ANAT activity assays. Two activity assays are used in this study. A) In pyruvate dehydrogenase based coupling assay, acetyl-coenzyme A was recycled and the resulting NADH was measured at 340 nm. B) In the DTNB based assay the production of

TNB was measured at 420 nm.

Figure 3-11: Reciprocal plot for MBP-ANAT-his fusion enzyme.

Substrate concentrations of L-aspartate were tested from 0.01-2 mM and acetyl-CoA concentration from 2.5-50 µM, with the data fitted to the equation for a sequential kinetic mechanism (Eq. 1).

75 Table 3.2: Substrate Kinetic Parameters for Aspartate N-acetyltransferase

relative Substrates kcat (U/mg) Km (mM) kcat/Km (%) Physiological substrates acetyl-CoA 0.0031 ± 0.0012 L-aspartate 0.071 ± 0.006 0.16 ± 0.05 100

Alternative substrates β-methylaspartatea 0.018 ± 0.002 0.36 ± 0.13 11.2 2,3-diaminosuccinatea 0.035 ± 0.005 0.92 ± 0.19 8.6 L-glutamate 0.023 ± 0.004 8.6 ± 1.6 0.59 a measured with the racemic mixture and adjusted for activity with only the L-isomer.

3.10 Detergents affect the enzyme activity of Aspartate N-acetyltransferase

Membrane proteins are proteins containing hydrophobic transmembrane segments or domains, with this class of proteins playing a vital role in life. Membrane-associated enzymes, receptors, ion and small molecule channels are known to participate in many crucial biological processes, and the malfunctioning of any of these proteins is associated with various diseases ranging from heart disease and obesity to cancer. The common practice for membrane protein structural studies involves extraction of the membrane protein out of its native lipid environment using detergents. As mentioned in the introductory section in this chapter, two difficulties have been reported with this approach related to the biochemical characterization of ANAT. First, it has been found to be difficult to extract this protein from the lipid environment. Second, the enzyme activity of ANAT is sensitive to detergents. We addressed the first question by performing detergent screening and identified several detergents that can extract higher levels of ANAT out of the membrane fractions. It is also necessary to test which

76 detergent has less denaturing effect on ANAT. Twenty-one detergents were prepared in stocks at concentrations equal to 10 times their critical micelle concentrations (CMC).

The ANAT activity was measured in the presence of each detergent at its CMC level using the DTNB based activity assay. All measurements were conducted at least in duplicate and the concentrations of L-aspartate and acetyl-CoA were kept at saturating levels. These 21 different detergents tested have been categorized into 4 groups based on their effect on ANAT activity (Figure 3-12). Group 1 detergents, such as Triton X-100 and Tween 20, have the least impact on ANAT activity. Treatment with group 2 detergents, such as DDM and DM, retains about 30 to 70% of the ANAT activity. Less than 20% of ANAT activity is retained after treatment with Group 3 detergents. Finally, group 4 detergents, such as SDS, completely eliminate ANAT activity.

Figure 3-12: Effects of detergents on ANAT activity.

77 The 21 detergents tested are categorized into 4 groups based on their effect on ANAT activity. Group 1 detergents (blue) have least impacts on ANAT activity. Group 2 detergents (green) retain about 30 to 70% of ANAT activity. Less than 20% of ANAT activity was retained by Group 3 detergents (red), while Group 4 detergents (black) eliminate ANAT activity completely.

3.11 Other factors affecting ANAT activity

In addition to these detergent effects, the effects of solvent, ionic strength, reaction temperature, and pH on ANAT activity was also tested. It is well known that organic solvents can decrease enzyme stability and activity. Dimethyl sulfoxide (DMSO) is one of the commonly used organic solvents in kinetic assays for screening inhibitor libraries.

The level of residual enzyme activity after DMSO treatment can be viewed as a measurement of general enzyme stability toward organic solvent perturbation. In this study, DMSO was added into the assay at various concentrations and the relative ANAT activities were recorded, either in the presence or absence of L-aspartate in the incubation mixture. ANAT is able to tolerant at least 20% DMSO before the activity begins to decline, as shown in Figure 3-13.

The effect of ionic strength on ANAT activity was evaluated by running kinetic assays at increasing NaCl concentrations. The highest activity was observed at 180 to 200 mM

NaCl. A significant loss of activity was seen when the NaCl concentration was below 50 mM, and the activity dropped as the NaCl concentration increased above 200 mM (data not shown).

78 ANAT activity assays were performed at four temperatures (4 oC, 22 oC, 37 oC and 50 oC) to test the temperature effect on enzyme activity. As expected, the highest activity was observed at 37 oC. As reaction temperature decreases below this value, the ANAT activity drops. ANAT has 6% activity at 4 oC and 23% activity at 22 oC, when compared to the value at 37 oC. If the reaction temperature is increased from 37 oC to 50 oC, only 41% of the activity is retained. Although this trend with temperature is only preliminary, it is consistent with the expectation that, because ANAT catalyzes its reaction in the human brain where the temperature is 37 oC 115, its activity should be optimized at this temperature.

Figure 3-13: Effect of organic solvent on ANAT activity. ANAT activity is compared in the presence of increasing amounts of DMSO in the reaction mixture.

79 3.12 pH activity profiles of ANAT

pH activity profile studies were used to measure the effects of pH on ANAT activity, and also to determine any potential functional groups which must be in the correct protonation state for enzyme activity. It is possible to identify certain functional groups in the enzyme or in L-aspartate that contribute to substrate binding or catalysis from a V/K profile of the enzyme. The Vmax profile leads to the identification of changes in functional group ionization in the enzyme-substrate complex. pH profile studies were conducted between pH 6 to 9 with 0.5 pH unit intervals. 100 mM ACES buffer (pH 6 – 6.5), 100 mM HEPES buffer (pH 7 - 8), and 100 mM TAPS buffer (pH 8.5 - 9) were used to maintain the assay pH. The potential inhibitory effects of buffers were tested using different buffers at overlapping pH values. Acetyl-coenzyme A was kept constant at 100

µM, which is much higher than its calculated Km value. The Vmax and V/K values were calculated at each pH unit. The resulting data were fitted to equation 2:

log Vmax (or Vmax/Km) = log [ C / (1 + [H]/Ka) ] (Eq. 2)

where C is the pH independent rate and Ka is the equilibrium constant for protonation of a single functional group.

It was observed that the ANAT activity decreases with decreasing pH, from 7.5 to 6.

Although the Vmax profile appears to be pH independent in the tested pH range, the V/K profile suggests a single functional group that, if protonated, leads to a loss of ANAT activity. From a fit of the data to Eq. 2 the pK value was determined to be 6.8, which

80 could reflect an active site histidine residue functioning as a general base. This is consistent with the proposed function of a catalytic histidine in other acetyltransferases, such as chloramphenicol acetyltransferase116 and supported by structural studies of an arylamine N-acetyltransferase.117

Figure 3-14: V/K pH profile of MBP-ANAT-his fusion enzyme.

The decreasing of ANAT activity with decreasing pH was modeled by a fit of the data to

Eq. 2 (dashed line). The de-protonation of a single functional group is required for optimal activity.

81 3.13 Crystallization trials of MBP-ANAT-his fusion

To provide molecular insights about the structure and function of ANAT, crystallization trials were conducted on the purified MBP-ANAT-his fusion enzyme. The

MBP-ANAT-his fusion protein was concentrated up to 7 mg/ml without any obvious precipitation.

Sparse matrix screening was performed using several commercial and home-made kits, without any success in obtaining crystallization conditions. The commercial kits used were crystal screens from Jena Bioscience, Hampton crystal screen 1, Hampton PEG/ion screen, Hampton MembFrac screen and Hampton Index. As a common phenomenon, more than half of the conditions led to precipitation within one hour after plate setup. A few conditions eventually precipitated within a week. There were very few clear drops remaining after several months, and none of these conditions showed any evidence of crystal growth.

To determine the pH effect on ANAT solubility, an in-house pH screen covering pH 4 to 9 and 5% to 30% PEG 6000 was screened. As shown in Figure 3-15 (panel A), ANAT precipitates appeared immediately across the pH range in just 10% PEG 6000. In the row with 5% PEG, ANAT was seen to be more soluble at pH values higher than 7. To further determine the proper concentration range for PEG in crystallization, an expansion screen using 5% to 10% PEG 6000 in 100 mM HEPES, pH 7 and 100 mM Tris, pH 8 was performed. It was noticed that precipitates started to show up even at 5.5% PEG 6000

(Figure 3-15, panel B). Further optimization using even finer grids at lower PEG concentrations was not successful.

82 There is a possibility that MBP can interfere with ANAT crystallization. An attempt was made to remove MBP by protease digestion. Significant loss of protein was noticed during concentration process.

To understand more about the solution properties of MBP-ANAT-His fusion for crystallization, Dynamic Light Scattering (DLS) experiment was conducted. MBP-

ANAT-His fusion was found to have a hydrodynamic radii of more than 40 nm, as shown in Appendix B1. In contrast, the hydrodynamic radii of a globular protein with molecular weight around 70 kDa is expected to be around 4 nm. This suggests that the MBP-

ANAT-His protein behaves like a large complex or heterogeneous complexes in solution.

Further optimization of the solution properties of ANAT is needed for successful crystallization.

Figure 3-15. pH and buffer screen for crystallization.

83 A) An in-house pH screen covering pH 4 to 9 and 5% to 30% PEG 6000 was tested for

ANAT. These results suggest that ANAT is very sensitive to PEG concentrations and is more likely to precipitate at lower pH.

B) An expansion screen using 5% to 10% PEG 6000 in 100 mM HEPES, pH 7 and 100 mM Tris, pH 8 was performed. Precipitates started to show up even at very low PEG concentrations.

3.14 Summary and Future Directions

Recently, aspartate N-acetyltransferase has been shown to play a critical role in

Canavan disease, which is caused by a disruption of the N-acetyl-L-aspartate metabolic pathway through mutations in the gene that codes for aspartoacylase. ANAT is a membrane enzyme that is sensitive to detergents. After extensive protein engineering efforts, we are now able to express human ANAT in E .coli, to solubilize it with the help of fusion partners, and to purify it to high purity.

To characterize the reaction catalyzed by human ANAT, the kinetic properties and substrate specificity has been determined. The ANAT activity has also been examined under different perturbations, such as detergents, organic solvents, temperature and pH.

To continue the crystallization studies, further experiments are needed to optimize the solution properties of ANAT.

Chapter 4

Inhibitor Development for Aspartate-N- acetyltransferase

4.1 Introduction

Before this study commenced, there was only one set of inhibitors that had been reported in the literature for the NAA biosynthetic activity. After recognizing that the process of NAA synthesis in rat brain is bio-catalyzed and that acetyl-coenzyme A is one of the substrates, Goldstein tested a series of coenzyme A derivatives for competitive inhibition using the ammonium sulfate precipitated protein from the supernatant of rat brain homogenate28. Among the coenzyme A analogs tested, butyryl-CoA has the lowest

Ki value, while phenylacetyl-CoA, benzoyl-CoA, propionyl-CoA and fluoroacetyl-CoA are each weaker inhibitors. It is important to note here that Goldstein’s experiments were performed in the 1950s, at a time when no one had any knowledge about the NAT8L gene or the aspartate-N-acetyltransferase enzyme.

As a purified version of ANAT has become available, it is now possible to examine more compounds as possible inhibitors, with any new inhibitors then subjected to

85 modifications to achieve enhanced selectivity and potency. Currently, the ANAT inhibitors that have been identified have at least two potential applications.

Firstly, ANAT inhibitors can be used in Substrate Reduction Therapy for the treatment of Canavan disease. As described in Chapter 1, enhanced knowledge about NAA metabolism in the brain, and a series of gene knock-out studies in rat and mouse models, suggested that the combined effects upon disruption of the NAA metabolic pathway are the underline causes of Canavan disease. Some of the disease symptoms can be explained by lack of NAA derived acetate, while the other symptoms, such as seizures and spongiform degeneration20, can potentially be explained by the toxic accumulation of

NAA molecules, which is a precursor for a dipeptide neurotransmitter, NAAG. There is accumulating evidence that the acetate deficiency can be compensated for by dietary supplements that enhance the intermediates in the TCA cycle and by increased acetyl-

CoA concentrations in oligodendrocytes. This approach has been designated as

Metabolite Therapy for Canavan disease. On the other hand, the NAA toxicity can be reduced with specific ANAT inhibitors. This approach has been designated as Substrate

Reduction Therapy for Canavan disease. We are proposing that the combination of metabolite supplementation and NAA reduction by ANAT inhibitors can provide an efficient clinical therapy for Canavan disease.

Secondly, ANAT inhibitors have potential to be developed as anti-cancer agents.

Recently, overexpression of ANAT has been discovered in non-small cell lung cancer, with NAA detected in blood from these patients118. A gene expression study using multiple cancer type cells suggests that NAT8L overexpression is common in ovarian cancer, melanoma cancer, breast cancer, colon cancer, and uterine cancers119. There is a

86 correlation between poor treatment prognosis and NAA concentration in ovarian cancer patients. Also, knockdown of NAT8L expression has been shown to reduce cell proliferation in a specific lung cancer cell, called A549 120. These exciting discoveries suggest that the development of ANAT inhibitors could also serve as a novel anti-cancer therapy.

4.2 Assay development for inhibitor screening

To identify initial inhibitors of ANAT, which hopefully will lead to an effective drug for Canavan disease, we adopted an approach that combined fragment library screening and structure based rational design.

Heterologously expressed human aspartate N-acetyltransferase (MBP-ANAT-his) was produced as described in Chapter 398. ANAT activity was tested using an established

DTNB-based assay in 96 well plates on a SpectraMax 190 UV plate reader (Molecular

Devices, CA). The assay conditions have been published 121 and those details are reproduced here. A typical activity assay mixture contains 20 mM HEPES, pH 7.4, 150 mM NaCl, 5% glycerol, 40 µM DTNB, 40 µM acetyl-CoA, 0.2 mM L-aspartate, 25 µg of enzyme and various amount of inhibitors in a total volume of 200 µl. Each possible inhibitor was prepared as a 200 mM stock in water and compounds from our in-house library were kept at 4 ºC. Acetyl-CoA was prepared as 10 mM stock in water and stored at -20 ºC to minimize degradation. Before each set of assays, acetyl-CoA was diluted into a 2 mM working solution using HEPES buffer. 2 mM DTNB was freshly prepared and kept on ice during the kinetic assay. For compound library screening, 2 µl of 200 mM L- aspartate, 2 µl of inhibitor, and 116 µl of buffer were added into each reaction well. 80 µl

87 of freshly prepared pre-mix, which includes 4 µl of acetyl-CoA, 4 µl of DTNB, 5 µl of enzyme and 67 µl of buffer, was added to start the reaction. The reaction time course was

-1 -1 monitored at 412 nm (ε = 14.15 mM cm ) for at least 15 minutes. For Ki determinations a two-fold serial dilution of each inhibitor was performed starting from either a 2 mM,

0.2 mM, or 0.02 mM stock solution, with the starting concentration determined according to the initial measured potency of the inhibitors. A negative control, in which no inhibitor was added, and a background signal control, in which neither L-aspartate nor inhibitor was added, were included in each set of assays. The pH of the reaction wells with the highest concentration of inhibitor was tested to rule out any possible false positive signals due to pH variations.

There are several variables that could potentially interfere with the assay. Firstly, since the assay signal comes from a thiol-exchange reaction, thiol-based reducing agents, such as dithiothreitol or β-mercaptoethanol, must be avoided during enzyme preparation or must be diluted into a very low concentration in the final assay condition. Secondly, a control in which ANAT enzyme is omitted must be included for every compound tested in order to identify any reagent that will react with DTNB. Thirdly, the ANAT protein itself contains cysteine side chains that can react with DTNB. This interference from the enzyme is minimal if the final enzyme concentration is less than 5 µg/ml. Lastly, some compounds will absorb at a wavelength close to 412 nm, which may increase the background absorption somewhat even at dilute concentrations.

To evaluate the quality of the developed DTNB assay and its suitability for high throughput screening, the Z’ factor was calculated as described by Zhang et al 122. The equation (Eq. 3) for Z’ calculation is

�! = 1 − (!!!!!!!) (Eq 3) |!!!!!|

where σ+ and σ- are standard deviations for positive controls and negative controls, respectively. µ+ and µ- are the mean signals from positive control reactions and negative control reactions.

If the Z’ factor is equal to or higher than 0.5, the separation band of the assay is large and the assay is a suitable assay for high throughput screening. The Z’ factor for the

DTNB assay was determined to be 0.60. The initial rates for positive and negative controls are shown in Appendix C1. It is therefore suitable to use this assay to screen for

ANAT inhibitors.

4.3 Initial inhibitors identified by Library screening

A total of 333 compounds from 4 different small molecule compound libraries that had been prepared in-house were initially screened against ANAT. There are 96 compounds from an amino acid library, 64 compounds from a second amino acid library,

96 compounds from a metabolic library, and 77 compounds belonging to constrained aspartic acid analogs, selected with the aim to reduce the conformational flexibility. An initial hit was defined as any compound having noticeable inhibitory effect (>50% inhibition) at 2 mM concentration. In addition to the commercial compound libraries mentioned above, a total of 128 additional compounds were synthesized by Vinay

Mutthamsetty and Dr. Bharani Thangavelu, with these compounds categorized into two series, dioic acid derivatives (D series) and phthalate derivatives (P series), based on their

89 structural similarities within each group. The results with this initial set of compounds is summarized in Table 4.1121. A representative kinetic absorbance curve for ANAT inhibitor is shown in Appendix C2.

The hit rates for each library are: 3% for the first amino acid library, 1.5% for the second amino acid library, 7.8% for the constrained aspartic acid analogs and 13.5% for the metabolic library. The overall hit rate is 7% for the entire library screen. There were also 38 compounds (11%) excluded from testing due to strong background absorption.

ANAT is highly specific for its natural substrate L-aspartic acid. As shown in Chapter

3, only three alternative substrates have been identified. As can be seen from the structures of these alternative substrates only small modifications attached to the β- carbon are tolerated to some extent without dramatically losing their binding affinity.

Since ANAT has a really selective substrate-binding site it is likely that the aspartate- binding site may also be very selective for inhibitor development. It is thus not surprising that all three moderate inhibitors identified from the amino acid libraries are N- derivatized aspartic acids, among which N-chloroacetyl-L-aspartic acid has the lowest inhibition constant value (Ki = 200 ± 40 µM).

An unexpected, significantly higher hit rate was observed from the metabolite library.

Eleven moderate inhibitors were identified, with Ki values ranging from 0.37 mM to 1.83 mM. It is interesting that all of the hit compounds have at least two carboxylate functional groups that are separated from each other by 2-4 chemical bonds (Figure 4-1).

These results suggest that both carboxylates are likely contributing to inhibitor binding to

ANAT. Based on the results from this small dataset, we hypothesized that the two carboxylate functional groups should be positioned approximately three carbon-carbon

90 bonds away from each other for optimal binding. Although fumarate is not an inhibitor for ANAT, 2-bromofumarate and 2-chlorofumarate are found to be good inhibitors. The fumarate derived inhibitors have a slightly better potency compared to the succinic acid derived inhibitors, which suggests the existence of two positively charged amino acid residues of ANAT in a planar geometry that are positioned to interact with these functional groups.

The screening of a constrained analog library led to the identification of the first strong inhibitor for ANAT. N-[(benzyloxy)carbonyl]-L-aspartic acid (Cbz-aspartic acid) was tested and the inhibition constant was determined to be 17 ± 3 µM.

To investigate how much the Cbz group itself contributes to the enhanced inhibition, a series of N-Cbz derived amino acids were then synthesized. A wide range of inhibition constants were measured for this series, which indicates that the higher potency likely comes from the combined effect of the core amino acid structure and the carboxybenzyl functional group (Table 4.2). N-Cbz itself clearly contributes to inhibitor binding to

ANAT because the ((benzyloxy)carbonyl)-L-alanine derivative is a weak inhibitor of

ANAT, while L-alanine itself is not an inhibitor.

L-glutamate, which is one carbon longer than L-aspartate, is a weak substrate for

ANAT with a relative catalytic efficiency (V/K) of 0.59% compared to the physiological substrate. However, N-[(benzyloxy)carbonyl]-L-glutamic acid is a slightly better inhibitor

(Ki = 17 ± 3 µM) than its aspartic acid counterpart. It is likely that the presence of two carboxylates along with the position of the phenyl ring on Cbz-L-aspartic acid contribute to the inhibition. This interesting discovery led to the systematic optimization of this dicarboxylic acid core structure in a search for improved ANAT inhibitors.

Table 4.1. Summary of Compound Library Screening and Hit Optimization against ANAT

number moderate strong a b Library of Most potent (Ki value) compounds inhibitors inhibitors Amino acids 96 3 0 N-chloroacetyl-L-aspartic acid (K = 200 µM) i Metabolites 96 13 0 2-bromofumarate (K = 367 µM) i Amino acids 64 1 0 N-alanyl-L-aspartic acid (K = 1.6 mM) II i Constrained 77 5 1 N-carbobenzyloxy-L-aspartic acid analogs (K = 17 µM) i Screened 22 1 compounds. 333 (6.6%) (0.3%) (hit rate) N-carbobenzyloxy-L-glutamic acid dioic acids 68 4 (K = 12 µM) (D series) i 4-aminomethyl(N-carboethyl,N-4- phthalates carboxy-2,6-dichlorobenzyl) phthalate (P series) 60 11 (K = 0.6 µM) i Total Number 461 16

a at least 50% inhibition when tested at 2 mM concentration b compounds with Ki values less than 100 µM

92 O O

A) HO OH

OH 2-hydroxymalonic acid 1.83 ± 0.55 mM M1

O O O

HO HO HO B) OH OH OH

O Br O Cl O 2-bromofumaric acid 2-chlorofumaric acid 2-methylfumaric acid 0.37 ± 0.11 mM 0.41 ± 0.05 mM 1.34 ± 0.15 mM M2 M3 M4

Br O O O

HO HO HO OH OH OH

O Br O Br O 2,3-dibromosuccinic acid 2-bromosuccinic acid methylsuccinic acid 0.71 ± 0.08 mM 0.89 ± 0.13 mM 0.94 ± 0.17 mM M5 M6 M7

O O O O

HO HO OH OH HO OH

O Cl O Cl O D-2-chlorosuccinic acid L-2-chlorosuccinic acid trans-epoxysuccinic acid

1.01 ± 0.14 mM 1.06 ± 0.15 mM 1.41 ± 0.23 mM M8 M9 M10

O OH O

O OH O HO O O O HO

C) HO OH HO OH O OH 3-hydroxy-3-methylpentanedioic acid cis-aconitic acid trans-aconitic acid 1.55 ± 0.14 mM 1.37 ± 0.20 mM 1.73 ± 0.92 mM M11 M12 M13

Figure 4-1 Structures of moderate inhibitors identified from the metabolite library.

The determined Ki values are listed under each molecule (numbered as M1-M13). All identified inhibitors have at least two carboxylate functional groups and they are 2 (A), 3

(B), or 4 (C) bonds separated from each other.

93 Name of inhibitors Ki (mM) ((benzyloxy)carbonyl)-L-glutamic acid 0.012±0.001 ((benzyloxy)carbonyl)-L-aspartic acid 0.017±0.003 ((benzyloxy)carbonyl)-D-phenylalanine 0.32±0.03 ((benzyloxy)carbonyl)-L-alanine 1.47±0.20 ((benzyloxy)carbonyl)-L-valine >2 Table 4.2: Compounds with a carboxybenzyl group and their inhibition constants.

4.4 Inhibitor optimization starting from di-carboxylic acid analogue core structures

Inspired by the unexpectedly low Ki value of Cbz-aspartic acid, a series of additional compounds were synthesized based on this core structure (Figure 4-2). First, the distance between two carboxylates was varied from 3-5 bond lengths. Second, the distance between the phenol ring and amide nitrogen was varied. As shown in Figure 4-2, compounds (3-phenylpropanoyl)-L-aspartic acid (D4) and (3-phenylpropanoyl)-L- glutamic acid (D9) were found to have significant lower Ki values than the other compounds in this series. These results indicate that the optimal distance between the phenol ring and the amide nitrogen is 4 bonds and that the optimal distance between the two carboxylates is 3-4 bonds.

94 OH OH O O O NH

1.4 ± 0.2 mM D1

O O O O HO HO HO HO OH OH OH OH O HN O O HN O O HN O O HN O

> 2 mM 0.46 ± 0.08 mM 0.031 ± 0.002 mM 0.20 ± 0.02 mM D2 D3 D4 D6 O O O O O O O O HO OH HO OH HO OH HO OH HN O HN O HN O HN O

0.13 ± 0.01 mM 0.25 ± 0.03 mM 0.038 ± 0.002 mM 0.61 ± 0.12 mM D7 D8 D9 D11

O O O O HO HO HO HO OH OH OH OH O HN O O HN O O HN O O HN O

1.60 ± 0.34 mM 1.16 ± 0.24 mM 2.90 ± 0.34 mM 0.74 ± 0.17 mM D12 D13 D14 D16

Figure 4-2: Di-carboxylic acid core: optimization of the distance between two carboxylates, as well as the distance between the phenol ring and the amide nitrogen.

From top to bottom: the distance between two carboxylates was elongated from 2-5 bonds; From left to right: the distance between the phenol ring and amide nitrogen was elongated from 2-5 bonds. The determined Ki values are listed under each molecule.

95 4.5 Inhibitor optimization starting from the phthalic acid core structure

At the same time while we were developing the di-carboxylic core structures, a library of phthalic acid analogs were being synthesized 123. These compounds were originally being developed and synthesized for a completely different aim, which was to develop selective inhibitors for aspartate semialdehyde dehydrogenase (ASADH), EC 1.2.1.11, from pathogenic bacteria as an approach for the production of new antibiotic agents.

ASADH catalyzes the reversible formation of L-aspartate-4-semialdehyde, NADP+ and inorganic phosphate from L-4-aspartyl phosphate and NADPH.

Since the phthalate library is the product of diversity-oriented synthesis124, 125 and because both L-aspartate and aspartate-4-semialdehyde have structural similarity, it was deemed possible that some of these compounds could be repositioned as ANAT inhibitors. Not surprisingly, the screening of the same family of compounds led to completely different inhibition patterns between the previously identified ASADH inhibitors and the ANAT inhibitors. For example, compounds P3, P5, P6, P10 and P11 are moderate inhibitors of ASADHs with Ki values ranging from 250 to 680 µM, but none of these compounds are ANAT inhibitors (Figure 4-3). The results from the phthalate library screening started to become more interesting as a π ring system was introduced onto the secondary nitrogen. The introduction of acetaldehyde group on the parent compound P3 led to a weak inhibitor (P12) with a 1.2 mM Ki value. Moving the π bond one bond away from the secondary nitrogen and replacing the carbon-oxygen double bond with a carbon-carbon double bond led to increased ANAT inhibition (P13).

Replacing the allyl group with a benzyl group created a compound with a weak 0.4 mM

Ki value (P14). While a big leap forward was not always rewarded (P30 and P48),

96 slightly increasing the aromaticity with the correct angle (P33 vs. P32) led to a much better inhibitor with an improved Ki value (P33). A one-carbon elongation on the alkyl carboxylate from compound P33 provided the first compound with an inhibition constant below 100 µM (P49). This improvement also suggested that the parent compound could be further optimized.

To test the difference between 4-aminomethylphthalate and 4-aminoethylphthalate as core structure, a series of compounds from both parent cores were synthesized. It was observed that while 4-aminomethylphthalate (P3) is not an inhibitor for ANAT, the 4- aminoethylphthalate compound (P4) itself is a weak inhibitor (Ki = 1.8 ± 0.3 mM). As seen in Figure 4-4, 4-aminoethylphthalate as the core structure produces consistently better inhibitor derivatives than 4-aminomethylphthalate, except for the ortho substituted bromobenzene (P18 and P37), which may be due to steric hindrance. Compared to the un-substituted compound P14, a mono-methyl substitution on the phenyl ring reduced the potency (P15, P16, P17, P43, P44, and P45) while mono-bromo substitution increased inhibitors’ potency (P18, P19, P37, P38, and P39), except for compound P20. Generally, methyl and bromo substitution at both the ortho and meta positions was seen to be better than at para position on the aryl side chain.

A trifluomethyl substitution also led to better inhibitors (Figure 4-5), with values that are similar to the bromo substitution. The best compound in this series, with bromo substitution at meta position (P22), resulted in up to a 4-fold increase in binding affinity compared to the un-substituted compound (P14). Since substitution at para position with these functional groups always led to weaker inhibitors, other effects such as hydrophobicity were considered. Introducing a hydrophobic t-butyl group at the para

97 position decreased the binding affinity (P28 and P46), while p-carboxylbenzyl derivatives (P34 and P50) also increased inhibition, with Ki values of 104 and 29 µM. To test if the increased inhibition was indeed due to the hydrophilic substitution or due to a potential negative charge generated at the para position, the p-carboxyl group was replaced by a carboxamide group (P35) and a ketone (P36), which are polar functional groups but without a formal charge. These two derivatives were found to be much worse inhibitors. This finding suggested that there is a positive charged amino acid residue close to the binding site for the aryl side chain that is making a favorable electrostatic interaction with the para carboxyl group.

After completion of the optimization experiments described above, one question arose about the geometry of the binding pocket of these benzylaminoethylphthalates. On the one hand, the observed effects from perfluoromethyl, halogen or 1-napthyl derivatives at the ortho and meta positions suggested a hydrophobic binding site; while on the other hand the p-carboxybenzyl derivatives suggested a positive charge in the inhibitor-binding site. Without further knowledge, it could be possible that the two modes of enhanced inhibition happened because this aromatic side chain might be binding in two different locations. To answer this question, nine additional compounds were synthesized by combining the best functional groups at the ortho, meta, or para positions on the benzene ring. As shown in Figure 4-6, a combination of carboxylate at para position and perfluoromethyl at meta position decreased the inhibition, while the same substitution at the ortho position created a compound (P53) with Ki value of 17 µM, which is better than the values for single substitutions (88 µM for perfluoromethyl derivative and 29 µM for p-carboxybenzyl derivative). Although an additional aromatic ring decreased the affinity

98 of the derivatives (P54 and P51), halogen substitution increased the inhibition constants dramatically (P57, P56, P55). In particular, dibromo (P59) or dichloro (P58) compounds gave an additional several-fold increase in inhibition compared to the mono substituted compounds. These results suggest that the two modes of enhanced inhibition are likely occurring through a set of interaction at same binding site. The di-bromo or di-chloro substituted benzylaminoethylphthalates are examples of the first set of sub-micromolar inhibitors of ANAT that have been produced.

4.6 Summary and Future Directions

Because of the recently discovered significance of ANAT in human brain function and

Canavan disease, and the correlation of NAA accumulation in the proliferation of multiple types of tumor cells, there is an urgent need to develop specific ANAT inhibitors that could potentially lead to new treatment therapies. This chapter has presented the identification and development of two series of ANAT inhibitors through a combination of fragment-based library screening, followed by targeted inhibitor optimizations.

Several initial inhibitors were identified by screening focused amino acid, metabolic, constrained analog and phthalate libraries. The surprisingly potent hit with carboxybenzyl aspartic acid lead to an exploration of di-carboxylic acids with optimized chain lengths, resulting in inhibitors with low micromolar inhibition constants. A rescreening of a previous synthesized library for a different enzyme target lead to a series of new findings and subsequent optimizations of that class of inhibitors. Finally, from this phthalate library optimization several sub-micromolar inhibitors of ANAT have now been developed.

99 To continue moving towards the Substrate Reduction Therapy as a treatment for

Canavan disease, we are now modifying these developed ANAT inhibitors so that they can more readily traverse bio-membranes to reach their target cells.

100 O CH O OH 3 O HO HO N OH N OH HO N OH O H OH O H OH O H OH O O O > 2000 µM > 2000 µM > 2000 µM P3 P5 P6 O O O HO HO HO N OH N OH N OH O CH3 OH O OH O OH N O O O O > 2000 µM > 2000 µM 1289 ± 240 µM P10 P11 P12 O O HO HO N OH N OH O OH O OH O O

719 ± 143 µM 420 ± 86 µM P13 P14 O O O HO N OH HO N OH O OH OH O O

953 ± 173 µM 550 ± 103 µM P30 P48 O O O O HO HO N OH N OH HO N OH O OH O OH OH O O O

538 ± 75 µM 115 ± 12 µM 34 ± 5 µM P32 P33 P49

Figure 4-3: Initial ANAT inhibitors identified from screening a 4-aminomethylphthalate derivatives. Ki values of the compounds are listed. Compounds are labeled as P series.

101 A) O O O HO HO HO N OH N OH N OH O OH O OH O OH CH O O O 3 H3C

H3C 1404 ± 260 µM 1702 ± 223 µM 4230 ± 770 µM P15 P16 P17 O O O O O O HO N OH HO N OH HO N OH OH OH OH

O H3C O O CH3 H3C

724 ± 91 µM 563 ± 89 µM 2750 ± 450 µM P43 P44 P45 B) O O O HO HO HO N OH N OH N OH O OH O OH O OH Br O Br O O Br 114 ± 15 µM 269 ± 31 µM 1200 ± 50 µM P18 P19 P20 O O O O O O HO N OH HO N OH HO N OH OH OH OH O O O Br Br Br 173 ± 21 µM 101 ± 4 µM 370 ± 60 µM

P37 P38 P39

Figure 4-4. Benzyl substituents: methyl and bromo functional groups.

4-aminoethylphthalate is generally better than 4-aminomethylphthalate as a parent structure. The effects of both methyl (A) and bromo (B) substitution on benzyl were tested.

102 O O O HO HO HO N OH N OH N OH O OH O OH O OH O O O CF3 F3C

F3C 390 ± 50 µM 104 ± 9 µM 791 ± 51 µM P21 P22 P23 O O O O O O HO N OH HO N OH HO N OH OH OH OH O O O CF3 F3C CF3 88 ± 6 µM 37 ± 1 µM 186 ± 16 µM

P40 P41 P42

O O O HO HO HO N OH N OH N OH O OH O OH O OH O O O F C 3 O F CF3 OH 687 ± 66 µM 426 ± 68 µM 104 ± 9 µM

P28 P29 P34

O O O O O O HO N OH HO N OH HO N OH OH OH OH O O O F3C O F CF3 OH 1660 ± 326 µM 587 ± 72 µM 29 ± 2 µM

P46 P47 P50

O O HO HO N OH N OH O OH O OH O O

O O NH2 CH3 1884 ±380 µM 2541 ± 219 µM

P35 P36

Figure 4-5. Trifluoromethyl effect and the additional influence of a hydrophilic functional group.

103 A) O O O O HO N OH HO N OH OH OH

F3C O O CF3 O O OH OH

930 ± 151 µM 17.0 ± 0.4 µM P52 P53

B) O O O O HO N OH HO N OH OH OH O O O OH O OH

47.4 ± 1.4 µM 187 ± 22 µM P54 P51

C) O O O O O O HO N OH HO N OH HO N OH OH OH OH F O Cl O Br O O O O OH OH OH

4.06 ± 0.08 µM 1.70 ± 0.04 µM 2.36 ± 0.11 µM

P57 P56 P55

D) O O O O HO N OH HO N OH Cl OH Br OH Cl O Br O O O OH OH

0.61± 0.01 µM 0.77 ± 0.01 µM P59 P58

Figure 4-6. Substitutions on benzylaminoethylphthalates.

To combine the knowledge learned from individual substitutients, perfluoromethyl (A), additional aromatic ring (B), mono-substituted halogens (C) and di-substituted halogens

(D) were attached at either the ortho or meta positions.

104

References

[1] Canavan, M. M. (1931) Schildler's encephalitis periaxialsis diffusa Report of A case in a child aged sixteen and one-half months, Archives of Neurology and Psychiatry 25, 200-308.

[2] van Bogaert, L., and Bertrand, I. (1949) Sur une idiotie familiale avec de´ge´ne´rescence spongieuse du ne´vraxe (Note pre´liminaire), Acta neurologica et psychiatrica belgica 49, 572-587.

[3] van Bogaert, L. (1963) Familial spongy degeneration of brain (complementary study of family R), Acta Psychiatrica et Neurologica Scandinavica 39, 107-113.

[4] Breitbach-Faller, N., Schrader, K., Rating, D., and Wunsch, R. (2003) Ultrasound findings in follow-up investigations in a case of aspartoacylase deficiency (Canavan disease), Neuropediatrics 34, 96-99.

[5] Kumar, S., Mattan, N. S., and de Vellis, J. (2006) Canavan disease: A white matter disorder, Mental Retardation and Developmental Disabilities Research Reviews 12, 157-165.

[6] Hoshino, H., and Kubota, M. (2014) Canavan disease: clinical features and recent advances in research, Pediatrics International 56, 477-483.

[7] Gowda, V. K., Bhat, M. D., Srinivasan, V. M., Prasad, C., Benakappa, A., and Faruq, M. (2016) A case of Canavan disease with microcephaly, Brain and Development 38, 759-762.

[8] Carpinelli, M. R., Voss, A. K., Manning, M. G., Perera, A. A., Cooray, A. A., Kile, B. T., and Burt, R. A. (2014) A new mouse model of Canavan leukodystrophy

105 displays hearing impairment due to central nervous system dysmyelination, Disease Models & Mechanisms 7, 649-657.

[9] Janson, C. G., McPhee, S. W. J., Francis, J., Shera, D., Assadi, M., Freese, A., Hurh, P., Haselgrove, J., Wang, D. J., Bilaniuk, L., and Leone, P. (2006) Natural history of Canavan disease revealed by proton magnetic resonance spectroscopy (H-1- MRS) and diffusion-weighted MRI, Neuropediatrics 37, 209-221.

[10] Zeng, B. J., Wang, Z. H., Torres, P. A., Pastores, G. M., Leone, P., Raghavan, S. S., and Kolodny, E. H. (2006) Rapid detection of three large novel deletions of the aspartoacylase gene in non-Jewish patients with Canavan disease, Molecular Genetics and Metabolism 89, 156-163.

[11] Janson, C. G., Kolodny, E. H., Zeng, B. J., Raghavan, S., Pastores, G., Torres, P., Assadi, M., McPhee, S., Goldfarb, O., Saslow, B., Freese, A., Wang, D. J., Bilaniuk, L., Shera, D., and Leone, P. (2006) Mild-onset presentation of Canavan's disease associated with novel G212A point mutation in aspartoacylase gene, Annals of Neurology 59, 428-431.

[12] Matalon, R., Michals, K., Sebesta, D., Deanching, M., Gashkoff, P., Casanova, J., Optiz, J. M., and Reynolds, J. F. (1988) Aspartoacylase deficiency and N- acetylaspartic aciduria in patients with canavan disease, American Journal of Medical Genetics 29, 463-471.

[13] Chakraborty, G., Mekala, P., Yahya, D., Wu, G., and Ledeen, R. W. (2001) Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase, Journal of Neurochemistry 78, 736-745.

[14] Namboodiri, A. M. A., Peethambaran, A., Mathew, R., Sambhu, P. A., Hershfield, J., Moffett, J. R., and Madhavarao, C. N. (2006) Canavan disease and the role of N-acetylaspartate in myelin synthesis, Mol Cell Endocrinol 252, 216-223.

[15] Mehta, V., and Namboodiri, M. A. A. (1995) N-Acetylaspartate as an acetyl source in the nervous system, Molecular Brain Research 31, 151-157.

[16] Madhavarao, C. N., Arun, P., Moffett, J. R., Szucs, S., Surendran, S., Matalon, R., Garbern, J., Hristova, D., Johnson, A., Jiang, W., and Namboodiri, M. A. A. (2005) Defective N-acetylaspartate catabolism reduces brain acetate levels and myelin lipid synthesis in Canavan's disease, Proceedings of the National Academy of Sciences of the United States of America 102, 5221-5226.

106 [17] Namboodiri, A. M. A., Moffett, J. R., Arun, P., Mathew, R., Namboodiri, S., Potti, A., Hershfield, J., Kirmani, B., Jacobowitz, D. M., and Madhavarao, C. N. (2006) Defective myelin lipid synthesis as a pathogenic mechanism of Canavan disease, Adv Exp Med Biol 576, 145-163.

[18] Baslow, M. H., and Guilfoyle, D. N. (2013) Canavan disease, a rare early-onset human spongiform leukodystrophy: Insights into its genesis and possible clinical interventions, Biochimie 95, 946-956.

[19] Akimitsu, T., Kurisu, K., Hanaya, R., Iida, K., Kiura, Y., Arita, K., Matsubayashi, H., Ishihara, K., Kitada, K., Serikawa, T., and Sasa, M. (2000) Epileptic seizures induced by N-acetyl-L-aspartate in rats: in vivo and in vitro studies, Brain Research 861, 143-150.

[20] Kitada, K., Akimitsu, T., Shigematsu, Y., Kondo, A., Maihara, T., Yokoi, N., Kuramoto, T., Sasa, M., and Serikawa, T. (2000) Accumulation of N-acetyl-L- aspartate in the brain of the tremor rat, a mutant exhibiting absence-like seizure and spongiform degeneration in the central nervous system, Journal of Neurochemistry 74, 2512-2519.

[21] Neale, J. H., Bzdega, T., and Wroblewska, B. (2000) N-Acetylaspartylglutamate, Journal of Neurochemistry 75, 443-452.

[22] Xuan, J., Pan, G., Qiu, Y., Yang, L., Su, M., Liu, Y., Chen, J., Feng, G., Fang, Y., Jia, W., Xing, Q., and He, L. (2011) Metabolomic Profiling to Identify Potential Serum Biomarkers for Schizophrenia and Risperidone Action, Journal of Proteome Research 10, 5433-5443.

[23] Pouwels, P. J., and Frahm, J. (1997) Differential distribution of NAA and NAAG in human brain as determined by quantitative localized proton MRS, Nmr Biomed 10, 73-78.

[24] Becker, I., Lodder, J., Gieselmann, V., and Eckhardt, M. (2010) Molecular Characterization of N-Acetylaspartylglutamate Synthetase, Journal of Biological Chemistry 285, 29156-29164.

[25] Tallan, H. H., Moore, S., and Stein, W. H. (1956) N-acetyl-L-aspartic acid in brain, Journal of Biological Chemistry 219, 257-264.

107 [26] Baslow, M. H. (1997) A review of phylogenetic and metabolic relationships between the acylamino acids, N-acetyl-L-aspartic acid and N-acetyl-L-histidine, in the vertebrate nervous system, Journal of Neurochemistry 68, 1335-1344.

[27] Nordengen, K., Heuser, C., Rinholm, J., Matalon, R., and Gundersen, V. (2013) Localisation of N-acetylaspartate in oligodendrocytes/myelin, Brain Struct Funct, 1-19.

[28] Goldstein, F. B. (1959) Biosynthesis of N-Acetyl-l-aspartic Acid, Journal of Biological Chemistry 234, 2702-2706.

[29] Wiame, E., Tyteca, D., Pierrot, N., Collard, F., Amyere, M., Noel, G., Desmedt, J., Nassogne, M.-C., Vikkula, M., Octave, J.-N., Vincent, M.-F., Courtoy, P. J., Boltshauser, E., and Van Schaftingen, E. (2010) Molecular identification of aspartate N-acetyltransferase and its mutation in hypoacetylaspartia, Biochemical Journal 425, 127-136.

[30] Bzdega, T., Turi, T., Wroblewska, B., She, D., Chung, H. S., Kim, H., and Neale, J. H. (1997) Molecular Cloning of a Peptidase Against N-Acetylaspartylglutamate from a Rat Hippocampal cDNA Library, Journal of Neurochemistry 69, 2270- 2277.

[31] Bzdega, T., Crowe, S. L., Ramadan, E. R., Sciarretta, K. H., Olszewski, R. T., Ojeifo, O. A., Rafalski, V. A., Wroblewska, B., and Neale, J. H. (2004) The cloning and characterization of a second brain enzyme with NAAG peptidase activity, Journal of Neurochemistry 89, 627-635.

[32] Berger, U. V., Luthi-Carter, R., Passani, L. A., Elkabes, S., Black, I., Konradi, C., and Coyle, J. T. (1999) Glutamate carboxypeptidase II is expressed by astrocytes in the adult rat nervous system, The Journal of Comparative Neurology 415, 52- 64.

[33] Fujita, T., Katsukawa, H., Yodoya, E., Wada, M., Shimada, A., Okada, N., Yamamoto, A., and Ganapathy, V. (2005) Transport characteristics of N-acetyl-l- aspartate in rat astrocytes: involvement of sodium-coupled high-affinity carboxylate transporter NaC3/NaDC3-mediated transport system, Journal of Neurochemistry 93, 706-714.

[34] Blüml, S., Wisnowski, J. L., Nelson, M. D., Jr., Paquette, L., and Panigrahy, A. (2014) Metabolic Maturation of White Matter Is Altered in Preterm Infants, PLOS ONE 9, e85829.

108 [35] Baslow, M. H., Suckow, R. F., Gaynor, K., Bhakoo, K. K., Marks, N., Saito, M., Saito, M., Duff, K., Matsuoka, Y., and Berg, M. J. (2003) Brain damage results in down-regulation of N-acetylaspartate as a neuronal osmolyte, Neuromolecular medicine 3, 95-104.

[36] Burlina, A. P., Schmitt, B., Engelke, U., Wevers, R. A., Burlina, A. B., and Boltshauser, E. (2006) Hypoacetylaspartia: Clinical and Biochemical Follow-Up of a Patient, In Adv Exp Med Biol (Moffett, J. R., Tieman, S. B., Weinberger, D. R., Coyle, J. T., and Namboodiri, A. M. A., Eds.), pp 283-287, Springer US, Boston, MA.

[37] Maier, H., Wang-Eckhardt, L., Hartmann, D., Gieselmann, V., and Eckhardt, M. (2015) N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time, The Journal of Neuroscience 35, 14501-14516.

[38] Steen, R. G., and Ogg, R. J. (2005) Abnormally High Levels of Brain N- Acetylaspartate in Children with Sickle Cell Disease, American Journal of Neuroradiology 26, 463-468.

[39] Yeh, Y.-C., Yen, C.-F., Li, C.-W., Kuo, Y.-T., Chen, C.-H., Lee, C.-C., Liu, G.-C., Huang, M.-F., Liu, T.-L., and Chen, C.-S. (2014) Altered neurochemical metabolites in Alzheimer's disease patients with unawareness of deficits, International Psychogeriatrics 26, 393-402.

[40] Steen, C., D’haeseleer, M., Hoogduin, J. M., Fierens, Y., Cambron, M., Mostert, J. P., Heersema, D. J., Koch, M. W., and De Keyser, J. (2013) Cerebral white matter blood flow and energy metabolism in multiple sclerosis, Multiple Sclerosis Journal 19, 1282-1289.

[41] Kaul, R., Gao, G. P., Balamurugan, K., and Matalon, R. (1993) Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease, Nat Genet 5, 118-123.

[42] Le Coq, J., An, H. J., Lebrilla, C., and Viola, R. E. (2006) Characterization of human aspartoacylase: The brain enzyme responsible for Canavan disease, Biochemistry 45, 5878-5884.

[43] Le Coq, J., Pavlovsky, A., Malik, R., Sanishvili, R., Xu, C. F., and Viola, R. E. (2008) Examination of the mechanism of human brain aspartoacylase through the binding of an intermediate analogue, Biochemistry 47, 3484-3492.

109 [44] Birnbaum, S. M., Levintow, L., Kingsley, R. B., and Greenstein, J. P. (1952) Specificity of amino acid acylases, Journal of Biological Chemistry 194, 455-470.

[45] Miller, Y. E., Drabkin, H., Jones, C., and Fisher, J. H. (1990) Human aminoacylase- 1: Cloning, regional assignment to distal chromosome 3p21.1, and identification of a cross-hybridizing sequence on chromosome 18, Genomics 8, 149-154.

[46] Newman, D., Abuladze, N., Scholz, K., Dekant, W., Tsuprun, V., Ryazantsev, S., Bondar, G., Sassani, P., Kurtz, I., and Pushkin, A. (2007) Specificity of Aminoacylase III-Mediated Deacetylation of Mercapturic Acids, Drug Metabolism and Disposition 35, 43-50.

[47] Moore, R. A., Le Coq, J., Faehnle, C. R., and Viola, R. E. (2003) Purification and preliminary characterization of brain aspartoacylase, Archives of Biochemistry and Biophysics 413, 1-8.

[48] Rual, J.-F., Venkatesan, K., Hao, T., Hirozane-Kishikawa, T., Dricot, A., Li, N., Berriz, G. F., Gibbons, F. D., Dreze, M., Ayivi-Guedehoussou, N., Klitgord, N., Simon, C., Boxem, M., Milstein, S., Rosenberg, J., Goldberg, D. S., Zhang, L. V., Wong, S. L., Franklin, G., Li, S., Albala, J. S., Lim, J., Fraughton, C., Llamosas, E., Cevik, S., Bex, C., Lamesch, P., Sikorski, R. S., Vandenhaute, J., Zoghbi, H. Y., Smolyar, A., Bosak, S., Sequerra, R., Doucette-Stamm, L., Cusick, M. E., Hill, D. E., Roth, F. P., and Vidal, M. (2005) Towards a proteome-scale map of the human protein-protein interaction network, Nature 437, 1173-1178.

[49] Bitto, E., Bingman, C. A., Wesenberg, G. E., McCoy, J. G., and Phillips, G. N. (2007) Structure of aspartoacylase, the brain enzyme impaired in Canavan disease, Proceedings of the National Academy of Sciences of the United States of America 104, 456-461.

[50] Wijayasinghe, Y. S., Pavlovsky, A. G., and Viola, R. E. (2014) Aspartoacylase Catalytic Deficiency as the Cause of Canavan Disease: A Structural Perspective, Biochemistry 53, 4970-4978.

[51] Zano, S., Wijayasinghe, Y. S., Malik, R., Smith, J., and Viola, R. E. (2013) Relationship between Enzyme Properties and Disease Progression in Canavan Disease, Journal of Inherited Metabolic Dsease 36, 1-6.

[52] Stenson, P., Mort, M., Ball, E., Shaw, K., Phillips, A., and Cooper, D. (2014) The Human Gene Mutation Database: building a comprehensive mutation repository

110 for clinical and molecular genetics, diagnostic testing and personalized genomic medicine, Hum Genet 133, 1-9.

[53] Tacke, U., Olbrich, H., Sass, J. O., Fekete, A., Horvath, J., Ziyeh, S., Kleijer, W. J., Rolland, M. O., Fisher, S., Payne, S., Vargiami, E., Zafeiriou, D. I., and Omran, H. (2005) Possible genotype-phenotype correlations in children with mild clinical course of Canavan disease, Neuropediatrics 36, 252-255.

[54] Hussain, R., Daud, S., Kakar, N., Ahmad, A., Baloch, A. H., Tareen, A. M., Kakar, M. A., and Ahmad, J. (2012) A missense mutation (p.G274R) in gene ASPA causes Canavan disease in a Pakistani family, Mol Biol Rep 39, 6197-6201.

[55] Hershfield, J. R., Pattabiraman, N., Madhavarao, C. N., and Namboodiri, M. A. A. (2007) Mutational analysis of aspartoacylase: Implications for Canavan Disease, Brain Research 1148, 1-14.

[56] Zeng, B. J., Wang, Z. H., Ribeiro, L. A., Leone, P., De Gaspari, R., Kim, S. J., Raghavan, S., Ong, E., Pastores, G. M., and Kolodny, E. H. (2002) Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease, J Inherit Metab Dis 25, 557-570.

[57] Kots, E. D., Khrenova, M. G., Lushchekina, S. V., Varfolomeev, S. D., Grigorenko, B. L., and Nemukhin, A. V. (2016) Modeling the Complete Catalytic Cycle of Aspartoacylase, The Journal of Physical Chemistry B 120, 4221-4231.

[58] Herga, S., Berrin, J. G., Perrier, J., Puigserver, A., and Giardina, T. (2006) Identification of the zinc binding ligands and the catalytic residue in human aspartoacylase, an enzyme involved in Canavan disease, FEBS Letters 580, 5899- 5904.

[59] Wang, Q., and Viola, R. E. (2014) Reexamination of aspartoacylase: Is this human enzyme really a glycoprotein?, Archives of Biochemistry and Biophysics 548, 66- 73.

[60] Tallan, H. H. (1957) Studies on the distribution of N-acetyl-L-aspartic acid in brain, Journal of Biological Chemistry 224, 41-45.

[61] Boltshauser, E., Schmitt, B., Wevers, R. A., Engelke, U., Burlina, A. B., and Burlina, A. P. (2004) Follow-up of a child with hypoacetylaspartia, Neuropediatrics 35, 255-258.

111 [62] Knizley, H. (1967) The Enzymatic Synthesis of N-Acetyl-l-aspartic Acid by a Water-insoluble Preparation of a Cat Brain Acetone Powder, Journal of Biological Chemistry 242, 4619-4622.

[63] Truckenmiller, M. E., Namboodiri, M. A. A., Brownstein, M. J., and Neale, J. H. (1985) N-Acetylation of L-Aspartate in the Nervous System: Differential Distribution of a Specific Enzyme, Journal of Neurochemistry 45, 1658-1662.

[64] Sebastiano, V., Maeder, M. L., Angstman, J. F., Haddad, B., Khayter, C., Yeo, D. T., Goodwin, M. J., Hawkins, J. S., Ramirez, C. L., Batista, L. F. Z., Artandi, S. E., Wernig, M., and Joung, J. K. (2011) In Situ Genetic Correction of the Sickle Cell Anemia Mutation in Human Induced Pluripotent Stem Cells Using Engineered Zinc Finger Nucleases, STEM CELLS 29, 1717-1726.

[65] Tsen, A. R., Long, P. M., Driscoll, H. E., Davies, M. T., Teasdale, B. A., Penar, P. L., Pendlebury, W. W., Spees, J. L., Lawler, S. E., Viapiano, M. S., and Jaworski, D. M. (2014) Triacetin-based acetate supplementation as a chemotherapeutic adjuvant therapy in glioma, International Journal of Cancer 134, 1300-1310.

[66] Clanton, R. M., Wu, G., Akabani, G., and Aramayo, R. (2016) Control of seizures by ketogenic diet-induced modulation of metabolic pathways, Amino Acids, 1-20.

[67] Ahmed, S. S., Li, H., Cao, C., Sikoglu, E. M., Denninger, A. R., Su, Q., Eaton, S., Navarro, A. A. L., Xie, J., Szucs, S., Zhang, H., Moore, C., Kirschner, D. A., Seyfried, T. N., Flotte, T. R., Matalon, R., and Gao, G. (2013) A Single Intravenous rAAV Injection as Late as P20 Achieves Efficacious and Sustained CNS Gene Therapy in Canavan Mice, Mol Ther 21, 2136-2147.

[68] Zano, S., Malik, R., Szucs, S., Matalon, R., and Viola, R. E. (2011) Modification of aspartoacylase for potential use in enzyme replacement therapy for the treatment of Canavan disease, Molecular Genetics and Metabolism 102, 176-180.

[69] Ratko, T. A., Marbella, A., Godfrey, S., and Aronson, N. (2013) Enzyme- replacement therapies for lysosomal storage diseases, Technical Briefs 12.

[70] Rigat, B. A., Tropak, M. B., Buttner, J., Crushell, E., Benedict, D., Callahan, J. W., Martin, D. R., and Mahuran, D. J. (2012) Evaluation of N-nonyl- deoxygalactonojirimycin as a pharmacological chaperone for human GM1 gangliosidosis leads to identification of a feline model suitable for testing enzyme enhancement therapy, Molecular Genetics and Metabolism 107, 203-212.

112 [71] Assadi, M., Janson, C., Wang, D.-J., Goldfarb, O., Suri, N., Bilaniuk, L., and Leone, P. (2010) Lithium citrate reduces excessive intra-cerebral N-acetyl aspartate in Canavan disease, European Journal of Paediatric Neurology 14, 354-359.

[72] Baslow, M. H., Kitada, K., Suckow, R. F., Hungund, B. L., and Serikawa, T. (2002) The effects of lithium chloride and other substances on levels of brain N-acetyl-L- aspartic acid in Canavan disease-like rats, Neurochem Res 27, 403-406.

[73] Baslow, M. H., and Guilfoyle, D. N. (2014) Evidence that lithium Inhibits Export of N-Acetyl-L-Aspartate from Neurons: A Retrospective Study of Canavan Disease and Bipolar Disorder Patients, Journal of Genetic Disorders & Genetic Reports 3, 2.

[74] Guo, F., Bannerman, P., Ko, E. M., Miers, L., Xu, J., Burns, T., Li, S., Freeman, E., McDonough, J. A., and Pleasure, D. (2015) Ablating N-Acetylaspartate Prevents Leukodystrophy in a Canavan Disease Model, Annals of Neurology 77, 884-888.

[75] Tarentino, A. L., Trimble, R. B., and Plummer, T. H. (1989) Chapter 5 Enzymatic Approaches for Studying the Structure, Synthesis, and Processing of Glycoproteins, Methods in Cell Biology 32, 111-139.

[76] Matsui, T., Takita, E., Sato, T., Kinjo, S., Aizawa, M., Sugiura, Y., Hamabata, T., Sawada, K., and Kato, K. (2011) N-glycosylation at noncanonical Asn-X-Cys sequences in plant cells, Glycobiology 21, 994-999.

[77] Khoury, G. A., Baliban, R. C., and Floudas, C. A. (2011) Proteome-wide post- translational modification statistics: frequency analysis and curation of the swissprot database, Scientific Reports 1, 90.

[78] Zielinska, D. F., Gnad, F., Wiśniewski, J. R., and Mann, M. (2010) Precision Mapping of an In Vivo N-Glycoproteome Reveals Rigid Topological and Sequence Constraints, Cell 141, 897-907.

[79] Ballou, C. E. (1990) Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects, Methods in Enzymology 185, 440-470.

[80] Alagesan, K., Khilji, S. K., and Kolarich, D. (2017) It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment, Anal Bioanal Chem 409, 529-538.

113 [81] Tajiri, M., Yoshida, S., and Wada, Y. (2005) Differential analysis of site-specific glycans on plasma and cellular fibronectins: application of a hydrophilic affinity method for glycopeptide enrichment, Glycobiology 15, 1332-1340.

[82] Kaji, H., Saito, H., Yamauchi, Y., Shinkawa, T., Taoka, M., Hirabayashi, J., Kasai, K.-i., Takahashi, N., and Isobe, T. (2003) Lectin affinity capture, isotope-coded tagging and mass spectrometry to identify N-linked glycoproteins, Nature Biotechnology 21, 667-672.

[83] Zhang, H., Li, X.-j., Martin, D. B., and Aebersold, R. (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry, Nature Biotechnology 21, 660-666.

[84] Sun, S., Shah, P., Eshghi, S. T., Yang, W., Trikannad, N., Yang, S., Chen, L., Aiyetan, P., Hoti, N., Zhang, Z., Chan, D. W., and Zhang, H. (2016) Comprehensive analysis of protein glycosylation by solid-phase extraction of N- linked glycans and glycosite-containing peptides, Nature Biotechnology 34, 84- 88.

[85] Gustafsson, C., Govindarajan, S., and Minshull, J. (2004) Codon bias and heterologous protein expression, Trends in Biotechnology 22, 346-353.

[86] Burgess-Brown, N. A., Sharma, S., Sobott, F., Loenarz, C., Oppermann, U., and Gileadi, O. (2008) Codon optimization can improve expression of human genes in Escherichia coli: A multi-gene study, Protein Expression and Purification 59, 94- 102.

[87] Boël, G., Letso, R., Neely, H., Price, W. N., Wong, K.-H., Su, M., Luff, J. D., Valecha, M., Everett, J. K., Acton, T. B., Xiao, R., Montelione, G. T., Aalberts, D. P., and Hunt, J. F. (2016) Codon influence on protein expression in E. coli correlates with mRNA levels, Nature 529, 358-363.

[88] Poddar, N. K., Zano, S., Natarajan, R., Yamamoto, B., and Viola, R. E. (2014) Enhanced brain distribution of modified aspartoacylase, Molecular Genetics and Metabolism 113, 219-224.

[89] Hu, J., and Sebald, W. (2011) N-terminal specificity of PEGylation of human bone morphogenetic protein-2 at acidic pH, International Journal of Pharmaceutics 413, 140-146.

114 [90] Antonkine, M. L., Falzone, C., Hansen, A., Yang, F., and Golbeck, J. H. (1998) Chemical rescue of site-modified ligands to the iron-sulfur clusters of PsaC in photosystem I, Photosynthesis: Mechanisms and Effects, 659-662.

[91] Kozakov, D., Grove, L. E., Hall, D. R., Bohnuud, T., Mottarella, S. E., Luo, L., Xia, B., Beglov, D., and Vajda, S. (2015) The FTMap family of web servers for determining and characterizing ligand-binding hot spots of proteins, Nat Protoc 10, 733-755.

[92] Wijayasinghe, Y. S. (2014) Molecular Insights into N-acetylaspartate Metabolism in Canavan Disease, PhD dissertation, University of Toledo, Chemistry and Biochemistry.

[93] Trott, O., and Olson, A. J. (2010) AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading, Journal of Computational Chemistry 31, 455-461.

[94] Matalon, R., Rady, P. L., Platt, K. A., Skinner, H. B., Quast, M. J., Campbell, G. A., Matalon, K., Ceci, J. D., Tyring, S. K., Nehls, M., Surendran, S., Wei, J., Ezell, E. L., and Szucs, S. (2000) Knock-out mouse for Canavan disease: a model for gene transfer to the central nervous system, The Journal of Gene Medicine 2, 165-175.

[95] Goldstein, F. B. (1969) The Enzymatic Synthesis of N-Acetyl-l-aspartic Acid by Subcellular Preparations of Rat Brain, Journal of Biological Chemistry 244, 4257- 4260.

[96] Madhavarao, C. N., Chinopoulos, C., Chandrasekaran, K., and Namboodiri, M. A. A. (2003) Characterization of the N-acetylaspartate biosynthetic enzyme from rat brain, Journal of Neurochemistry 86, 824-835.

[97] Lu, Z.-H., Chakraborty, G., Ledeen, R. W., Yahya, D., and Wu, G. (2004) N- Acetylaspartate synthase is bimodally expressed in microsomes and mitochondria of brain, Molecular Brain Research 122, 71-78.

[98] Wang, Q., Zhao, M., Parungao, G. G., and Viola, R. E. (2016) Purification and characterization of aspartate N-acetyltransferase: A critical enzyme in brain metabolism, Protein Expression and Purification 119, 11-18.

115 [99] Zano, S. P. (2013) Therapeutic Approaches for the Treatment of Canavan Disease and Regulation of Bacterial Quorum Sensing Pathway, PhD dissertation, University of Toledo, Chemistry and Biochemistry.

[100] Tahay, G., Wiame, E., Tyteca, D., Courtoy, P. J., and Van Schaftingen, E. (2012) Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase, Biochemical Journal 441, 105-112.

[101] Benson, D. A., Cavanaugh, M., Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., and Sayers, E. W. (2013) GenBank, Nucleic Acids Research 41, D36- D42.

[102] Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., and Sayers, E. W. (2009) GenBank, Nucleic Acids Research 37, D26-D31.

[103] Benson, D. A., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., and Sayers, E. W. (2011) GenBank, Nucleic Acids Research 39, D32-D37.

[104] Benson, D. A., Karsch-Mizrachi, I., Clark, K., Lipman, D. J., Ostell, J., and Sayers, E. W. (2012) GenBank, Nucleic Acids Research 40, D48-D53.

[105] Benson, D. A., Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., and Sayers, E. W. (2014) GenBank, Nucleic Acids Research 42, D32-D37.

[106] Benson, D. A., Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., and Sayers, E. W. (2015) GenBank, Nucleic Acids Research 43, D30-D35.

[107] Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., and Sayers, E. W. (2016) GenBank, Nucleic Acids Research 44, D67-D72.

[108] Benson, D. A., Cavanaugh, M., Clark, K., Karsch-Mizrachi, I., Lipman, D. J., Ostell, J., and Sayers, E. W. (2017) GenBank, Nucleic Acids Research 45, D37- D42.

[109] Magrane, M., and Consortium, U. (2011) UniProt Knowledgebase: a hub of integrated protein data, Database 2011, bar009-bar009.

116 [110] Wain, H. M., Bruford, E. A., Lovering, R. C., Lush, M. J., Wright, M. W., and Povey, S. (2002) Guidelines for Human Gene Nomenclature, Genomics 79, 464- 470.

[111] Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M., and Seraphin, B. (1999) A generic protein purification method for protein complex characterization and proteome exploration, Nature Biotechnology 17, 1030-1032.

[112] Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., and Séraphin, B. (2001) The Tandem Affinity Purification (TAP) Method: A General Procedure of Protein Complex Purification, Methods 24, 218- 229.

[113] Shevchenko, A., Tomas, H., Havlis, J., Olsen, J. V., and Mann, M. (2007) In-gel digestion for mass spectrometric characterization of proteins and proteomes, Nat Protoc 1, 2856-2860.

[114] Toriumi, K., Kondo, M., Nagai, T., Hashimoto, R., Ohi, K., Song, Z., Tanaka, J., Mouri, A., Koseki, T., Yamamori, H., Furukawa-Hibi, Y., Mamiya, T., Fukushima, T., Takeda, M., Nitta, A., Yamada, K., and Nabeshima, T. (2014) Deletion of SHATI/NAT8L increases dopamine D1 receptor on the cell surface in the nucleus accumbens, accelerating methamphetamine dependence, The International Journal of Neuropsychopharmacology 17, 443-453.

[115] Wang, H., Wang, B., Normoyle, K. P., Jackson, K., Spitler, K., Sharrock, M. F., Miller, C. M., Best, C., Llano, D., and Du, R. (2014) Brain temperature and its fundamental properties: a review for clinical neuroscientists, Frontiers in Neuroscience 8, 1-17.

[116] Lewendon, A., and Shaw, W. V. (1993) The pKa of the catalytic histidine residue of chloramphenicol acetyltransferase, Biochemical Journal 290, 15-19.

[117] Sinclair, J. C., Sandy, J., Delgoda, R., Sim, E., and Noble, M. E. (2000) Structure of arylamine N-acetyltransferase reveals a catalytic triad, Nat Struct Mol Biol 7, 560-564.

[118] Lou, T.-F., Sethuraman, D., Dospoy, P., Srivastva, P., Kim, H. S., Kim, J., Ma, X., Chen, P.-H., Huffman, K. E., Frink, R. E., Larsen, J. E., Lewis, C., Um, S.-W., Kim, D.-H., Ahn, J.-M., DeBerardinis, R. J., White, M. A., Minna, J. D., and Yoo, H. (2016) Cancer-Specific Production of N-Acetylaspartate via NAT8L

117 Overexpression in Non–Small Cell Lung Cancer and Its Potential as a Circulating Biomarker, Cancer Prevention Research 9, 43-52.

[119] Zand, B., Previs, R. A., Zacharias, N. M., Rupaimoole, R., Mitamura, T., Nagaraja, A. S., Guindani, M., Dalton, H. J., Yang, L., Baddour, J., Achreja, A., Hu, W., Pecot, C. V., Ivan, C., Wu, S. Y., McCullough, C. R., Gharpure, K. M., Shoshan, E., Pradeep, S., Mangala, L. S., Rodriguez-Aguayo, C., Wang, Y., Nick, A. M., Davies, M. A., Armaiz-Pena, G., Liu, J., Lutgendorf, S. K., Baggerly, K. A., Eli, M. B., Lopez-Berestein, G., Nagrath, D., Bhattacharya, P. K., and Sood, A. K. (2016) Role of Increased n-acetylaspartate Levels in Cancer, Journal of the National Cancer Institute 108, djv426.

[120] Weindl, D., Cordes, T., Battello, N., Sapcariu, S. C., Dong, X., Wegner, A., and Hiller, K. (2016) Bridging the gap between non-targeted stable isotope labeling and metabolic flux analysis, Cancer & Metabolism 4, 1-14.

[121] Thangavelu, B., Mutthamsetty, V., Wang, Q., and Viola, R. E. (2017) Design and optimization of aspartate N-acetyltransferase inhibitors for the potential treatment of Canavan disease, Bioorganic & Medicinal Chemistry 25, 870-885.

[122] Zhang, J.-H., Chung, T. D. Y., and Oldenburg, K. R. (1999) A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays, Journal of Biomolecular Screening 4, 67-73.

[123] Thangavelu, B., Bhansali, P., and Viola, R. E. (2015) Elaboration of a fragment library hit produces potent and selective aspartate semialdehyde dehydrogenase inhibitors, Bioorganic & Medicinal Chemistry 23, 6622-6631.

[124] Tan, D. S. (2005) Diversity-oriented synthesis: exploring the intersections between chemistry and biology, Nat Chem Biol 1, 74-84.

[125] Galloway, W. R. J. D., Isidro-Llobet, A., and Spring, D. R. (2010) Diversity- oriented synthesis as a tool for the discovery of novel biologically active small molecules, Nature Communications 1, 80.

118

Appendices

Appendix A1

Table A.1: Typical components in de-glycosylation reaction.

Reaction Control-1 Control-2 (microliter) (microliter) (microliter)

H2O 5.7 10.7 5.7

buffer 3 3 3

100 mM DTT 0.3 0.3 0.3

ASPA 16 16 16

PNGase F 5 0 5* (500 U/µl)

Total Volume 30 30 30

* PNGase F storage buffer was prepared in lab and used to replace the PNGase F reagent.

119 Appendix A2

Figure A-1 Codon optimized nucleotide sequence for human aspartoacylase.

120 Appendix A3

Figure A-2: hASPA E. coli cloning construct synthesized by Life Technologies.

121 Appendix A4

Figure A-3: N-terminal PEGylation reaction of ASPA.

122 Appendix B1

Figure B-1: Hydrodynamic radius of MBP-ANAT-His fusion protein

DLS experiment was conducted on a Malvern Zetasizer system. Although the curve of the correlation function is smooth, the calculated hydrodynamic radii of MBP-ANAT-His fusion protein was more than 40 nm, suggesting very large aggregates.

123 Appendix C1

Figure C-1: Positive and negative controls for DTNB assay

4# 3.5# 3# 2.5# 2# 1.5# posi.ve#control# 1# nega.ve#control# mOD420/min* 0.5# 0# 0# 5# 10# 15# 20# 25# !0.5# !1# reac/on*

To calculate the Z’ factor for the DTNB assay for ANAT inhibitor screening, assays were conducted in 20 mM HEPES, pH 7.4, 150 mM NaCl, 5% glycerol, 40 µM DTNB,

40 µM acetyl-Coenzyme A, and 25 µg of ANAT enzyme in 200 µl total volume with

(positive control) and without (negative control) 0.2 mM L-aspartic acid. The separation band between the positive controls and negative controls is large, suggesting that the

DTNB assay is suitable for inhibitor screening.

124 Appendix C2

Figure C-2: Representative kinetic absorbance curve for ANAT inhibitor

125