Identification of Novel Monoamine Oxidase B Inhibitors from Ligand Based Virtual Screening

Home , Benserazide, Enzyme inhibitor, Mofegiline, Oxidative deamination, Rasagiline

Identification of novel monoamine oxidase B inhibitors from ligand based virtual screening

A thesis submitted

to Kent State University in partial

Fulfillment of the requirements for the

Degree of Master of Science

Mohammed Alaasam

August, 2014

Thesis written by

Mohammed Alaasam

B.S., University of Baghdad, 2007

M.S., Kent State University, 2014

Approved by

Werner Geldenhuys , Chair, Master’s Thesis Committee

Richard Carroll , Member, Master’s Thesis Committee

Prabodh Sadana , Member, Master’s Thesis Committee

Eric Mintz , Director, School of Biomedical Sciences

James Blank , Dean, College of Arts and Sciences

Table of Contents

List of figure ...... vi

List of tables ...... xiii

Acknowledgments...... xiv

Chapter one: Introduction ...... 1

1.1.Parkinson's disease ...... 1

1.2.Monoamine oxidase enzymes ...... 7

1.3.Structures of MAO enzymes ...... 12

1.4.Three dimentiona structure of MAO-B enzyme ...... 13

1.4.1. membrane binding domain ...... 14

1.4.2. FAD binding domain ...... 16

1.4.2.1.Role of aromatic cage in MAO-B catalysis ...... 22

1.4.3. Substrate binding domain ...... 27

1.5.Structural comparison between human MAO-B and human MAO-A ...... 34

1.5.1. Oligomeric state of MAO enzymes ...... 35

1.5.2. Membrane binding domain structural differences ...... 36

1.5.3. FAD binding domain structural differences ...... 36

1.5.4. Substrate binding domain structural differences ...... 36

1.6.Neuroprotective effect of MAO-B inhibitors ...... 39

iii

1.7.Oxidative deamination reaction of MAO enzymes ...... 41

1.7.1. Reductive half reaction ...... 42

1.7.1.1.Proposed mechanisms of Cα—H cleavage ...... 44

1.7.1.2.Polar nucleophilic mechanism ...... 47

1.7.2. Oxidative half reaction ...... 49

1.8.Structural mechanism of MAO inhibtion ...... 49

1.9.LSD1 enzyme ...... 54

1.9.1. Three dimensional structure of LSD1 ...... 56

1.9.2. Substrate specificity of LSD1 enzyme ...... 58

Chapter two: Methods and Materials ...... 62

2.1.Overview ...... 62

2.2.Monoamine oxidase inhibition assay ...... 68

2.3.LSD1 inhibitor screening assay ...... 71

2.4.Docking studies ...... 73

2.5.Bovine serum albumin binding assay ...... 73

2.6.Parallel Artificial Membrane Permeability Assay (PAMPA) ...... 75

Chapter three: Results ...... 78

3.1.MAO inhibition assay results...... 78

3.1.1. MAO-B inhibition assay results ...... 84

3.1.2. MAO-A inhibition assay results ...... 84

3.2. MAO-B docking studies results ...... 90

3.3. LSD1 inhibition assay results ...... 102

3.4. LSD1 docking studies results...... 104

3.5. Bovine serum albumin binding assay results ...... 115

3.6. Parallel artificial membrane permeability assay results ...... 118

Chapter four: ...... 121

4.1.Discussion...... 121

4.2.Structure activity relationships ...... 140

Bibliography ...... 147

List of figures

Figure (1): Simple oxidative deamination reaction ...... 9

Figure (2): MAO enzyme specifities for amine substrates...... 10

Figure 3: 3D structure of MAO-A as monomer and MAO-B as dimer ...... 13

Figure (4): Three dimensional structure of MAO-B enzyme ...... 15

Figure (5): Covalent binding of MAO enzyme to isoalloxazine ring of FAD cofactor ... 16

Figure (6): Two dimesional representation of FAD incorporation in MAO-B enzyme ... 18

Figure (7): Three dimensional demonstration of phenolic side chain of Tyr398 in

perpendicular position to flavin ring of MAO-B enzyme...... 20

Figure (8): Covalent flavinylation of MAO-B enzyme ...... 21

Figure (9): Three dimensional representation of aromatic cage in MAO-B enzyme crystal

structure (top view) ...... 22

Figure (10): Proposed nucleophilic mechanism of reductive phase of MAO catalysis .... 23

Figure (11): Three dimensional representation of bent form of reduced flavin ring of

MAO-B crystal structure ...... 24

Figure (12): Simplified demonstration of suggested role of aromatic cage in MAO-B

catalysis ...... 25

Figure (13): P-nitrobenzylamine and P-nitrophenethylamine structures ...... 26

Figure (14): Three dimentional image of MAO-B enzyme crystal to illustrate loop99-112,

gating residues, entrance and substrate cavities ...... 28

Figure (15): Three dimensional reoresentation of Ile199 and Tyr326 ...... 29 vi

Figure (16): Three dimensional representation of (A) open and (B) close conformation of

Ile199 side chain of MAO-B enzyme ...... 31

Figure (17): Comparison of MAO-A monopartite and MAO-B dipartite active sites

cavities ...... 33

Figure (18): Metabolic pathway of dopamine with its precursors and metabolites ...... 40

Figure (19): Metabolic action of MAO-B enzyme on MPTP ...... 41

Figure (20): Oxidation-reduction reaction of flavin ring of FAD cofactor ...... 42

Figure (21):Oxidative deamination reaction mediated by MAO enzymes ...... 43

Figure (22): Proposed hydride mechanism of Cα—H cleavage reaction of reductive half

reaction in flavoenzymes ...... 44

Figure (23): Proposed heterolytic proton abstraction (single electron transfer) of Cα—H

cleavage reaction of reductive half reaction in flavoenzymes ...... 45

Figure (24): Proposed heterolytic hydrogen abstraction of Cα—H cleavage reaction of

reductive half reaction in flavoenzymes ...... 46

Figure (25): Proposed polar nucleophilic mechanism of reductive half reaction in MAO

enzymes catalysis ...... 48

Figure (26): Rasagiline, its methyl derivatives and its flavin adduct ...... 51

Figure (27): Suggested mechanism of Mofegiline for irreversible inhibition of MAO-B

enzyme ...... 52

Figure (28): Chemical structure of Tranylcypromine ...... 53

vii

Figure (29): Proposed oxidative demethylation reaction of LSD1 enzyme on Lys4 of

Histone 3 ...... 55

Figure (30): Three dimensional structure of LSD1-CoREST complex enzyme ...... 57

Figure (31): Mechanism of flavin-tranylcypromine adduct formation in MAO and LSD1

enzyme ...... 60

Figure (32): Mechanism of flavin adduct formation for propargyline containing

compounds in MAO and LSD1 enzymes ...... 61

Figure (33): Catalytic conversion of Kynuramine into 4-hydroxyquinoline ...... 69

Figure (34): Suggested mechanism of Mofegiline for irreversible inhibition of MAO-B

enzyme ...... 71

Figure (35): Functional groups with established inhibitory effect on MAO enzymes

ctalytic activity ...... 78

Figure (36): Inhibitory effect of the most active test compounds on MAO-B enzyme

activity ...... 88

Figure (37): Inhibitory effect of the most active test compounds on MAO-A enzyme

activity ...... 89

Figure (38): Inhibitory effect of the most active test compounds on both MAO-A and

MAO-B enzyme activity ...... 90

Figure (39): Two dimentional demonstration of docking of test compound (6366286) in

crystal structure of MAO-B enzyme ...... 93

viii

Figure (40): Three dimentional demonstration of docking of test compound (6366286) in

crystal structure of MAO-B enzyme ...... 94

Figure (41): Two dimentional demonstration of docking of test compound (6373721) in

crystal structure of MAO-B enzyme ...... 95

Figure (42): Three dimentional demonstration of docking of test compound (6373721) in

crystal structure of MAO-B enzyme ...... 96

Figure (43): Two dimentional demonstration of docking of test compound (7138125) in

crystal structure of MAO-B enzyme ...... 97

Figure (44): Three dimentional demonstration of docking of test compound (7138125) in

crystal structure of MAO-B enzyme ...... 98

Figure (45): Two dimentional demonstration of docking of test compound (6636424) in

crystal structure of MAO-B enzyme ...... 99

Figure (46): Three dimentional demonstration of docking of test compound (6636424) in

crystal structure of MAO-B enzyme ...... 100

Figure (47): Two dimentional demonstration of docking of test compound (7320244) in

crystal structure of MAO-B enzyme ...... 101

Figure (48): Three dimentional demonstration of docking of test compound (7320244) in

crystal structure of MAO-B enzyme ...... 102

Figure (49): Inhibitory effect of most active compounds on LSD1 enzyme activity ..... 103

Figure (50): Two dimentional demonstration of docking of test compound (6366286) in

crystal structure of LSD1 enzyme ...... 105

Figure (51): Three dimentional demonstration of docking of test compound (6366286) in

crystal structure of LSD1 enzyme ...... 106

Figure (52): Two dimentional demonstration of docking of test compound (6373721) in

crystal structure of LSD1 enzyme ...... 107

Figure (53): Three dimentional demonstration of docking of test compound (6373721) in

crystal structure of LSD1 enzyme ...... 108

Figure (54): Two dimentional demonstration of docking of test compound (6636424) in

crystal structure of LSD1 enzyme ...... 109

Figure (55): Three dimentional demonstration of docking of test compound (6636424) in

crystal structure of LSD1 enzyme ...... 110

Figure (56): Two dimentional demonstration of docking of test compound (7138125) in

crystal structure of LSD1 enzyme ...... 111

Figure (57): Three dimentional demonstration of docking of test compound (7138125) in

crystal structure of LSD1 enzyme ...... 112

Figure (58): Two dimentional demonstration of docking of test compound (7320244) in

crystal structure of LSD1 enzyme ...... 113

Figure (59): Three dimentional demonstration of docking of test compound (7320244) in

crystal structure of LSD1 enzyme ...... 114

Figure (60): Effect of most active compounds on percentage of free BSA ...... 116

Figure (61): Three dimentional demonstration of orientation of test compound (6366286)

in crystal structure of MAO-B enzyme ...... 130

Figure (62): Three dimentional demonstration of orientation of test compound (6373721)

in crystal structure of MAO-B enzyme ...... 131

Figure (63): Three dimentional demonstration of orientation of test compound (6636424)

in crystal structure of MAO-B enzyme ...... 132

Figure (64): Three dimentional demonstration of orientation of test compound (7138125)

in crystal structure of MAO-B enzyme ...... 133

Figure (65): Three dimentional demonstration of orientation of test compound (7320244)

in crystal structure of MAO-B enzyme ...... 134

Figure (66): Three dimentional demonstration of orientation of test compound (6366286)

in crystal structure of LSD1 enzyme ...... 135

Figure (67): Three dimentional demonstration of orientation of test compound (6373721)

in crystal structure of LSD1 enzyme ...... 136

Figure (68): Three dimentional demonstration of orientation of test compound (6636424)

in crystal structure of LSD1 enzyme ...... 137

Figure (69): Three dimentional demonstration of orientation of test compound (7138125)

in crystal structure of LSD1 enzyme ...... 138

Figure (70): Three dimentional demonstration of orientation of test compound (7320244)

in crystal structure of LSD1 enzyme ...... 139

Figure (71): Chemical structures of lead compound Pioglitazone and its derivatives

6366286 and 6838234 ...... 142

Figure (72): Three dimentional representation of docking Pioglitazone and its derivatives

6366286 and 6838234 in crystal structure of MAO-B enzyme ...... 143

Figure (73): Chemical structures of lead compound Pioglitazone and its derivatives

6636424 and 6634507 ...... 144

Figure (74): Chemical structures of lead compound Pioglitazone and its derivatives

6373721 and 6634507 ...... 144

Figure (75): Three dimentional representation of docking Pioglitazone and its derivatives

6373721 and 6634507 in crystal structure of MAO-B enzyme ...... 145

Figure (76): Chemical structures of test compounds 5472855 and 6209863 ...... 146

xii

List of tables

Table (1): Modified Hoehn and Yahr scale 2004 ...... 3

Table (2): MAO inhibitors: their selectivites, reversiblities and therapeutic uses ...... 8

Table 3: Two dimensional structures, molecular weights, and chemical name and formula

for all test compounds ...... 64

Table (4): Monoamine oxidase inhibition assay results for all test compounds ...... 80

Table (5): MAO-B inhibitory concentrations (IC50), inhibition curves, quantitative

values of inhibitory effects of most active test compounds on MAO-B

enzymatic activity (%)...... 86

Table (6): MAO-A inhibitory concentrations (IC50), inhibition curves, quantitative

values of inhibitory effects of most active test compounds on MAO-A

enzymatic activity (%)...... 89

Table (7): Estimated free binding energy of most active test compounds, estimated and

experimental inhibition constant of top test compounds ...... 91

Table (8): quantitative values of inhibitory effects of most active test compounds on

LSD1 enzymatic activity (%)...... 103

Table (9): BSA EC50, BSA binding curves, for most active test compounds ...... 117

Table (10): permeabilty vlaues (Pe) and LogPe for most active test compounds ...... 120

xiii

Acknowledgments

I would like to express my deep gratefulness to my wife, parents, brothers and sisters for their endless support, help and encouragement throughout my life journey.

I would like to thank my advisor (mentor) Dr. Werner Geldenhuys for his priceless support and substantial guidance throughout research course and thesis writing.

I would also like to express my gratefulness to Dr. Richard Carroll and Dr. Prabodh

Sadana for serving on my thesis committee and for their vital contribution to my project and thesis.

Finally, I would like to extend my gratitude to Dr. Eric Mintz and Mrs. Judith

Wearden for their precious support.

xiv

Chapter one: introduction

1.1. Parkinson’s disease:

Parkinson disease is the second most common age related neurodegenerative disorder after Alzheimer’s disease with prevalence of 0.5-1% among patients older than

65 years old and up to 4% among those older than 80 years of age in the united states1,2.

Its characteristic clinical symptoms include; Motor symptoms such as tremor, bradykinesia, rigidity and postural instability in addition to hypomimia, dysphagia, micrographia3 and non-motor symptoms; such as Neuropsychiatric symptoms that include the disturbances of the cognition, speech and mood behavior in addition to other behavioral and psychiatric problems3,4. Other symptoms are apathy, pain dysautonomia and sleep disorders3,4.

On the basis of disease origin; Parkinson disease can be classified into four types: idiopathic Parkinsonism, acquired Parkinsonism, heredodegenera-tive

Parkinsonism, and Parkinsonism plus syndromes3. On the basis of disease progression; Parkinson disease can also be divided into several stages of progression according to Hoehn and Yahr modified staging scale3 in 20045 as shown in table (1).

Pathologically Parkinson’s disease is caused by loss of the dopamine producing neurons in the pars compacta of the substantia nigra of mid brain6-8.

The main pathologic feature of the PD is the presence of cytoplasmic inclusions called lewy bodies. The main components of these inclusions are presynaptic proteins called alpha-synuclein that are in its fibrils form9,10. The function of synuclein is still unclear; but it may have a synaptic role (vesicular transport and trafficking role) due to their localization near the presynaptic terminals11. Alpha-synuclein aggregation to form lewy bodies is the hallmark of the PD and other neurodegenerative diseases such as lewy body’s dementia which together with PD are called synucleinopathy disorders12. Alpha- synuclein can increase the risk of familial and non-familial PD by the duplication and triplication mutation of the normal alpha-synuclein protein and by point mutation of the gene (SNAC) that codes for it13-19.

There is at least 70% decrease in dopamine release from the dopaminergic neurons in the substantia nigra before early clinical onset of the Parkinsonism that progress over time20. These estimations along with the observation of the substantial dopaminergic neurons loss at the late stages of the disease lead to a conclusion that there is a substantial neuronal cell death throughout the progression of the disease19. These suggest that striatal dopaminergic neurons have a relative vulnerability to cell death when compared to other neuronal cells21. The dopaminergic neurons at the substantia nigra are particularly vulnerable to oxidative stress due to the low level of the glutathione and the high nigral iron level22.

Oxidative stress and subsequent damaged neuronal cells can provoke microglial activation23. Microglial cells are the immune cells of the central nervous system that normally phagocytize and remove damaged neuronal cells24. Chronic microglial

activation lead to release reactive oxygen species that further damage neuronal cells23,24.

There are evidences support that; release of the nitrated, oxidized and aggregated alpha- synuclein from the affected dopaminergic neurons may result in further inflammation and oxidative stress25.

Parkinson disease is mostly idiopathic3 but there are a number of environmental factors that associated with its increased risks such as: pesticides exposure and head injuries26. Genetic factors are only account for 5% of PD patients27: Parkinson’s disease is found to be associated with number of genetic mutations that affect one of several specific genes such as: synuclein alpha (non-A4 component of amyloid precursor)

(SNCA) (gene codes for α-synuclein), leucine rich repeat kinase 2 (LRRK2)26, E3 ubiquitin ligase Parkin28, PETN-induced novel kinase 1 (PINK1)29.

Modified Hoehn and Yahr scale 2004

Stage 1.0 Unilateral involvement only.

Stage 1.5 Unilateral and axial involvement.

Stage 2.0 Bilateral involvement without impairment of balance.

Stage 2.5 Mild bilateral disease with recovery on pull test.

Stage 3.0 Mild to moderate bilateral disease; some postural instability; physically independent.

Stage 4.0 Sever disability; still able to walk or stand unassisted.

Stage 5.0 wheelchair bound or bedridden unless aided

Table (1): Stages of Parkinson disease as suggested by modified Hoehn and Yahr staging scale5

There are several suggested preventive measures that lower risk of Parkinson’s disease where the disease risk is lowered by 25% in Caffeine-consuming patients30 and

by one third in tobacco smoker as compared to non-smokers2. Antioxidant such as vitamin A and D are suggested to reduce the risk of PD but they are not proven yet2.

Parkinson’s disease cannot be permanently cured because pharmacological and non-pharmacological interventions do not arrest or reverse progression of the disease, but they only relieve motor and non-motor symptoms of the disease31. The symptomatic treatment of the PD alleviates the disease symptoms but they do not necessarily slow the disease progression. The pharmacological treatments are classified according to the disease stages at which the patient may experience specific clinical manifestations that require a pharmacological intervention32 into; Early pharmacological therapy: At which patients with PD developed a functional disability that require symptomatic pharmacological treatment and Late pharmacological therapy: At which patients who are already on Levodopa develop a motor complications32.

Medications act either by increase dopamine level at the dopaminergic receptors in the brain or mimic its effect31. Levodopa is considered as the standard symptomatic treatment of the PD32. Since dopamine cannot cross blood brain barrier, levodopa

(dopamine precursor) is given orally to cross BBB and increase dopamine concentration in the neuronal tissues. When given orally, it is readily converted into dopamine in the periphery by dopa decarboxylase enzyme and only small amount cross the blood brain barrier unchanged. Therefore; it is administered with peripheral dopa decarboxylase inhibitors such as benserazide and carbidopa to reduce its peripheral conversion into dopamine and thus reducing peripheral side effects and increase the amount of the drug that crosses blood brain barrier31,32. Levodopa alleviates many motor symptoms of the PD

but it has no effects on the non-motor symptoms33. It does not halt degeneration of the dopaminergic neurons in the substantia nigra31. Dopamine agonists have the same effect of the dopamine on its receptors. They were used as adjuvant therapy to levodopa but recent studies have shown that the dopamine agonists can be used as monotherapy to avoid or delay the motor complications of levodopa 32. Dopamine receptor agonists can be classified into two groups: ergot derivatives that include carbergoline, pergolide bromocriptine and lisuride and non-ergot derivatives that include pramipexole, peribidil ropinirole and apomorphine. Several studies proposed that these agents are neuroprotective34-37. Amantadine is an antiviral agent that is discovered by chance as effective treatment of PD. it is used to alleviate motor symptoms (especially dyskinesia) of the PD and drug induced dyskinesia. Its exact mechanism of action is unclear but it is thought that its action is attributed to its antiglutamate action 38. Anticholinergics or cholinergic receptors antagonists have been shown to be effective in reducing the resting tremor39. The mechanism of action is unknown but depletion of dopamine lead to loss of the Muscarinic 4 auto-receptors at the ventral putamen that causes acetylcholine release

40. Evidence based medicines reviews approve that M4 muscarinic receptors antagonist such as trihexyphenidyl or benztropin are effective particularly for PD tremor41.

Catechol-O-methyl transferase inhibitors are usually used in combination with levodopa (combined with carbidopa or benserazide) to further reduce the peripheral metabolism of levodopa into O-methyldopa by catechol-O-methyl transferase. This will increase the levodopa half-life by 25-100%32. This class of drug includes entacapone and tolcapone.

Monoamine oxidase inhibitors are used as adjuvant therapy with levodopa.

These drugs inhibit oxidative deamination reaction which is the main catabolic pathway of the dopamine in the striatum42. This result in increasing endogenous dopamine level and reduce required dose of levodopa43. MOA inhibitors are classified into non-selective

MAO inhibitors, selective MAO-B inhibitors and selective MAO-A inhibitors. Clinical and preclinical studies have shown that MAO-B inhibitors have a neuroprotective effects that may delay or halt neuronal cell death43. MAO inhibitor drugs are summarized in table (2) with their selectivity and therapeutic use44,45.

Recently; there are several neuroprotective agents that delay or halt neuronal cell death in Parkinson’s disease such as Non-steroidal anti-inflammatory drugs (Aspirin),

Selenium, Nicotine, Melatonin, Iron chelators, Vitamins. Cyclooxygenases and nitric oxides are reported to be increased in PD46-49. Aspirin (non-selective COX inhibitor) may act as neuroprotective in MPTP-induced PD50. In vivo studies have shown that salicylic acid can act as free radical scavengers in the brain51. Selenium has a neuroprotective effect due to its role in the functioning glutathione peroxidase (an antioxidant enzyme) that leads to delay the neuronal degeneration at the substantia nigra52. Nicotine is nicotinic receptors agonist that stimulate the striatum to release dopamine that lead to delay the neuronal degeneration53. The mechanism of action is not yet established but nicotine may stimulate the presynaptic receptors aα4, α7and β2 in the corpus striatum54. Nicotine may provoke the release of neuroprotective factor (fibroblast growth factor-2) from the dopaminergic neurons in the substantia nigra55. Melatonin is a hormone secreted by pineal gland. Melatonin has an antioxidant effect by inhibition of

free radicals activity56. Since α-synuclein aggregation is increased in the brain in the presence of iron57, so that Iron chelators (such as desferoxamine) may have neuroprotective effect when used concomitantly with MAO-B inhibitors via lowering the iron content in the substantia nigra58. Vitamin A, E, and C are approved as antioxidants that inhibit the oxidative stress by its action as free radicals scavengers. Many studies have shown a relationship between these vitamins deficiency and the risk of PD59-61.

Non-pharmacological treatments of Parkinson’s disease include; Cell transplantation62,

Vaccination63, Gene therapy64 and Surgery31.

1.2. Monoamine oxidase enzymes:

MOAs are flavin dependent mitochondrial enzymes (bound to the outer mitochondrial membrane) that oxidatively deaminate primary and secondary aromatic amine neurotransmitters such as adrenaline, noradrenaline, serotonin, dopamine, histamine, and other trace amines through formation of intermediate imines and production of ammonia (for primary amine) or substituted amine (for secondary amines) and aldehydes 45,65. This reaction is coupled with a reduction of molecular oxygen in the

FAD cofactor into hydrogen peroxides that may contribute to the pathogenesis of

Parkinson’ disease and other neurodegenerative diseases44.

Degradation of neurotransmitters by MAO results in regulation of their level in the central nervous system. Figure (1) show the simplified oxidative deamination reaction catalyzed by MAO enzymes44.

Table (2): MAO enzyme inhibitors: their different selectivity, reversibility and therapeutic uses44,45.

Type of inhibitor MAO inhibition reversibility Therapeutic uses

Non-selective MAO inhibitors

Phenelzine (FDA) Irreversible MAO inhibition. Anxiety/ Depression

Tranylcypromine (FDA) Irreversible MAO inhibition. Anxiety/ Depression

Iproniazid (FDA) MAO Irreversible inhibition Depression

Ladostigil MAO Irreversible inhibition in Depression, parkinsonism

the brain only. and Alzheimer.

MAO-B inhibitors

Selegiline (L-deprenyl) Irreversible MAO-B inhibition. Parkinsonism

(FDA)

Rasagiline (FDA) Irreversible MAO-B inhibition. Parkinsonism

Lazabemide (FDA) Reversible MAO-B inhibition. Antiparkinsonian in

clinical trials.

Safinamide (FDA) Reversible MAO-B inhibition. Antiparkinsonian in

clinical trials.

Zonisamide (FDA) Reversible MAO-B inhibition. Antiepileptic and

antiparkinsonian in Japan.

MAO-A inhibitors

Meclobemide(FDA) Reversible MAO-A inhibition. Not approved in US.

Clorgyline Irreversible MAO-A inhibition. Depression

Figure (1): Simple oxidative deamination reaction catalyzed by MAO enzymes. The figure is

44 adapted from Binda et.al. and modified by Marvin Sketch software v.6.2. MAO substrate (R1-NHR2)

is deaminated into ammonia (NH3) and aldehyde (CHO) through imine formation (not shown). R1 is

aromatic moiety. R2 is hydrogen or methyl group.

There are two isoforms of MAO enzymes in mammals; MAO-A and MAO-B that are encoded by different genes65,66. They are differentiated by their different sensitivity to the MAO inhibitors Clorgyline and L-deprenyl67 where MAO-A is inhibited by very low concentration (in nanomolar) of Clorgyline and MAO-B is inhibited by very low concentration (in nanomolar) of L-deprenyl67,68. MAO-A preferentially metabolizes serotonin, adrenaline and noradrenaline; whereas MAO-B preferentially metabolizes phenylethylamine and benzylamine65 with an overlapping metabolizing activity of both enzymes on adrenalin and dopamine69. The high affinity of MAO-B isozyme for phenylethylamine is thought to contribute in the ability of the MAO-B in degradation of exogenous amines to prevent their possible function as false neurotransmitters69. MAO-A and MAO-B have approximately 70% of sequence identity (amino acid identity) and substrate specificity as shown in figure (2)44.

Figure (2): MAO enzymes specifities for aromatic amine substrates. 3,4-methylenedioxymeth-

amphetamine and 1-methyl-4-phenyl-1,2,3,6-tetreahydropyridine are exogenous amines. The figure is

adapted from Binda et. al.44 and modified by Marvin sketch v.6.2.

In humans both MAO isoforms are found in all tissues except blood platelets that only contain MAO-B enzyme70. In regards to their distribution in the periphery; MAO-A is predominantly (highest level) found in the liver and placenta and partly (lowest level) in the spleen70 in addition to the lungs and small intestine while MAO-B are mainly found in the blood platelets71. In the central nervous system; Both MAO enzymes are found in the neurons and astroglial cells. MAO-A distribution in the C.N.S. is slightly different from that of MAO-B where MAO-A is mainly found at the locus coerulus whereas MAO-B is mainly found in the dorsal raphi nuclei72-74.

Distribution of MAO enzymes in the C.N.S and in the periphery reflect their primary functions75. In the liver, gastrointestinal tract and blood circulation, the MAO enzymes degrade the exogenous amines to regulate their level that may affect circulatory system76. MAO-B localization at dopaminergic neurons support its involvement in dopamine metabolism; whereas MAO-A localization at the adrenergic, noradrenergic and serotonergic neurons support its involvement in serotonin and adrenaline metabolism that are involved in stress and mood behavior44. Several studies have shown that inactive

MAO-A enzymes due to mutations result in an aggressive behavior in male mice under stress77.

Most of amine neurotransmitters such as noradrenalin, adrenalin, serotonin and dopamine are stored in synaptic vesicles, thus they are protected from the deamination effect of MAO78. When these neurotransmitters released into the synaptic cleft; most of them will bind to their receptors on the postsynaptic neurons to provoke a stimuli. Only small amount of these neurotransmitters that are not bound or been reuptake are free out of the synaptic cleft and these are subject to metabolic action of MAOs and other enzymes such as COMTs and aldehyde dehydrogenases78.

By inhibition of MOA enzymes catalytic activity on neurotransmitters such as dopamine, serotonin, norepinephrine, epinephrine and other aromatic neurotransmitters and thus increasing their level at the synaptic cleft in the central nervous system; MOA inhibitors are widely used in treatment of Parkinson’s disease and depression. Non- selective and selective MAO-A inhibitors are approved to have antidepressant effect that result from their ability to increase serotonin and norepinephrine concentration at the

synaptic clefts through inactivation of their degradation by MAO enzymes. Reduced level of serotonin and norepinephrine are considered as the characteristics of depression79.

MAO inhibitors are associated with undesirable and potentially fatal adverse effect called hypertensive crisis (also named cheese reaction). Hypertensive crisis occur in those patients on non-selective MOA inhibitors after consumption of tyramine containing foods such as cheese and/or wine. Tyramine is absorbed into blood circulation due to lack of MAO-A (and to lesser extent lack of MAO-B)79. Tyramines induce norepinephrine release in the medulla that in the absence of MAO-B leads to greatly activate the sympathetic system that causes a sudden increase in the blood pressure79.

Selective MAO-B inhibitors do not cause hypertensive crises80,81. Hydrazine containing

MAO inhibitors are associated with liver toxicity which is the main reason of this drug withdrawal45.

1.3. Structures of MAO enzymes:

MAO enzymes are mitochondrial enzymes linked through their C-termini to the outer mitochondrial membrane82. Both MAO enzymes are covalently linked to FAD cofactor by thioether linkage between the Cysteinyl residue of the MAOs (cysteine 397 in

MAO-B and cysteine 406 in MAO-A) and 8α-methylene of the flavin ring (isoalloxazine ring)82. The FAD covalent linkage is directed towards the C–terminal of MAO enzymes and it is essential for their catalytic activity83 although another studies have shown that the mutant MAO-A with Cys406Ala can function with noncovalent FAD linkage84.

MAO-A crystallizes as monomer while MAO-B crystallizes as dimer as shown in figure

385. The surface contact between the MAO-B monomers constitutes about 15% of the total monomeric surface area85.

Figure 3: Three dimensional structures of both MAO-A as monomer and MAO-B enzyme as

dimer. For both MAO enzymes; The membrane binding domain (C-terminal) is shown in green, FAD

binding domain is shown in blue, the substrate binding domain is shown in red and the FAD cofactor

is shown in yellow86,87. The MAO-B and MAO-A structures were obtained from protein data bank

(2BK3) and (2BSX) respectively and modified by Chimera software v.1.8.1.

1.4. Three dimensional structure of MAO-B:

MAO-B is a dimeric protein. Each globular monomer consists of 520 amino acids residues and embedded in the outer mitochondrial membrane by hydrophobic helix (C- terminal) of (489-500) residues69,83. Each monomer consists of three structural domains:

(i) Membrane binding domain (residues 489-515), (ii) FAD binding domain (residues 4-

79, 211-285 and 391-453) and (iii) Substrate binding domain (residues 80-210, 286-390 and 454-488) that includes gating residues, entrance cavity and substrate cavity86.

1.4.1. Membrane binding domain:

Each monomer of the dimeric MAO-B structure has a C-terminus (residues 461-

520) that extend towards mitochondria to be inserted in the outer mitochondrial membrane88. Sequence analysis of MAO-B by Toppred14 predicted that C-terminal is a helix of 27 amino acids long (residues 489-515) with a hydrophobic surface that allow its insertion in to lipid bilayer of the mitochondrial membrane83. The membrane binding domain extends from a polypeptide chains (residues 461-488) followed by an α-helix that starts at Val489 to be embedded in the mitochondrial surface83. Figure (4) show three dimensional structure of MAO-B83.

MAO-B has other attachment sites to the outer mitochondrial membrane other than C-terminal. C-terminal truncation mutagenesis experiments have shown that deletion of the C-terminal will not completely detach the enzyme (MAOs) from its binding sites on mitochondria89. The other interaction sites of MAO-B to the outer mitochondrial membrane are; (i) Hydrophobic amino acid side chains (Phe481, Leu482, Leu486 and

Pro487) of the amino acid residues (481-488) that are oriented towards the outer mitochondrial membrane90, (ii) Loop 99-112 ends that are embedded in the outer mitochondrial membrane90 and (iii) Exposed hydrophobic surface of Pro109, Ile11090,

Trp15769 that are attached to the mitochondrial membrane. These structural data support

the suggestion that binding of MOA-B enzymes to the outer mitochondrial membrane is not confined to C-terminal only90.

Figure (4): Three dimensional structure of MAO-B enzyme. The membrane binding domain is

green, FAD binding domain is blue, Substrate binding domain is red, FAD cofactor is yellow and loop

99-112 is s black. The C-terminal of MAO-B is embedded in the outer mitochondrial membrane. The

structure of MAO-B was obtained from protein data bank (2BK3) and modified by Chimera software

v.1.8.1.83. The dimensions of the mitochondrial membrane contact are not exact.

The effects of the mitochondrial membrane interactions other than C-terminal on the catalytic activity of the enzyme are still unclear69. Loop 99-112 interactions with the

outer mitochondrial membrane may affect function of the loop in gating entrance cavity69. Hydrophobic interactions of amino acid residues (side chains and exposed surfaces) of MAO-B enzyme with the outer mitochondrial membrane near entrance cavity may play a role in attracting positively charged (protonated) amine substrates and subsequent increase in its concentration at the active site of the enzyme by this generated electrostatic interactions69.

1.4.2. FAD binding domain:

MAO-B (and MAO-A) are covalently bound to FAD as a cofactor by thioether linkage located between Cysteinyl residue (Cys397 in MAO-B and Cys406 in MAO-A) and 8α-methylene of the flavin ring (isoalloxazine ring)66 as shown in figure (5)82.

Figure (5): Covalent binding of MAO-B enzyme to isoalloxazine ring of FAD cofactor. This

figure is adapted from Edmondson et. al. 86.

The thioether linkage between MAO-B and flavin ring is close to the C-terminal although most flavoprotein enzymes have their FAD linkage close to the N-terminal. This structural observation suggest that MAO-B enzyme (and MAO-A) undergo extensive folding to allow this unique covalent FAD-protein linkage that appear to be essential for their catalytic activity86.

The cofactor FAD is incorporated in the hydrophobic region of MAO-B enzyme; linked either to the hydrophobic side chains of amino acids and/or to the peptide bonds of the proteins through H-bonds as illustrated in figure (6)86. There is an electrostatic interaction between positively guanidine group of Arg42 and the anionic pyrophosphate of FAD86,91. The pyrophosphate also linked by H-bond to a water molecule (not shown) and to a peptide bond of the Thr426 and Ser1586. FAD ribose ring is H-bonded to a water molecule, guanidino group of Arg36 and carboxyl group of Glu3486. Mutation of Glu34 residue in the dinucleotide binding motif of FAD-dependent enzymes by Asp, Gln, or Ala lead to more than 90% loss of catalytic activity92; thus the H-bond between FAD ribose ring and carboxyl group of Glu34 is absolutely essential in FAD binding to maintain the integrity of the covalent FAD structure in MAO-B enzyme86. The FAD adenine ring is H- bonded to the peptide bond of the Val235 and H-bonded to a water molecule (not shown)86. The 3`-OH of the FAD ribityl side chain is H-bonded to the carbonyl oxygen of

Gly434 (not shown)86.

Figure (6): 2D image illustrates incorporation of FAD in MAO-B enzyme. Green dashes are H- bonds, carbons are black, nitrogens are blue, oxygens are red, phosphates are purple and sulfur is yellow. Short red lines represent the electrostatic interactions. H-bonds between FAD and MAO-B enzyme with water molecules are not shown. The figure was obtained from protein data bank (1GOS) and modified by Ligplot software v.1.4.5.

The orientation of ribityl side chain toward substrate active site is essential for

MAO enzymatic catalysis where it enable amine substrate to identify the flavin ring86.

Isoalloxazine ring has numerous H-bonds between; 2-carbonyl oxygen of the pyrimidine ring, N-H of Met436 peptide bond and a water molecule, N3-H of isoalloxazine ring and carbonyl group of Tyr60, 4-carbonyl oxygen of isoalloxazine ring and N-H of Tyr60, 4- carbonyl oxygen of isoalloxazine ring and N-H of Ser59, 4-carbonyl oxygen of isoalloxazine ring and N(5)-H on the same ring and between a water molecule and N(1) of Lys296 (not shown)86. There are no positively charged amino acids located near N1 of the isoalloxazine ring (flavin ring) which is considered to be essential for its stabilization.

These interactions between flavin ring and MOA-B amino acid residues have steric effects that influence flavin reactivity and enzyme catalytic activity86. These steric effects are caused by: (i) energetically unfavorable cis-conformation of amide linkage between Cys397 and Tyr39893 rather than the energetically favorable trans-conformation.

This enables isoalloxazine ring to form an ‘’aromatic cage’’ by being perpendicular with the phenolic side chain of Tyr39893. ‘’Aromatic cage’’ play an important role in amine substrates oxidation catalyzed by MAO-B94. Figure (7) show the perpendicular orientation of the flavin ring with phenolic side chain of Tyr398. (ii) The reactive region of the flavin ring (including N5and C4a atoms) assumes SP3 configuration rather than

SP2. This is attributed to a deformation or ((puckering)) about the pyrimidine portion of the isoalloxazine ring. This puckering of pyrimidine ring allow the formation of flavin adduct easily at N5 and C4a positions93. This is observed with tranylcypromine and N-(2- aminoethyl)-P-chlorobenzamide that are reversible inhibitors of MAO enzymes93.

Figure (7): (Top view) Three dimensional demonstration of phenolic side chain of Tyr398 that is

perpendicular on the flavin ring of MAO-B enzyme. The nitrogen is in blue, oxygen is red and

sulfur is yellow. The structure of MAO-B was obtained from protein data bank (2BK3) and the figure

is adapted from Edmondson et. al.86 and modified by Chimera software v.1.8.1.

The covalent thioether linkage is absolutely required for the catalytic activity of

FAD-dependent MAO-B and MAO-A enzymes. Mutation studies illustrate that mutant

MAO-B enzymes (with Cys397Ser) and mutant MAO-A (with Cys406Ser) are inactive when Serine of Cys397 of MAO-B (or of Cys406 in MAO-A) cannot form a covalent thioether linkage with flavin (FAD) due to insufficient nucleophilicity of its hydroxyl group95. Another study has shown that the catalytic activity of MAO-A enzymes is not absolutely dependent on the FAD covalent linkage84 where mutant MAO-A (Cys406Ala) is synthesized as apoenzyme and folded into the original conformation and inserted into outer mitochondrial membrane with no catalytic activity detected. When FAD is added the catalytic activity can be detected and it depends on the concentration of the added

FAD that reaches to saturation level with Kd 62 ± 5nM84. The synthesized mutant MAO-

A is unstable to detergent solubilization in contrast to the covalently linked MAO-A

enzymes which support the prediction that covalent linkage is essential for stabilization of the MAO-A enzymes86.

Mechanism of covalent flavinylation (also called Quinone-methide mechanism) occurs as in the following steps96; (i) Loss of proton occur at 8α-position on the isoalloxazine ring (flavin ring) is followed by formation of quinone methide tautomer. (ii)

Nucleophilic attack of thiol groups (SH) of Cysteinyl residue of MAO-B enzyme on 8α- methide of the flavin. (iii) Oxidation of the reduced 8α-flavin adducts to produce covalently bound FAD as shown in figure (8)86.

Figure (8): Covalent flavinylation of MAO-B enzyme. The figure is adapted from Edmondson et. al.

86and modified by Marvin sketch v.6.2.

1.4.2.1. Role of aromatic cage in MAO-B catalysis:

FAD dependent MAO enzymes have aromatic amino acids residues (two phenyl rings of the tyrosyl residues Tyr398, Tyr435 of MAO-B and Tyr407, Tyr444 of MAO-A) located on the re face of the flavin ring (isoalloxazine ring) and oriented in perpendicular position to form the ‘’aromatic cage’’ in which the distance between these two phenyl rings is 7.8 Aº90 as shown in figure (9). Location and orientation of the ‘’aromatic cage’’ suggests its functional role in MAO enzymes catalytic reaction. The ‘’aromatic cage’’ exerts a steric effect upon orientation of the amine substrates at the substrate active site in

MAO enzyme near flavin ring that lead to provides a path for the oriented amine substrate to reach flavin active site.

Figure (9): Three dimensional representation of aromatic cage in MAO-B crystal structure (Top

view). The phenolic side chains of tyrosyl residues (Tyr398 and Tyr435) are perpendicular to the plane

of the flavin ring. Black line represents the center of the aromatic cage. Nitrogen atom (N5) and (N10)

are blue, oxygen atoms are red and carbon atoms are green. MAO-B structure was obtained from

protein data bank (2BK3) and the figure was adapted from Li et. al.97 and modified by Chimera

software v.1.8.1.

‘’Aromatic cage’’ also increases amine substrate (N) nucleophilicity to promote flavin adduct formation97 where it enhance nucleophilic attack of (N) atom of amine substrate (in deprotonated form) on C4a of the isoalloxazine ring to generate a flavin adducts. This nucleophilic attack lead to generate a basic (N5) of the flavin ring that subsequently abstract (H) from the amine substrate to form an imine98,97 as in figure (10).

Figure (10): Proposed Polar Nucleophilic Mechanism for the Reductive Phase of Monoamine

Oxidase A and B Catalysis. This figure is adapted from Miller et. al.98 and modified by Marvin Sketch

software v.6.2.

The conformation of the flavin ring is bent (30º bent from planarity) when it is in reduced form and planar in its oxidized form (ground state form)93 as shown in figure

(11). This bending makes (N5) of flavin ring more nucleophile and (C4a) more electrophile (better target for nucleophilic attack)99; thus formation of flavin adduct at

(C4a) will generate strong base at (N5) that withdraw proton from the substrate easily97.

Figure (11); three dimensional representation of bent form of reduced FAD flavin ring of MAO-B

crystal structure. The MAO-B structure was obtained from protein data bank (2BK3) and the figure is

created by using Chimera v.18.1.

The space (distance of 7.8 Aº) between phenyl rings of the Tyr398 and Tyr435 at the ‘’aromatic cage’’ of MAO-B enzyme provides an appropriate environment to polarize amine substrate lone pair to increase substrate (N) nucleophilicity required for polar nucleophilic attack on (C4a) of the flavin ring and to increase the number of the activated molecules that are ready for enzymatic catalysis97. Figure (12)97 show the space between the phenyl rings that required for polarization of the amino substrates required for nucleophilic attack at (C4a) of flavin ring. The dipolar effect exerted by the phenyl rings of MAO-B residues Tyr398 and Tyr435 may play a role in MAO-B substrate specificities where it may affect the orientation of amine substrate at the substrate binding site in a

manner that decrease the distance between (N) of amine substrate and (C4a) of the flavin ring which in required for the nucleophilic attack to form the flavin adduct in MAO-B catalysis97. This dipolar effect of the two phenyl rings in the aromatic cage (figure (11)) is similar to the effect of the electron withdrawal group at para- position of the amino substrate97.

Figure (12): simplified demonstration of proposed role of the aromatic cage in amine oxidation.

This figure is adapted from Li et. al.97.

These structural data may explain why p-nitrobenzylamine is weak substrate for

MAO-B although its analogue p-nitrophenethylamine is strong substrate that is shown in figure (13). The substrate affinity may depend on the orientation of the amine substrate in substrate binding site that may be determined by the ‘’aromatic cage’’97. The short side chain of p-nitrobenzylamine (one methyl group) is not sufficient to decrease the distance between (N) of the amine substrate and (C4a) of the flavin ring that is necessary for the

(N) nucleophilic attack; So that (N) of the amine substrate is not close enough to the

(C4a) of the flavin ring to perform polar nucleophilic attack required for MAO-B catalysis97. Addition of another methyl group to the side chain as in its analogue (p- nitrophenethylamine) elongate side chain to enable the nucleophile (N) of amine substrate to be close enough to (C4a) of the flavin ring to perform polar nucleophilic attack required for MAO-B catalysis97. These structural data also explain why p- nitrophenethylamine is MAO-B strong substrate even with mutant Tyr435 forms of

MAO-B97. The lost dipolar effect of the phenyl group of the tyrosyl residues (Tyr398 and

Tyr435) of the aromatic cage that is normally exist in wild type MAO-B is overcome by the strong electron withdrawal group (NO2) at para- position that achieve similar effect in polar nucleophilic mechanism of WT MAO-B97.

Figure (13): p-nitrobenzylamine and p-nitrophenethylamine structures are created by Marvin

Sketch software ver. 6.2.2.

1.4.3. Substrate binding domain:

The substrate binding domain involves the following components90: (i) Entrance cavity which is a hydrophobic cavity with small volume of 290Aº: this cavity is lined by

Phe103, Pro104, Trp119, Leu164, Leu167, Phe188, Leu171, Ile199, Ile316 and Tyr326,

(ii) Substrate cavity which is also hydrophobic cavity with large volume of 420Aº, and

(iii) Loop 99-112 (also called ‘’Gating switch’’) as shown on figure (14). The hydrophobic cavities (small entrance and large substrate cavity) (in MAO-B) are separated by four amino acids residues side chains (Tyr326, Ile199, Leu171 and Phe168) and shielded from the solvent by loop 99-112 ‘’gating switch’’ that is located at the protein surface90. The entrance and substrate cavities are lined by aromatic and aliphatic amino acids that provide a highly hydrophobic path that connect protein surface to the flavin active site83.

Loop 99-112 or ‘’gating switch’’ is a flexible loop located at the protein surface and control gating of the entrance cavity83. The ends of loop 99-112 are embedded in the outer mitochondrial membrane as shown in figure (14)90. The hydrophobic residues

(negatively charged) of the mitochondrial surface are thought to exert an electrostatic attraction on the protonated (positively charged) amine substrates83. Loop 99-112 must transiently move on the protein surface to allow substrate entry in to the entrance cavity then the four residues (especially Ile199 and Tyr326) that separate the two cavities are also displaced to allow substrate entry into the flavin active site90; therefore substrates move a distance of about 20Aº from the protein surface to reach the flavin active site83.

Figure (15)100 show Ile 199 and Tyro 326 geometry and position at the MAO-B active site.

Figure (14): Three dimensional images of MAO-B enzyme illustrate: loop 99-112 (gating switch),

gating residues (mainly Ile199 and Tyr326), entrance cavity and substrate cavity of MAO-B

structure. The membrane binding domain (C-terminal) is green, FAD binding domain is blue,

substrate binding domain is red, loop99-112 is black, Ile199 and Tyr326 in gating residues are cyan

and FAD cofactor is yellow. The MAO-B structure was obtained from protein data bank (2BK3) and

the figure is created by using Chimera v.18.1.

Figure (15): Three dimensional representation of the gating residues (mainly Ile199 and Tyr326)

in the MAO-B structure. The FAD binding domain is shown as blue, the substrate binding domain is

shown as red, the gating switch that gate entrance cavity is shown as black , Ile199 and Tyr326 are

shown as cyan and FAD is shown as yellow. The MAO-B structure was obtained from protein data

bank (2BK3), adapted from Milczek et. al. 100 and modified by Chimera v.1.8.1.

Ile199 residue of MAO-B (correspond to Phe208 in MAO-A) is one of the four residues that separate entrance cavity from substrate cavity. Ile199 residue side chain act as ‘’gating residue’’ that gate substrate cavity entrance83 and can adopt two conformations (‘’open’’ or ‘’closed’’) depending on the chemical nature and size of the substrate or inhibitor93. For example; Ile199 residue side chain adopt ‘’closed’’ conformation (in which the side chain gate substrate cavity) with Pargyline, Isatin and tranylcypromine that are small inhibitors of MAO-B enzyme as shown in figure (16a)

and adopt an ‘’open’’ conformation (normal resting conformation) with N-(2- aminoethyl)-p-chlorobezamide or 1,4-diphenyl-2-butene that are large inhibitors of

MAO-B enzymes83 as shown in figure (16b). These data support the suggestion that

Ile199 residue play an important role in identification or recognition of substrates in

MAO-B. There is a steric effect between Ile199 ‘’gating residue’’ and loop 99-

112 ‘’gating switch’’:

 When Ile199 side chain is rotating in an open conformation such as with large

inhibitor 1,4-diphenyl-2-butene the Phe103 side chain on loop 99-112 experience a

steric effect and closes the entrance cavity100.

 When Ile 199 side chain adopt ‘’closed’’ conformation (such as with small inhibitor

Isatin), Phe103 side chain on loop 99-112 experience no-steric effect and this result

in opening of the entrance cavity100. Phe 103 side chain on loop 99-112 ‘’gating

switch’’ and Ile 199 side chain ‘’gating residue’’ conformations are in interconverting

manner in a way that provide an access channel to the active site in ligand free

state100.

 When Mutation of Ile199 residue by Ala residue occurs; Phe103 side chain will adopt

open conformation that result in opening of the entrance cavity100.

In addition to Ile199 side chain role in substrate recognition for MAO-B; there is another gating residue which is Tyr326 that have a role in substrate or inhibitor recognition100,101. Tyr326 residue is a component of the hydrophobic residues that line substrate cavity near the junction between entrance and substrate cavity.

Figure (16): 3D representations of (A) ‘’close’’ and (B) ‘’open’’ conformation of Ile199 side chain in MAO-B complex with Isatin (small inhibitor) and 1,4-diphenyl-2-butene (large inhibitor) respectively. Isatin and 1,4-diphenyl-2-butene molecule are green, Tyr326 and Ile199 residues are red, loop99-112 is black and FAD is yellow. MOA-B complex with Isatin is (1OJA) and (1OJ9) with 1,4- diphenyl-2-butene were obtained from protein data bank and modified by Chimera software v.1.8.1.

In the ground state (ligand free enzyme) of MAO-B enzyme, the phenolic side chains of Tyr326 reduce steric effect of substrate cavity on Phe103 of loop99-112. This steric effect is reduced when large inhibitors occupy MAO-B substrate binding site98,102.

MAO-A active site (substrate binding site) is monopartite in which the entrance cavity is not separated from substrate cavity by amino acid residues while MAO-B active site can be dipartite in which the two cavities are separated by four amino acid residues

(mainly Ile199 and Tyr326) as shown in figure (17)85, or monopartite when large inhibitors are bound where they force Il199 side chain to adopt an open conformation.

When small MAO-B inhibitor such as Isatin binds within substrate cavity; the Ile199 side chain adopt ‘’closed’’ conformation (its side chain rotate into closed conformation) that create a dipartite active site100. When large substrate or MAO-B inhibitor such as

1,4-diphenyl-2-butene bind within the substrate binding site; the Ile199 side chain adopt an ‘’open’’ conformation that result in fusion of the entrance (290Aº) and substrate cavity (420Aº) to create a monopartite active site. The fusion of the two cavities results in one large cavity with 700Aº100.

Several mutational studies support the role of Ile199 in MAO-B substrates or inhibitors recognition by MAO-B active site103. Mutant MAO-B with replacement of Ile

199 and Tyr326 side chains (isopropyl and phenolic side chain respectively) with methyl group of alanyl residue show no major effect on the enzyme structure or the conformation of other residues in the active site of mutant MAO-B compared to WT

MAO-B but these mutation can change the dipartite cavity of MAO-B in to monopartite cavity with volume of 732Å100.

Figure (17): Comparison of MAO-A monopartite (top) and MAO-B dipartite (bottom) active site cavities. The structures are obtained from protein data bank (2BK3) for MAO-B and (1OJA) for MAO-

A. The FAD is shown as yellow and gating residues (Ile199 and Tyr326 in MAO-B and corresponding

Phe208 and Ile335 in MAO-A) are shown as red, the loop 99-112 is shown in black. This figure was adapted from Edmondson et. al. 85 and modified by Chimera software v.1.8.1.

This causes shift of Phe103 by about 1Å in the direction of the Ile 199 side chain100 So that the Phe103 will have a conformation similar to ‘’closed’’ conformation of Ile 199 that is adopted when small inhibitors are bound93. In other study in which mutant MAO-B with mutation of Ile 199 to Ala; Ile199 to adopt permanent

‘’open’’ conformation lead to decrease small MAO-B inhibitors (such as Isatin) binding affinity to 3-4 folds that normally favor ‘’closed’’ conformation and increase large MAO-

B inhibitors binding affinity to 24 folds that favor ‘’open’’ conformation in contrast to that of wild type MAO-B enzymes100. Double mutant MAO-A (mutation to Ile199Ala and Tyr326Ala) decrease inhibitor binding affinity of small inhibitors such as Isatin100.

Double mutant MAO-B (mutation to Ile199Ala and Tyr326Ala) decrease by 10 folds or abolish small MAO-B inhibitor binding affinity because the double mutant MAO-B will have a permanent monopartite cavity and small inhibitors such as Isatin favor the dipartite cavity100.

1.5. Structural comparison of hMAO-A, hMAO-B and rMAO-A

enzymes:

Studying structural differences between MAO enzymes provides a molecular basis to design new specific (selective), reversible or irreversible inhibitors for each enzyme. HMAO-A have sequence identity of about 70% with hMAO-B and 92% with rMAO-A. There are important structural differences and similarities between them that may or may not affect their catalytic activity and substrates specifity.

1.5.1. Oligomeric state of enzymes:

The important structural difference among MAO enzymes is that; h MAO-B and rMAO-A are dimers, while h MAO-A is monomer. The surface contact between two monomers in the dimeric structure of hMAO-B and rMAO-A is 15% of the total monomeric surface85. A survey104 have been shown that all MAO-enzyme exist in oligomeric states mainly as dimers in their mitochondrial bound forms but modeling and mutagenic studies105 have shown that hMAO-A existence in monomeric state is attributed to the selective mutation of hMAO-A Glu151 to Lys151 at the site near the protein surface and close to the charged amino acids residue that are involved in the monomer- monomer contact. Lys151 residue may affect this contact resulting in disruption of the dimeric interface. Other MAOs (rMAO-A and hMAO-B) have highly conserved amino acid residue including glutyl residues at the dimeric contact; therefore, they retain their dimeric forms106.

Recently published pulsed EPR data (a special technique that is used to know whether the crystallographic structure of a protein determined by X-crystallography is similar to the structure of the same protein in its membrane bound form); have shown that all MAO enzymes (rMAO-A, hMAO-A and hMAO-B) are existed in their dimeric form when they are bound to mitochondrial membrane independent on the glutyl residues replacement107. The pulsed EPR data for detergent solubilized forms of MAO enzymes show that both hMAO-B and rMAO-A are in their dimeric from and about 50% of hMAO-A exist in dimeric form108. The monomeric hMAO-A are more likely to crystalize

than its dimeric form while the dimeric form of rMAO-A are more likely to crystallize than its monomeric form106.

1.5.2. Membrane binding domain structural differences:

Both MAO enzymes are mitochondrial enzymes anchored to the outer mitochondrial membrane by their C-terminal. Several studies109,110 suggest that chimeric enzyme that is built from fragment of MAO-A and MAO-B that have ‘’swaps’’106 of their

C-terminals are inactive, this suggest that there are differences in the membrane binding domain between two enzymes106.

1.5.3. FAD binding domain structural differences:

FAD binding sites are highly conserved106. FAD cofactor structure is identical in both MAO-A and MAO-B enzymes87. Since FAD active sites are identical, thus mechanisms of oxidative deamination in both isozymes are similar too111. Flavin ring is bent (30º from planarity) in all MAO-enzyme (hMAO-A, hMAO-B and rMAO-A) and ribityl side chain of the flavin ring is extended106. FAD covalent thioether linkage is located between thiol group of MAO enzymes and Cys396 of hMAO-B and Cys406 of hMAO-A106. The ‘’aromatic cage’’ residues Tyr398, Tyr435 residues of MAO-B and

Tyr407, Tyr444 in MAO-A are identical in both hMAO-A and hMAO-B enzymes.

1.5.4. Substrate binding domains structural differences:

The substrate binding domain cavities are hydrophobic in all MAO enzymes106.

The MOA-B cavity may be a large single cavity with volume 700Å3 when large inhibitors are bound (such in deprenyl adduct of MAO-B) or bipatric cavity when small inhibitors are bound (such as Isatin adduct of MAO-B) in which entrance cavity (290

Aº3) is separated from the substrate cavity (420Å)85,87. These different conformations are dependent on the size and chemical nature of the substrate or MAO-B inhibitors85,87. In contrast, hMAO-A and rMAO-A have monopartic cavity with volume of 550 Aº3 (for

Clorgyline adduct of hMAO-A) and 450 Aº3 respectively85,87.

The entrance cavity in all MAO enzymes is shielded from solvent movement by loop 99-112 ‘’gating switch’’ and substrate cavity is gated by Ile199 residues ‘’gating residues’’85,87,100. Phe208 in MAO-A corresponds to the Ile 199 ‘’gating residue’’ in hMAO-B100,106 and Tyr326 in hMAO-B corresponds to Ile335 in hMAO-A that occupy the same position. Ile 199 and Tyr326 in MAO-B and corresponding Phe 208 and Ile 335 in MAO-A significantly affect the shape of substrate cavities and determine each enzyme substrate specifity87,100,106. For example: Deprenyl (selective MAO-B inhibitor) will collide with Phe208 in MAO-A to exert its inhibitory action. Similarly Clorgyline

(selective MAO-A inhibitor) will have to displace Tyr326 in MAO-B enzyme to inhibit the enzymatic activity87.

Non conserved residue Cys172, Leu171 in hMAO-B corresponds to Asn181 and

Ile180 in hMAO-A and they do not affect shape of cavities significantly106. The substrate active site cavity in hMAO-A is short and wide while those of MAO-B are long and

narrow. The cavity shaping loop (210-216 residues) in hMAO-A is different from that of all other MOA enzyme (hMAO-B and rMAO-A). The cavity shaping loop of rMAO-A is more similar to that of hMAO-B rather than that of hMAO-A106 and thus rMAO-A can oxidize MAO-B substrates more efficiently than MAO-A substrates112. The shape and size of substrate binding site of MAO-A differs from that of MAO-B in87: (i) (7) of (20) amino acids of the substrates binding sites are different between MAO enzymes. (ii)

Cavity shaping loop (210-216 residues) adopts different conformations in hMAO-A that result in movement of Cα of flavin ring to a distance of 6 Aº which confer the hMAO-A enzyme its extended form.

In general, MAO-A enzymes (hMAO-A and rMAO-A) are more flexible than

MAO-B in that; the cavity shaping loop (210-216 residues) adopts different conformations when substrates or inhibitor are bound. For example; Clorgyline binding to hMAO-A causes hMAO-A substrate active site to adopt an extended helical conformation106 and it’s binding to rMAO-A causes the rMAO-A to adopt a folded conformation106. The cavity shaping loop (210-216 residues) in hMAO-A make Glu216 residues in the substrate binding site in direct contact with clorgyline and push Glu216 out of the substrate active site87. In rMAO-A the cavity shaping loop brings the Glu215 in contact with clorgyline and push Glu216 out of the substrate active site. This make clorgyline binds the MAO-A in extended conformation and MAO-A in folded conformation87.

In hMAO-A, there are two Cysteinyl residues (Cys321 and Cys323) are located close to the entry of the active catalytic site87. In Clorgyline adduct of hMAO-A; the side

chains of these residues are in contact with inhibitor aromatic ring87 but mutational studies95 have shown that mutations of these Cysteinyl residues of hMAO-A do not affect their catalytic activity.

1.6. Neuroprotective effect of MAO-B inhibitors:

A MAO-B level increase with age by 4-5 folds and it is considered as the main

MAO enzyme in the substantia nigra. The products of oxidative deamination reactions catalyzed by MAO-B in brain may contribute in neuronal cell degeneration45. Dopamine

(amine neurotransmitter abundant at the dopaminergic neurons of the substantia nigra) is deaminated in the substantia nigra by MAOs (mainly MAO-B) into H2O2 and 3,4 dihydroxyphenylacetylaldehyde (DOPAL) that is further oxidized by aldehyde dehydrogenases into 3,4 dihydr-oxyphenylacetic acid (DOPAC) then DOPAC is methylated by COMT into Homovanillic acid as in figure (18)113,114. Thus, oxidative deamination of dopamine in the substantia nigra by MAOs result in production of H2O2 that in the presence of ferrous (Fe+2) is reduced in to hydroxyl radicals (•OH) through

Fenton reaction115. The hydroxyl radicals is the most damaging radical for almost all cells115. DOPAL have been shown to be involved in the alpha-synuclein aggregation which is the main pathologic feature of Parkinson’s disease116.

MAO- B inhibitors play a neuroprotective role in preventing neuronal cell death induced by MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)117. MPTP can causes irreversible parkinsonian symptoms attributed to its neurotoxic metabolite MPP+ (N- methyl-4-phenylpyrid-inium) which has selective neurotoxicity towards dopaminergic

neurons of pars compacta of substantia nigra. Several studies have demonstrated that

MAO enzymes (mainly MAO-B) are responsible for this selective neuronal death. MPTP is not neurotoxic by its self but it is lipophilic compound that cross the blood brain barrier117.

Figure 18: Metabolic pathway of dopamine with it’s the precursors and metabolites. COMT

reactions occur in the postsynaptic neurons and glial cells whereas those mediated by MAO occur in

pre-, postsynaptic neurons and glial cells. The figure is adapted from Finberg et. al. 114 and modified

by Marvin Sketch software v.6.2.

In the brain, MAO enzymes (mainly MAO-B) catalyze C6 (allylic) α-carbon oxidation of MPTP into MPDP+ (2,3-dihydropyrid-inium) which is an intermediate species that undergo further oxidation into MPP+117,118 as shown in figure (19). MPP+ selectively kill dopaminergic neurons of pars compacta of substantia nigra by inhibiting

or inactivating the complex I of the mitochondrial electron transport chains which is essential for oxidative phosphorylation and ATP production. Depletion of ATP and generation of free radicals lead to neuronal cell destruction117,118. MOA-B inhibitors such as Selegiline inhibit the action of MAO-B enzymes on MPTP substrates and thus protect dopaminergic neurons from the damaging effects of its metabolites (MPP+)117,118.

Figure (19): Metabolic action of MAO-B on MPTP. This figure is adapted from Kalgutkar et. al. 71

and modified by Marvin Sketch v.1.6.3.

1.7. Oxidative deamination reactions of MAO enzymes:

MAO-A and MAO-B are mitochondrial enzymes that catalyze oxidative deamination of primary, secondary, and partially tertiary amine substrates that include neurotransm-itters and exogenous amines106. Oxidative deamination is a redox reaction that involves: (i) Reductive half reaction; in which oxidized flavin cofactor coupled to

MAOs is reduced by two electrons derived from Cα-H cleavage of the amine substrate as

shown in figure (20). (ii) Oxidative half reaction; in which the reduced flavin linked to

MAOs is oxidized using O2 as electron acceptor to generate H2O2, imine and oxidized flavin cofactor. Imine subsequently hydrolyzed by aldehyde dehydrogenase to produce

106 ammonia (NH4) and aldehyde .

Figure (20): Oxidation-reduction reaction of flavin ring of FAD cofactor of MAO enzymes. The

figure is adapted from Binda et. al. 93 and modified by Marvin Sketch software v.6.2. Oxidation

involves addition of 2 electrons (2H).

1.7.1. Reductive half reaction:

In which the deprotonated amine substrates bind to the active site on the of MAOs enzymes to be oxidized into protonated imine (N==H)106 using FAD as an oxidizing agent. The reductive half reaction involves a cleavage of (Cα—H) of the amine substrate and transfer of two reducing equivalents (any species that carry an electron such as H) to the isoalloxazine ring to form protonated imine and reduced form of the flavin (flavin hydroquinone)119. Figure (21) show the proposed mechanism of MAOs catalysis based on analysis of para-substituted benzylamine binding to MAO-A98 and on analysis of para-

substituted dimethylbenzylamine or para-substituted α-methyl benzylamine analogs to

MAO-B120.

Figure (21): Oxidative deamination mechanism mediated by MAO enzymes. Both MAO enzymes

follow the same reductive half reaction with all amine substrates and lower loop of oxidative half

reaction for most amine substrates except phenylethylamine for which MAO-B follow the upper loop

of oxidative half reaction. The figure is modified and adapted from Edmondson et. al. 106.

Both MAOs enzymes follow the same reductive half reaction with all substrates in which all amine substrates react with the active site of the oxidized isoalloxazine

(flavin) ring to generate protonated imine and reduced form of flavin ring FADH2 (flavin hydroquinones)106. For the oxidative half reaction; for most amine substrates, MAOs follow the lower loop of the oxidative reaction shown in figure (21) except for phenethylamine in which MAO-B follow the upper loop. In oxidative half reaction; the

reduced flavin is oxidized by O2 to yield oxidized flavin—imine adduct and H2O2. Imine will be released from the enzyme adduct and subsequently hydrolyzed by aldehyde dehydrogenase into ammonia (NH4) and aldehyde106.

1.7.1.1. Proposed mechanisms of Cα—H cleavage in reductive half reaction:

There are three possible mechanisms for Cα—H cleavage in amine substrates in the reductive half reaction106,119:

1. Heterolytic hydride transfer:

In which hydrogen is abstracted from amine substrates as hydride ion (ion that carry two electrons) to its acceptor (N5 of the flavin ring) to produce reduced flavin

(FADH2) and imine substrate106,119 as shown in figure (22)106.

Figure (22): Proposed hydride mechanism of C-H Bond cleavage Reactions in Flavoenzymes. The

figure is adapted from Edmondson et. al. 106 and modified by Marvin Sketch software v.6.2.

2. Heterolytic proton (H+) abstraction (single electron transfer SET):

This mechanism is based on the following statement: the PKa of Cα—H proton of amine substrate is acidic that require very strong base to abstract proton from it. There are no strong basic amino acid residues in the MAOs that can abstract proton from Cα—H bond. Thus amine substrates must be first oxidized by a single electron to amine radical cations that increase C—H liability (lower PKa) to donate proton for abstraction106 121 as shown in figure (23)106. There are several evidences against this single electron transfer mechanism of Cα—H bond. The most important evidence against this mechanism122 has shown that; oxidation potential of amine into amine cation radical is (1.5+V) while the redox potential of FAD is (0.004V) which is too low to oxidize the amine in to amine cation radical. Other studies have shown that there are no experiments proved that both

MAO enzymes follow proton abstraction mechanism and no experiment detects the flavin radical intermediate formation98,102,123. Other evidence against this mechanism is that ; magnetic field has no effect on the rate of enzyme reduction124.

Figure (23): Proposed heterolytic proton abstraction (single electron transfer) of C—H cleavage.

The figure is adapted from Edmondson et. al.106 and modified by Marvin Sketch v.6.2.

3. Homolytic hydrogen (H•) abstraction:

This involve homolytic cleavage of (C—H) bond that result in formation of substrate radical and hydrogen atom (radical with one electron) on the (N5) of flavin ring106,119. Figure (24) show the proposed mechanism of homolytic hydrogen abstraction of (C—H) cleavage. Bissel et. al. studies125 do not support that MAOs oxidative deamination is initiated by homolytic single electron transfer mechanism.

Figure (24): Proposed mechanism of homolytic hydrogen abstraction of (C—H) cleavage. The

figure is adapted from Edmondson et. al.106 and modified by Marvin Sketch software v.6.2.

The Cα—H cleavage of the reductive half reaction is considered to be the rate limiting step in MAOs catalysis98,102. For hMAO-A; para-substituted analogs are used to illustrate the effect of electron donating or electron withdrawal substituent power on the rate of the reduction reaction MAO-A119 and it has been found that; (i) If that rate limiting step ‘’Cα—H cleavage’’ follow hydride ion transfer mechanism, Thus the rate of reduction reaction of hMAO-A catalysis will increase with decreasing electron

withdrawal power of substituent at para- position of the amine substrate102,119. (ii) If

(Cα—H ) cleavage occur by proton abstraction reaction ,so the rate of reaction of hMAO-

A catalysis will increase with increasing the electron withdrawal power of para- substituted group of amine substrates98. (iii) If (Cα—H) cleavage follow homolytic hydrogen abstraction, so there is a little effect of electron withdrawal power on the rate of reduction reaction102.

The wide catalytic site of MAO-A allow aromatic ring of substituted benzylamine to interact with the flavin active site in different conformation required for transmission of electronic effect from the para-substituted group to the benzyl carbon where Cα—H cleavage occur85 whereas the narrow catalytic site of MAO-B reduce number of conformations required for the bound benzyl ring and inhibit the electronic effect exerted by para-substituted group on (Cα—H) cleavage85. Analyses of MAO-B oxidative deamination have shown that rate of reaction reduction depend mainly on steric effect with no detectable influence of para-substitution group102.

1.7.1.2. Polar nucleophilic mechanism:

Most investigators accept the heterolytic proton (H) abstraction mechanism for

(Cα—H) cleavage. Since MAO enzyme have no very basic amino acid residues to abstract proton from (Cα—H); therefor, isoalloxazine ring must be activated to be a strong base that is able to abstract a proton from the amine substrates106. The bent conformation of the resting form (oxidized) isoalloxazine ring (~30 bent from planarity) causes C4a to be more electrophilic (low electron density) and N5 more nucleophilic

(high electron density)126. The dipole-dipole interaction of the ‘’aromatic cage’’ will result in disruption of lone pair of electrons of (N) of amine that increase the (N) nucleophilicity. Both bent conformation of isoalloxazine and dipole-dipole interaction of the ‘’aromatic cage’’ will facilitate the polar nucleophilic attack of the N of substrate on the C4a of the flavin ring resulting in flavin adduct formation106. Figure (25) show the proposed polar nucleophilic mechanism of reductive half reaction of oxidative deamination catalyzed by MAO enzymes.

Figure (25): Suggested polar nucleophilic mechanism of the reductive half reaction in MAO

enzymes oxidative deamination reaction. The figure is adapted from G.A. Hamilton et. al.127 and

modified by Marvin Sketch v.6.2.

Recent studies have been shown that reduced flavin ring is very basic and this is estimated from the PKa of (N5) proton where it is equal to ~24128, so that strong base

(N5) of flavin ring will abstract a proton from (Cα—H) of amine substrate to form the flavin adduct.

1.7.2. Oxidative half reaction:

In which reduced flavin ring is oxidized by O2 to produce H2O2 and oxidized flavin as shown in oxidation reduction reaction in figure (20). MAO-B km for O2 is ~240

μM102 while those of MAO-A is ~12μM98, thus saturated concentration of O2 is required to operate MAO-A with less than half saturated required for MAO-B106. The reason still unknown, but if may attributed to that, O2 can bind MAO-B in both entrance and substrate cavity106.

Lys residue of (MAOs) is H-bonded through H2O molecule to (N5) of flavin ring.

This linkage was suggested to be involved the oxidative reaction of MAOs129. The reaction of triplet (O2) with singlet reduced flavin must involve single electron transfer to form flavin semiquinone and superoxide anion130. Then, two subsequent proposed mechanisms; the first involve formation of radical intermediate (C4a -flavin peroxide) that release H2O2 and oxidized flavin. The second reaction do not involve any intermediate formation, but involve reaction of superoxide with neutral flavin radical to

106 form H2O2 and oxidized flavin in (proton -electron coupled transfer step) .

1.8. Structural mechanism of MAO inhibition:

The substrate binding cavity is elongated in MAO-B and compact in MAO-A106.

Mutagenesis studies131 have shown that the conserved Gly110 is essential for entrance loop flexibility that should be transiently displaced to allow substrates entry and the cavity shaping loop (residues 210-216) in MAO-A play a key role in the substrate specifity more than that of MAO-B. The substrate cavities of MAO-B are hydrophobic with only small hydrophilic area near the re-face of the flavin ring93 that allow the entry of substrates with different sizes to reach the active site and form flavin adduct106.

Binding of different inhibitors (and substrates) to hMAO-B suggest its active site plasticity where inhibitors with aromatic rings, aliphatic ligands (e.g. trans, trans farnesol), oleamide, biocide (di(2-hydroxyethyl)methyldodecyl ammonium ion) has affinity to its active site132.

Both Small inhibitors such as tranylcypromine and Isatin and large inhibitors such as deprenyl (Selegiline)87 and Rasagiline133, trans,trans-farnesol and safinamide can bind to MAO-B active site with different forms depending on the gating residues (Ile199) conformations134. Binding of large inhibitors such as deprenyl (Selegiline)87 and

Rasagiline133 rotate the Ile199 side chains into an open conformation that result in large monopartite (single) cavity with volume of 700 A while small inhibitors binding to the

MAO-B active site rotate the Ile199 side chain into closed conformation that result in bipartite cavity of entrance cavity with volume of 290 A and substrate cavity of 420 A.

This MAO-B cavity plasticity has important pharmacological applications106 where small

inhibitors have similar affinity to both MAO-A and MAO-B enzymes while large inhibitors are highly selective to MAO-B enzymes.

Rasagiline is highly specific for MAO-B even it only occupies half of the entire cavity of MAO-B106. There is an evidence demonstrates that: the loss of MOA-B specifity for Rasagiline (N-propargyl-1(R)-aminoindan) result from methylation of its propargyline side chain that usually recognized by the flavin moiety on the enzyme135.

Figure (26) shows Rasagiline, Rasagiline-flavin adduct and methyl derivative of

Rasagiline.

Figure (26): Rasagiline, methyl derivative of Rasagiline and Rasagiline-flavin adduct. These

chemical structures are adapted from Binda et. al.135 and modified by Marvin Sketch software v.6.2.

Mofegiline is powerful, selective and irreversible MAO-B inhibitor that is used for study of covalent inhibition of MAO-B enzymes136. Mofegiline forms covalent flavin adduct with MAO-B enzyme and associated with non-covalent inhibition of MAO-A136.

Figure (27): Suggested Mechanism for irreversible Mofegiline Inhibition of Human MAO-B

enzyme. The figure is adapted from Milczek et. al.136 and modified by Marvin Sketch software v.6.2.

Mofegiline form covalent flavin adduct by binding of its vinyl (ethylene —

HC==CH2) side chain to the N5 of the flavin ring where the flavin ring of the MAO-B is alkylated by the vinyl side chain of Mofegiline137 with loss of the fluoride ion. Figure

(27) illustrates the mechanism of flavin adduct formation with Mofegiline136.

Mofegiline cannot irreversibly inhibit MAO-A (cannot form covalent flavin adduct) because the isoalloxazine ring of MAO-A enzyme is unable to cleave Cα—H bond of the extended arylalkylamine of amine inhibitors that is required to form reduced flavin to able to nucleophilically attack the vinyl side chain of the Mofegiline to form flavin adduct123.

All irreversible MAO inhibitors form covalent flavin adduct at the (N5) position of the flavin ring of MAO enzymes except tranylcypromine which is an irreversible

MAO inhibitor that form covalent flavin adduct at C4a position of flavin ring of MAO-B enzyme93. Structural and mechanistic studies have shown that; inhibition of MAOs are attributed to the flavin adduct formation at N5 position of the flavin ring137.

Figure (28) structure of tranylcypromine. This figure is created by Marvin Sketch software v.6.2.

Another structural data have shown that inhibition of other amino oxidase enzymes such as LSD1 by tranylcypromine (structure shown in figure (28)) is attributed

to the opening of the cyclopropyl ring of the inhibitor and formation of flavin adduct at both N5 and C4a positions of flavin ring138.

1.9. LSD1 enzyme:

Nucleosomes are the building blocks of chromatin molecules. Each nucleosome consists of segments of packaged DNA that are wrapped around eight core histone proteins (octamer) that include two copies of each of the following; H2A, H2B, H3, and

H4139. These histones proteins have amine termini (N) that protrude from the surrounding

DNA and subjected to different post-translational modifications such as methylation, ubiquitination, phosphorylation and acetylation. These modifications can affect several

DNA regulatory processes such as DNA replication, transcriptional activation and repression and repair processes139. Methylation and acetylation of histones may lead to inactivate tumor suppressor genes and thus lead to cancer progression139.

LSD1 is flavin dependent enzyme that belongs to amino oxidase superfamily. It is histone demethylase enzyme that selectively (with the aid of CoREST) demethylates or removes methyl groups from Lys4 of the histone 394,140. It is a component of several transcriptional co-repressor complexes such as CoREST (multidomain corepressor protein required for interaction of LSD1 with its target proteins) and HDAC (histone deacetylase enzyme that remove acetyl from Lys of histone) that repress DNA transcription141. LSD1 also can demethylate (without aid of CoREST) specific methyl of

Lys residues of P53 (tumor suppressor protein) that lead to control its activity (apoptosis inducer)142.

LSD1 uses its non-covalently bound FAD cofactor as an oxidizing agent to remove one or two methyl groups from Lys4 of Histone 3 oxidatively with production of formaldehyde (CHO) and H2O2 and consumption of O2 molecule that is required to oxidize one flavin molecule per one methyl removal cycle94,140. There are several lysine residues that can be mono- or di-methylated by LSD1 enzyme (H3K4, H3K9, H3K27,

H3K36, H3K79, and H4K20)113,143-145. Figure (29) show the oxidative demethylation reaction induced by LSD194,140. LSD1 require 15 amino acids residues to demethylate its substrates (Lys4 of histone 3)140,141.

Figure (29): Oxidative demethylation reaction of FAD-dependent LSD1 on Lys4 of histone 3.

First; histone containing substrate bind through its 20 amino acid side chain of Lys residue into to the

FAD as oxidizing agent that oxidize the Lys4 side chain with reduction of O2 into H2O2. The produced

imine intermediate then is hydrolyzed to produce demethylated histone 3 and formaldehyde. The figure

is adapted from Huang et. al.146 and modified by Marvin Sketch v.6.2.

Inhibition of LSD1 has important therapeutic implications in cancers and neurodegenerative diseases where its inhibition may reactivate gene expression that inactivated or silenced by LSD1 in those diseases146,147. LSD1 inhibition may inhibit other components of the transcriptional co-repressor complexes such as HDAC proteins.

Since the mechanism of oxidative cleavage and the active site of LSD1 and MAOs enzymes are identical, many MAO inhibitors are tested as LSD1 inhibitors147 where

Phenelzine (hydrazine (H2N==NH2) containing compound), pargyline, and propargyline have been established to have an inhibitory effect on LSD1 enzyme141,147.

1.9.1. Three dimensional LSD1 structure:

The asymmetric structure of LSD1 consists of three domains: (i) Swirm domain

(residues 172-270) that consists of six α helix arranged as SWα4 in the center surrounded by five helixes SWα1, SWα2, SWα3, SWα5, and SWα6. There are two β-sheets between

SW4, SW5 and C-terminal of the Swirm domain that play roles in the hydrophobic interaction of Swirm domain with the amino oxidase like domain148. They contribute in establishing LSD1 stability but their function is not established yet. Swirm domain may act as a binding site for histone 3 N-terminal required for demethylation of its Lys4148.

(ii) Amino oxidase like domain consists of two subdomains; FAD- binding subdomain

(residues 271-365, 559-657 and 770-833) and substrate binding subdomain (residues

357-417, 523-558 and 658-769). These two subdomains create large catalytic center at their interior with volume of 1245Aº148. The distance from the protein surface in to flavin active site is 23A which is different from that of MAOs that is 20A148. One side of the

catalytic center is flat and mainly composed of highly conserved hydrophobic residues while the other side is mainly composed of conserved acidic residues. Both sides are essential for LSD1 catalytic activity. There are two highly acidic α-helix residues (Sα1 and Sα3) at entrance of the catalytic center that act as a binding site for basic histone 3 tail148. (iii) Tower domain (residues 418-522) protrudes from the catalytic center of the amino oxidase like domain without any interaction with other LSD1 domains. Mutation studies have shown that it is essential for LSD1 demethylation activity in addition to its role in LSD1-CoREST corepressor complex in which it act as a binding site for the

CoREST protein in the corepressor complex. It may contribute with CoREST to bring substrate (containing Ly4 of histone 3) close to the catalytic center of LSD1148. Figure

(30) show the three dimensional structure of LSD1 enzyme.

Figure (30): 3D demonstration of LSD1-CoREST complex enzyme structure. FAD-binding

subdomain is blue, substrate binding subdomain is red, Swirm domain is cyan, Tower domain is green,

CoREST domain is black and FAD cofactor is yellow. The LSD1 structure was obtained from protein

data bank (2H94) and figure is adapted from Forneris et. al.149 modified by Chimera software v.1.8.1.

LSD1-CoREST- histone peptide complex (that is linked to the C-terminal amino oxidase domain) adopts specific conformations that enable the Lys4 side chain of the histone 3 N-terminal to be close to the flavin active site149 to undergo oxidative demethylation. Polar residues of histone peptide (Arg2, Thr6, Lys9, Arg8, and Thr11) and N-terminal amino acid groups have specific interactions with the protein residues of the enzyme150. The addition of epigenetic markers will produce an electrostatic and steric effect that affect previously established interactions that affect LSD1 binding to histone

3141,149. These specific interactions are unique for substrate recognition made by LSD1 enzyme.

1.9.2. Substrate specifity or recognition of LSD1 enzyme:

LSD1 enzyme requires histone substrate to have 20 amino acids N-terminal for its catalytic activity141 to bring Lys4 of histone 3 close to the active site located inside the amino oxidase like domain of the LSD1 enzyme. The long (20 amino acids) N-terminal amino acid is important for its substrate specifity151 where it also enable the LSD1 to sense the epigenetic markers encoded by histone tails141,152,153. There are several requirements for the substrates to be recognized and subjected to the LSD1 catalytic activity148: (i) the distance between the protein surface and flavin active site is about 23Aº so that substrate must enter the catalytic center to bind active site (flavin ring) of LSD1 enzyme. For Lys4 of histone 3 substrate; they must enter catalytic site in appropriate conformation to bring its methylated Lys4 into close contact with the catalytic site148. (ii)

The acidic surface of the entry (lined by highly conserved acidic residues mainly

glutamate and aspartate) to the catalytic center can easily interact with basic chemical groups or basic residues of the histone 3148. (iii) 10 residues polypeptide can fill the catalytic center of the LSD1 enzyme, so that the 21 (or 16) amino acid polypeptide of

Lys4 of histone 3 must be folded in loop like structure that can interact with the acidic residues and bring methylated lys4 into close contact to the active site of the catalytic center148.

LSD1 act efficiently on histone substrate that are free of other epigenetic modifications such ubiquitylation, acetylation, and phosphorylation since presence of such modifications decrease its catalytic activity154,155. This support the idea that LSD1 only demethylate already processed histone traits by enzymes such as serine phosphatase, histone deacetylase and arginine demethylases153. LSD1 inhibition result in reactivation of gene transcription but do not prevent other modifications such as histone deacetylation. Inhibition of HDAC enzyme increase H3K4 methylation156 147 so that

LSD1 demethylation effect will remove final epigenetic marker associated with genetic activation150.

LSD1 is flavin dependent enzyme share a 20% sequence identity with MAO enzymes94. MAO enzymes catalyze the oxidative deamination of amine substrates through formation of covalent flavin adduct at C4a position of the flavin ring while LSD1 enzymes catalyze the oxidative demethylation of Lys4 of histone by forming a noncovalent adduct at N5 position of the flavin ring. FAD folding in both LSD1 and MAOs is identical150 to enable the Lys side chain to be close to the active site of the flavin ring94.

FAD domain of LSD1 enzyme has two domains different from that of MAOs that are

SWIRM and tower domain that link LSD1 to the histone tail in the chromatin150.

MAO inhibitors such as tranylcypromine is able to inhibit LSD1 enzyme by different covalent modifications from that of MAO enzymes where the opening of its propyl ring can form flavin adduct at N5 and C4a positions of the flavin ring138,147,157 while in MAOs; tranylcypromine form covalent flavin adduct at only C4a position of the flavin ring93 as illustrated in figure (31).

Figure (31): Mechanisms of flavin-tranylcypromine adduct formation in MAO and LSD1

enzymes. The figure is adapted from Forneris et. al. 150 and modified by Marvin Sketch software v.6.2.

Other MAO inhibitors such as propargyline containing inhibitors (Selegiline and

Rasagiline) inhibit MAO enzymes by forming a covalent flavin adduct at N5 position of the flavin ring. In LSD1, these inhibitors do not form flavin adducts directly with the isoalloxazine ring but they bind to the Lys4 residues of the histone through their propargyline group. Propargyline modified Lys4 of histone tail will then interact and form a covalent adduct with the LSD1 flavin ring158-160. Figure (32) shows the difference in flavin adduct formation of propargyline containing compounds (Rasagiline and

Selegiline) between LSD1 and MAO.

Figure (32): Mechanisms of flavin adduct formation for Propargyline containing compounds in

MAO and LSD1 enzymes. Propargyl irreversible inhibition involves covalent bond formation on N5

of isoalloxazine ring of both enzymes. The figure is adapted from Forneris et. al.150 and modified by

Marvin Sketch software v.6.2.

Chapter two: methods and materials

2.1. Overview:

Using similarity analysis approach that depends on molecular shape comparison method (MSC) which compare the shapes of two or more molecules towards the active site of the target receptor (MAO-B)161; in addition to the matching method (Docking) that define the target protein active site geometry, hydrogen bonding, hydrophobicity and electrostatic interaction and then dock chemical compounds (ligands) that have geometry that match that of the protein active site162; sixteen (16) compounds are generated as alternative models from two different lead compounds (thiazolidinedione derivatives of pioglitazone and Zonisamide) that have potential inhibitory effect on MAOs catalytic activity.

All aromatic chemical compounds are purchased from Chembridge online chemical store (www.hit2lead.com) with a possible inhibitory effect on MAO enzymes.

The chemical structures of these compounds have functional groups that are established to play a key role in MAO inhibition such para-phenyl substituted groups, propargyline, thiazolidinedione and hydrazine. The 2D chemical structures, compounds ID, chemical name and molecular weight of test compounds are illustrated in table 3. Initially, these compounds are virtually screened by docking techniques (structure-based techniques) that involve a computational fitting or docking of each chemical

compound in the active site of MAO-B (and LSD1) with different conformations, positions, orientations, and electrostatic interaction163,164. This will provide an impression about the binding affinity and possible inhibitory effect for each compound to MAO-B enzyme. Then, these compounds are tested in the laboratory for their actual inhibitory effect and selectivity toward MAO enzymes, in addition to LSD1 enzyme inhibitory action that may have a therapeutic implication in certain tumors146,147 and neurodegenerative diseases165,166.

Test compounds are dissolved in appropriate volume of dimethyl sulfoxide

(DMSO) to prepare stock solutions of 10 mM and 20mM. Only 6373721 and 6366286 are prepared as stock solutions of 20mM concentration whereas the rest compounds are prepared as 10mM concentration stock solutions. All test compounds are stored at -80C (-

4F).

The aims of the study are to identify novel MAO-B inhibitors by ligand based virtual screening using Zonisamide and thiazolidinedione derivatives of pioglitazone as lead compounds and to evaluate the MAO-B inhibitory effect of two structurally different lead compounds derivatives and how these evaluations affect our choices in recognition of new compounds.

Table 3: 2D chemical structures, compounds ID, chemical name and molecular weight of the test compounds

Compound Chemical name and Chemical structure M.

ID formula Wt

1 4003841 [4-(2-methoxyetho-xy) 204

phenyl]amine

hydrochloride

(C9 H13NO2.CH)

2 5114860 4-(4-hydroxyphenyl)-3- 162

buten-2-one

(C10 H10 O2)

3 5119666 (4-hydroxybenz-ylidene) 170

malononitrile

(C10 H6 N2 O)

4 5472855 5-[4-(benzyloxy)-3- 357

methoxybenzylidene]-2- thioxo-1,3-thiazolidin-4-

one

(C18 H15 N O3 S2)

5 6209863 5-{3-methoxy-4-[(4- 371

methylbenzyl)oxy]benzylid

ene}-2-thioxo-1,3-

thiazolidin-4-one

(C19 H17 N O3 S2)

6 6364633 5-{3-ethoxy-4-[(3- 383

methylbenzyl)oxy]benzylid

ene}-3-methyl-1,3-

thiazolidine-2,4-dione

(C21 H21 N O4 S)

7 6366286 5-{4-[(3,4-dichloro- 394

benzyl)oxy]benzylidene}-

3-methyl-1,3-thiazolidine-

2,4-dione

(C18 H13 Cl2 N O3 S)

8 6373721 5-{4-[2-(2-chlorophe- 376

noxy)ethoxy]benzylidene}- 1,3-thiazolidine-2,4-dione

(C18 H14 Cl N O4 S)

9 6594612 5-{3-chloro-4-[(2- 408

chlorobenzyl)oxy]benzylid

ene}-3-ethyl-1,3-

thiazolidine-2,4-dione

(C19 H15 Cl2 N O3 S)

1 6634507 5-{4-[3-(2- 390

0 chlorophenoxy)propoxy]be

nzylidene}-1,3-

thiazolidine-2,4-dione

(C19 H16 Cl N O4 S)

1 6636424 5-{4-[3-(4-ethylphen- 383

1 oxy)propoxy]benzylidene}

-1,3-thiazolidine-2,4-dione

(C21 H21 N O4 S)

1 6836234 5-{4-[(3,4-dichlorob- 418

2 enzyl)oxy]benzylidene}-3-

(2-propyn-1-yl)-1,3-

thiazolidine-2,4-dione.

(C20 H13 Cl2 N O3 S)

1 7138125 N-(5-{3-chloro-4-[(3- 405

3 fluorobenzyl)oxy]benzylid

ene}-4-oxo-4,5-dihydro-

1,3-thiazol-2-yl)acetamide

(C19 H14 Cl F N2 O3 S)

1 7315349 Methyl (4-aminophen- 181

4 oxy)acetate

1 7320244 5-{3-chloro-4-[(4- 380

5 fluorobenzyl)oxy]benzylid

ene}-2-thioxo-1,3-

thiazolidin-4-one

1 9192036 4-[(2-oxo-1-pyrroli- 309

6 dinyl)methyl]-N-(2-

pyridinylmethyl)

benzamide

C18 H19 N3 O2

2.2. MAO inhibition assay:

It is a flourimetric assay used to measure potential inhibitory effect of test compounds towards MAO enzymes catalytic activity. The assay is based on the ability of each compound to inhibit oxidative metabolism of non-fluorescent and non-selective substrate (Kynuramine) mediated by MOA enzymes into its fluorescent metabolite (4- hydroxyq-uinoline) through formation of a transient aldehyde intermediate 3-(2- aminophenyl)-3-oxo-propionaldehyde167 as shown in figure (33)168. Thus MAO inhibitory effect of each test compound will be related to the amount of produced fluorescent 4-hydroxyquinoline which can be estimated from its flourscence intensity measurement.

96-well plates (Falcon Cat#353241) were used in the MAO-B inhibition assay and 384-well plates (Sigma Aldrich) were used in MAO-A inhibition assay with final volume of 200μl. The buffer used was phosphate buffer (KPO4) of 0.1mM concentration and 7.4 PH. The substrate required for the assay, Kynuramine, was ordered from Sigma-

Aldrich (Co. St. MO USA) and diluted with appropriate volume of distilled water to

obtain a stock solution of 25mM concentration. Both recombinant human MAO-A and human MAO-B enzymes of 5 mg/ml stock solution are ordered from (BD Gentest) and stored at –80ºC.

Figure (33): Catalytic conversion of Kynuramine into 4-Hydroxyquinoline through formation of

an intermediate 3-(2-aminophenyl)-3-oxo-propionaldehyde. The figure was adapted from Yan

et.al.168 and modified by Marvin Sketch software v.6.2.

Both MAO enzymes were diluted with 0.1M phosphate buffer (KPO4) to produce a final solution of 0.006 mg/ml of MAO-A enzyme for MAO-A assay and 0.015 mg/ml of MAO-B enzyme for MAO-B assay. In MAO-A inhibitory assay; 25 mM Kynuramine was further diluted with suitable volume of phosphate buffer (KPO4) (0.1M, 7.4PH) to give 40μM stock solution that is equal to the Km of the of reaction catalyzed by MAO-A enzyme. In MAO-B inhibitory assay; Kynuramine was prepared as 20μM stock solution

which is equal to the Km of the MAO-B catalyzed reaction. 3μl of each test compound was added to 147μl of phosphate buffer KPO4 (0.1M, PH 7.4) in duplicates of the first raw of the 96-well plate except two wells that are preserved for the vehicle control which is the dimethyl sulfoxide (DMSO) that reported to have the least MAO inhibitory effect

(100% MAO activity).

Serial dilution was performed across the plates by pipetting 50μl from the first raw into next seven wells with discarding of last 50 μl to create eight point dose inhibition curve. The plate was then covered and incubated for 10 minutes at 37ºC to allow inhibitors reach 37ºC for their maximum inhibitory effect. Prepared and pre- incubated Kynuramine of 40μM (or 20μM in case of MAO-B) was then added as

50μl/well in MAO-A assay. Reaction was initiated by addition of 50μl of 0.006μM

MAO-A (or 0.015μM of MAO-B in MAO-B assay) that kept in ice prior to use. The plate was covered and returned to incubator for additional 20 minutes at 37ºC. As a result the final volume of each well in the MAO inhibitory assay was 200μl in each well. After that

2N NAOH is added as 75μl to stop the reaction. The plate was then read at wave lengths

310/380 excitation/emission by a flourometer (Top reader BMG floustar) to measure flourscence intensity which reflect the amount of the 4-hudroxyquinoline produced (low flourscence intensity as compared to that of the control reflect the low amount of the reaction fluorescent product (4-hydroxyquinone) which indicate good inhibitory effect of the test compound). The flourscence intensity results are used to calculate IC50

(concentration of the test compound at which 50% of the maximum MAO activity is inhibited or lost) using Prism5 software (www.graphpad.com).

2.3. LSD1 inhibitor screening assay:

LSD1 assay is fluorescent based assay designed to screen the LSD1 inhibitory effects of the test compounds. It is based on the following principle: LSD1 catalyzes the oxidative demethylation reaction of mono- or di-methyl of the lysine residue of the histone 3 to produce H2O2 which in turn react with ADPH (10-acetyl-3,7-dihydroxy- phenoxazine) to produce fluorescent compound Resorufin in the presence of Horseradish peroxide (HRP) as shown in figure (34).

Figure (34): Chemical basis of the LSD1 inhibition assay. This figure was adapted from

(www.caymanchem.com) and modified by Marvin Sketch software V.6.2.

LSD1 inhibitory screening kit was ordered from Cayman chemicals (Ann, Arbor,

MI, USA) (www.caymanchem.com). Black 96-well plate (Falcon Cat#353241) was used

in the assay with a final volume of 200μl in each well. LSD1 assay buffer was diluted with appropriate volume of HPLC-grade water to get 50mM Hepes (PH 7.5). Only five compounds with pronounced MAO inhibitory effect were tested in the assay. LSD1 enzyme was kept in ice prior to use. The flourimetric substrate ADPH was prepared immediately before the assay by dissolving the assay vial content in 100μl of DMSO and then diluted in 400μl of assay buffer. The horseradish peroxide was prepared by dissolving one vial’s content in 1ml of assay buffer. Finally LSD1 assay peptide

(substrate for LSD1) was ready to use. To get 100μM concentration of the assay substrate, only 20μl is needed to be added. DMSO was used as vehicle control that result in 100% LSD1 enzyme catalytic activity.

For each test compound, nine wells were needed. The first three wells (also called

100% initial activity wells) contain the vehicle control (DMSO) and associated with

100% of enzyme activity. They contain 120μl of the assay buffer, 20μl of the LSD1, 20μl of horseradish peroxide, 10μl of flourimetric substrate and 10μl of DMSO. The second three wells (also called background wells) contain 140μl of assay buffer, 20μl of the

LSD1, 20μl of horseradish peroxide, 10μl of flourimetric substrate and 10μl of DMSO.

The third three wells (also called inhibitor wells) contain 120μl of the assay buffer, 20μl of the LSD1, 20μl of horseradish peroxide, 10μl of flourimetric substrate and 10μl of test compound. The reaction was started by addition of 20μl of the assay peptide to all wells except background wells. The plate was then covered and incubated for 30 minutes at

37ºC. After incubation, the plate was read at excitation 530-540nm and emission 585-

595nm by using a top reader flourometer (BMG-FlouStar).

2.4. Docking studies:

Sixteen (16) test compounds were prepared, in 2D and 3D structures using

Marvin Sketch software v.6.2.2. (www.chemaxon.com), for docking in different orientations, conformations, and positions in the active site of MAO-B crystal (2BK3) structure that was obtained from RCSB protein data bank and prepared as monomer by using UCSF Chimera software v.1.8.1 (www.cgl.ucsf.edu/chimera). LSD1 crystal

(2XAQ) was obtained from protein data bank as LSD1-CoREST complex with tranylcypromine derivative (MC2584, 13b) and processed by Chimera v.1.8.1.

The 3D structure of these compounds and MAO-B protein were initially processed by Autodock tools software v. 1.5.6 (http://mgltools.scripps.edu) then these compounds were docked into MAO-B active site by using Autodock 4.2 software

(http://autodock.scripps.edu). Docking results were then analyzed by Autodock tools

1.5.6 to obtain enzyme-inhibitor complexes in ranked and different conformations, binding affinities and orientations. Inhibitor-enzyme complexes with highest binding affinity were then processed by ligplot+ v 1.4.5 and Pymole v.1.3

(www.schrodinger.com) to obtain 2D and 3D structural images that illustrate their interactions with the active site of the enzyme.

2.5. Bovine Serum Albumin assay (BSA):

Human serum albumins have many essential physiological functions169. One of its important functions is serving as a transporter for organic and inorganic molecules into

various tissues in the body. Due to large structural homology between human and bovine serum albumin170, bovine serum albumin is used in many binding affinity measurement methods to assess test compounds ability to bind human serum albumin. Bovine serum albumin has been reported to exhibit a strong flourscence emission at 342nm when excited at 280nm. This emission efficiency is attributed to its aromatic amino acid residues tryptophane and tyrosine that act as intrinsic fluorophores171. Bovine serum albumin has 2 fluorophore tryptophan residues; tryptophane 143 that is located on the surface of the protein and tryptophane 212 that is located at the protein binding site170,172.

In comparison, human serum albumin possess only one fluorophore tryptophane residue

(Try214)170.

BSA binding assay is used to estimate the binding affinity of test compounds to the serum albumin that is essential for its distribution in various body tissues. The principle of this assay is that; the binding or interaction of the test compound to the bovine serum albumin quenches its intrinsic flourscence (i.e. decrease the emission efficiency (quantum yield) of the fluorophore residues)173,174.

Black 96-well plates (Falcon Cat#353241) were used with a final volume of

200μl/well. The buffer used in the assay was phosphate buffer KPO4 (0.1M and 7.4 PH).

BSA was ordered from Boston Bioproduct (Ashland, MA, USA). Prior to the test, BSA stock solution of 1mg/ml was prepared by solubilization of a specific amount of BSA with a similar volume of phosphate buffer. 3μl of each test compound was added to 147μl of the phosphate buffer KPO4 in duplicate of the first raw of 96 wells plate except two

wells where dimethyl sulfoxide was added as a vehicle control that have no binding affinity to BSA (100% flourscence intensity).

A serial dilution of test compounds was made by pipetting of 50μl/well into the next seven wells across the plate to create eight point dose inhibition curves. BSA stock solution 1mg/ml was then added as 100μl/well. Then, plate was covered and incubated for 30 minutes at 37ºC. After incubation, plate read at 280/340nm wave lengths using a flourometer (Top reader BMG-Floustar).

2.6. Parallel artificial membrane permeability assay (PAMPA):

It is a fluorescent, non-cell based assay designed to give a prediction about the transcellular permeability of the test compound through different biological membranes such as blood brain barrier, skin and gastrointestinal tract in early drug discovery175. It is high-through output screening assay, reproducible, of low cost, and associated with high success. In addition only small amount of the test compounds are used175. The assay tests permeability of compounds across biological membranes that is attributed to the passive diffusion only175 and it is unable to determine permeability that is attributed to the more complex active transport. Passive diffusion or permeability of compounds depends on their lipophilicity, PH, and solubility175.

HDM-PAMPA (HDM; hexadecane method) was carried out using 96-well multiscreen permeability plate to determine passive diffusion of test compounds through an artificial hexadecane liquid layer on polycarbonate membrane support that separates between donor compartment (contain test compound at specific concentration dissolved

in buffer media) and acceptor compartment (contain only the buffer medium). Only most active compounds with pronounced MAO inhibitory effects were tested for their passive permeability.

The assay buffer used was 5% (v/v) dimethyl sulfoxide in phosphate buffer (7.4

PH). The 96-well multiscreen permeability plate (donor plate) (Cat# MAPBMN310) and

(Cat# MSSACCEPTOR) were ordered from Millipore Corporation (Billerica, MA,

USA,). Hexadecane and hexane were ordered from commercial sources. The 5% (V/V) hexadecane in hexane was prepared in the laboratory. The five 10mM test compounds

(6366286, 7138125, 6636424, 6373721, and 7320244) were prepared in concentrations of

100μM by dilution in appropriate volume of assay buffer (5% (v/v) DMSO/phosphate buffer). The control vehicle used was verapamil (100μM) which is known to pass through lipophilic membranes at different concentrations. The Five most active compounds are tested for their flourscence intensities where they exhibited best flourscence intensity at

360/40 emission and 620/40 excitation.

Initially 15μl of 5% (v/v) hexadecane in hexane was pipetted carefully into the each needed well of the donor plate. Donor plate was then allowed to dry for one hour in a fume hood to ensure hexane evaporation and formation of uniform layer of hexadecane and then placed in the acceptor plate that already contains 300μl of assay buffer (5% (v/v)

DMSO/KPO4) in order to ensure liquid contact with the artificial polycarbonate membrane. 150μl/well of 100μM test compounds was added as duplicate in the donor plate. The plate was then covered and incubated at room temperature for four hours.

Equilibrium solutions were prepared for each test compound by simply dissolving 150μl

of test compound in 300μl of assay buffer. After that, 250μl/well of two wells for each test compound was pipetted from the acceptor plate and 250μl/well of equilibrium solution was pipetted into black 96-well plate (Falcon Cat#353241).

In order to determine the concentration of each test compound in the acceptor plate, test compounds were serially diluted with the assay buffer across the plate to generate eight point flourscence concentrations curve. Finally, the plate was read at

360/40 emission and 620/40 excitation by using FLX 800 microplate flourscence reader

(Biotek instrument) to determine flourscence intensity of each compound that reflect its concentration.

Chapter three: Results

3.1. MAO inhibition assay:

Since MAO-B enzyme crystallized structure is well analyzed, sixteen (16) test compounds are chosen on the basis of similarity analysis and docking studies and thought to have an inhibitory effect on MAO-A, MAO-B or both enzymes. All test compounds have chemical groups with proposed inhibitory effect on MAO catalytic activity such para-phenyl substituted groups, propargyline, thiazolidinedione and hydrazine that are shown in the figure (35).

Figure (35): Several chemical groups have an inhibitory effect on MAO enzyme activity. The

figure is created by Marvin Sketch v.6.2.

As compared to their lead compounds; several test compounds have shown a pronounced inhibitory effect on MAO-A, MAO-B, or both enzymes at micromolar or nanomolar concentrations. Other compounds show no or weak inhibition effect on MAO enzymes. Results of MAO inhibition assay for the all compounds are summarized in table

(4).

MAO inhibitors are of great importance to patients with Parkinson disease to alleviate their symptoms and provide a neuroprotective effects that may delay or halt disease progression. Selectivity of MOA inhibitors toward MAO-B is one of our goals for providing alternative models to decrease risk of adverse reactions that may develop with non-selective inhibitors.

We focused in this chapter on five test compounds (6366286, 6373721, 6636424,

7138125, and 7320244) that have shown pronounced inhibitory effect on MAO-B enzymes with extremely low IC50 at nanomolar and micromolar concentrations.

Table 4: Results from MAO inhibition assay for all test compounds.

Test 2D structure IC50 (μM) IC50 (μM)

compound for MAO-A for MAO-B

4003841 14.62 0.03103

5114860 45.94 40.75

5119666 86.09 28.51

5472855 - 8.429

6209863 - 1.586

6364633 65.86 4.188

6366286 5.331 0.02634

6373721 19.61 0.04826

6594612 176.6 15.84

6634507 9.709 3.979

6636424 5.773 0.2057

6836234 - 1.044

7138125 6.281 0.08108

7315349 0.3919 0.1935

7320244 25.07 0.3859

9192036 - -

3.1.1. MAO-B inhibition assay:

Based on MAO-B inhibition assay results; test Compound 6366286 (IC50 of

0.02634 μM) can be considered as the most active MAO-B inhibitor among the most active five compounds. It markedly decreases MAO-B activity into (34.84 ± 0.7348) of the control (100% activity) at very low concentration (0.056μM). Test compound

7320244 is the weakest MAO-B inhibitor among these test compounds with an IC50 of

(0.3859µM) that decreases MAO-B enzymatic activity into (30.91 ± 1.121) of the control

(100% activity) at (1.11μM) concentration. Compound 6373721 at 0.1μM concentration

(twice as that of 6366286) decrease MOA-B activity into (29.38 ± 0.09092) of the control. Compounds 7138125 at 0.56μM concentration (five times more than that of

6373721) and compound 6636424 at 1.11μM (same concentration as that of 7320244) decrease the MAO-B activity into (18.17 ± 0.3266) and (24.95 ± 0.5214) of the control respectively. Figure (36) show the inhibitory effect of top active test compounds

(6366286, 6373721, 6636424, 7138125, and 7320244) on MAO-B enzymes at different concentrations. MAO-B IC50, inhibition curves and the quantitative values for MAO-B enzyme activity inhibition for each test compounds are shown in table (5).

3.1.2. MAO-A inhibition assay:

Test compound 6366286 markedly decrease MAO-B activity at 0.0556μM concentration into (34.84 ± 0.7348) of the control (100%activity) while in MAO-A inhibition assay the same compound at concentration 5.556 (100 times more than that used in MAO-B) only decrease MAO-A activity into (76.72 ± 2.429). Compound

6373721 decrease MOA-B activity into (29.38 ± 0.09092) of the control (100% activity) at 0.11μM while 100 times of this concentration (i.e. 11.11μM) is required to inhibit

MAO-A activity (100%) into (96.83 ± 11.53) of the control. Compound 7138125 decrease MAO-B activity of the control (100%) into (18.17 ± 0.3266) at 0.5556μM whereas 10 times of that concentration (5.556μM) is needed to decrease MAO-A activity

(100%) into (96.76 ± 8.025) of the control. Similarly, test compounds 6636424 and

7320244 that decrease MAO-B activity (100%) into (24.95 ± 0.5214) and (30.91 ± 1.121) respectively at 1.111μM whereas 10 times concentrations (11.111μM) are required to decrease MAO-A activity into (44.06 ± 0.7687) and (82.77 ± 4.182) respectively. Figure

(37) show the inhibitory effect of the five test compounds (6366286, 6373721, 6636424,

7138125, and 7320244) on enzymatic activity of MAO-A. MAO-A IC50 (μM) and quantitative values for inhibitory effects on MAO-A enzyme activity of the compounds are shown in table (6). 100 or 10 times more concentrations required to inhibit MAO-A enzymatic activity than that required to inactivate MAO-B enzyme. These data suggest that these compounds have higher affinity toward MAO-B enzyme than that toward

MAO-A. Figure (38) show the inhibitory effects of test compounds on both MAO-A and

MAO-B enzymatic activity.

Table 5: MAO-B IC50, inhibition curves that are used to measure IC50 (nM) and the MAO-B inhibitory effects quantitative values for most active test compounds

Compound ID Mean ± SEM MAO-B Inhibition curve

1 6366286 (0.056) 34.84 ± 0.73

2 6373721 (0.1) 29.38 ± 0.09

3 7138125 (0.56) 18.17 ± 0.33

4 6636424 (1.11) 24.95±0.5214

5 7320244 (1.11) 30.91±1.121

Figure (36): Inhibitory effects of most active compounds on MAO-B enzymatic activity.

Flourscence intensity values are normalized by the control values and expressed as Mean ±

SEM.

Figure (37): Inhibitory effect of most active compounds on MAO-A enzymatic activity.

Flourscence intensity values are normalized by the control values and the results are expressed as

(Mean ± SEM).

Table (6): MAO-A IC50 (μM) and quantitative values for the effects of the most active compounds on MAO-A enzymatic activity.

Compound ID Mean ± SEM IC50 μM IC50 μM

(For MAO-A) MAO-B MAO-A

1 6366286 (0.056) 76.72 ± 2.429 0.02634 5.331

2 6373721 (0.11) 96.83 ± 11.53 0.04826 19.61

3 7138125 (0.56) 96.76 ± 8.025 0.08108 186.1

4 6636424 (1.11) 44.06 ± 0.7687 0.2057 5.773

5 7320244 (1.11) 82.77 ± 4.182 0.3859 25.07

Figure (38): Inhibitory effects of most active MAO-B inhibitors on both MAO enzymatic

activities. The concentrations used in MAO-B assay are lower by 100 or 10 times than that of

the same compounds used in MAO-A assay. The flourscence intensity values are normalized

by the control values and expressed as Mean ± SEM.

3.2. MAO-B docking studies:

These studies are made by using Autodock tools that is designed to predict the potential binding ability of small molecules such as our test compounds (drug candidates) or substrate to the 3D crystalized structure of the receptor protein (or enzymes) such as

MAO-B or LSD1. Docking studies can provide an impression on how these small molecules interact within the active sites of the enzymes in different conformations and provide a good correlation between inhibition constants calculated by these tools and experimental ones. Low MAO-B or LSD1 binding energy can give a prediction on how strong hydrophobic interactions are.

Based on docking studies; all top test compounds do not form hydrogen bond with MAO-B residues or peptides. The large chemical structures of test compounds

6366286, 6373721, 7138125, 6636424 and 7320244 occupy both entrance and substrate cavity of MAO-B enzyme by forcing the Ile199 residues (gating residues) side chain to adopt an open conformation.

Test compound conformation with an extremely low estimated binding free energy is proposed to have the strongest hydrophobic interaction within active site of

MAO-B enzyme. The estimated binding free energy of the most active test compounds are illustrated in table (7).

Table 7: Estimated Binding free energy of top test compound, estimated and experimental dissociation constants (Ki).

Compound ID MAO-B binding energy Estimated Ki Experimental Ki

1 6366286 -8.85 324.05nM 26.34nM

2 6373721 -9.71 76.61nM 48.26nM

3 7138125 -8.55 540.02nM 81.08nM

4 6636424 -5.68 68.46μM 0.2057μM

5 7320244 -8.21 952.19nM 385.9nM

As shown in 3D demonstrations (figures 40,42,44,46 and 48) of test compounds interactions with MAO-B crystal structure; all five test compounds are oriented in extended conformations within MAO-B enzyme active site. The orientation of test compounds 6366286, 6636424 and 7320244 are similar where tail aromatic ring of these compounds are facing isoalloxazine ring of the enzyme. Dichlorobenzyl ring (not the thiazolidinedione) of 6366286, 4-ethylphenoxy ring moiety of 6636424 and 4- fluorobenzyl of 7320244 are facing isoalloxazine ring of the flavin cofactor and are situated between the two tyrosyl residues of the aromatic cage (shown in red). Whereas thiazolidinedione rings (1,3-thiazolidine-2,4-dione) of the test compound 6373721 and 4- oxo-4,5-dihydro-1,3-thiazol-2-yl) acetamide of 7138125 are facing the flavin ring and positioned between tyrosyl residues of the aromatic cage. The hydrophobic interactions of test compounds with the amino acid residues of the enzyme are demonstrated in as 2D representations of test compounds as obtained from docking studies in figure (39, 41, 43,

45 and 47).

Figure (39): 2D demonstration of docking of the of test compound 6366286 into MAO-B enzyme. Carbon atom is in black; nitrogen atom is blue, oxygen atom is red, sulfur atom is yellow. The hydrophobic interactions are represented as small multiple red lines. This image was created by Ligplot v. 1.4.5.

Figure (40): 3D demonstration of docking of test compound 6366286 in the active site of crystalized MAO-B structure. Ligand is green, gating residues (Ile199 and Tyr326) are blue, aromatic cage residues (Tyr398 and Tyr435) are orange, carbon atoms are green, sulfur atom is yellow, chlorine atom is cyan oxygen atom is red and nitrogen atom is blue. The image was created by Pymol v 1.3.

Figure (41): 2D demonstration of docking of test compound 6373721 into MAO-B enzyme. Carbon atom is black; nitrogen atom is blue, oxygen atom is red, sulfur atom is yellow. The hydrophobic interactions are represented as small multiple red lines. This image is created by Ligplot v. 1.4.5.

Figure (42): 3D demonstration of docking of test compound 6373721 in the active site of crystalized MAO-B structure. Ligand is green, gating residues (Ile199 and Tyr326) are blue, aromatic cage residues (Tyr398 and Tyr435) are orange, carbon atoms are green, sulfur atom is yellow, chlorine atom is cyan oxygen atom is red and nitrogen atom is blue. The image was created by Pymol v 1.3.

Figure (43): 2D demonstration of docking of test compound 7138125 into MAO-B enzyme. Carbon atom is black; nitrogen atom is blue, oxygen atom is red, sulfur atom is yellow. The hydrophobic interactions are represented as small multiple red lines. This image was created by Ligplot v. 1.4.5.

Figure (44): 3D demonstration of docking of test compound 7138125 in the active site of crystalized MAO-B structure. Ligand is green, gating residues (Ile199 and Tyr326) are blue, aromatic cage residues (Tyr398 and Tyr435) are orange, carbon atoms are green, sulfur atom is yellow, chlorine atom is cyan, fluorine atom is brown, oxygen atom is red and nitrogen atom is blue. The image was created by Pymol v 1.3.

Figure (45): 2D demonstration of docking of test compound 6636424 into MAO-B enzyme. Carbon atom is in black; nitrogen atom is blue, oxygen atom is red, sulfur atom is yellow. The hydrophobic interactions are represented as small multiple red lines. This image was created by Ligplot v. 1.4.5.

100

Figure (46): 3D demonstration of docking of test compound 6636424 in the active site of crystalized MAO-B structure. Ligand is green, gating residues (Ile199 and Tyr326) are blue, aromatic cage residues (Tyr398 and Tyr435) are orange, carbon atoms are green, sulfur atom is yellow, chlorine atom is cyan oxygen atom is red and nitrogen atom is blue. The image was created by Pymol v 1.3.

101

Figure (47): 2D demonstration of docking of test compound 7320244 into MAO-B enzyme. Carbon atom is black; nitrogen atom is blue, oxygen atom is red, sulfur atom is yellow. The hydrophobic interactions are represented as small multiple red lines. This image was created by Ligplot v. 1.4.5.

102

Figure (48): 3D demonstration of docking of test compound 7320244 in the active site of

crystalized MAO-B structure. Ligand is green, gating residues (Ile199 and Tyr326) are blue,

aromatic cage residues (Tyr398 and Tyr435) are orange, carbon atoms are green, sulfur atom

is yellow, chlorine atom is cyan, fluorine atom is brown, oxygen atom is red and nitrogen

atom is blue. The image was created by Pymol v 1.3.

3.3. LSD1 inhibition assay:

LSD1 is flavin dependent enzyme that belongs to amino oxidase superfamily. It is histone demethylase enzyme that selectively demethylates or removes methyl groups from Lys4 of the histone 394,140. All five top test compounds do not significantly inhibit the LSD1 enzymatic activity. Figure (49) show the inhibitory effects of test compounds

103

6366286, 6373721, 7138125, 6636424 and 7320244 on the LSD1 enzyme. Table (8) shows the quantitative values of each test compound effect on LSD1 enzyme.

Figure (49) show the inhibitory effect of five test compounds on LSD1 enzyme activity. The

concentrations used are at 100μM. The results are expressed as Mean ± SD.

Table (8): Quantitative values of active test compound effect on LSD1 enzyme activity.

Compound ID Mean ± SD

LSD1 inhibitor LSD1-C12 43.13 ± 1.67

1 6366286 96.47 ± 14.53

2 6373721 92.69 ± 1.58

3 7138125 91.18 ± 7.65

4 6636424 94.08 ± 18.98

5 7320244 90.32 ± 12.97

104

3.4. LSD1 enzyme Docking studies:

All test compounds are oriented inside the LSD1 crystal in folded conformation except for the compound 7138125 that have an extended conformation that enable the side chain amine group to form a weak hydrogen bond with the N5 of the flavin ring.

Test compound 6366286 has a folded conformation in which the dichlorobenzyl ring is facing the flavin ring of LSD1 enzyme with binding energy of (-6.82) and inhibition constant Ki of (10.06μM). Figures (50, 51) show 2d and 3d representation of docking of compound 6366286 in the LSD1 crystal structure. The orientations, geometry, H-bonding are illustrated in the 2D and 3D representations of the test compounds docking into the active site in LSD1. Compound 6373721 has three hydrogen bonds; two of them are between (N) and (O) of the thiazolidinedione ring and the N of the Val764 of the enzyme.

The third H-bond is located between the (O) of the thiazolidinedione and the (O) of the

Tyr773 of the enzyme. Compound 6636424 has only one H-bond between (O) of the thiazolidinedione ring and (N) of the Val764 as shown in its 2D imaging. Compound

7138125 is the only test compound that form H-bond with (N5) of the flavin ring in addition to that formed between the chlorine moiety of its benzyl ring and (O) of Tyr773.

Compound 7320244 has only one H-bond between thiol group of its thiazolidinedione ring and the (N) atom of the Val764. The binding energies for all five compounds are between (-5) and (-7) and illustrated in 2D images as in figures (50, 52, 54, 56 and 58).

The 3D images for test compounds docking within LSD1 enzyme are shown in figures

(51, 53, 55, 57, and 59).

105

Figure (50): 2D demonstration of docking of test compound 6366286 into LSD1 enzyme.

The carbon atom is in black; nitrogen atom is in blue, oxygen atom in red, sulfur atom in yellow. The hydrophobic interactions are represented as small multiple red lines. This image is created by Ligplot v. 1.4.5.

106

Figure (51): 3D demonstration of docking of test compound 6366286 in the active site of

crystalized LSD1 structure. The ligand is in green. The image is created by Pymol v 1.3.

107

Figure (52): 2D demonstration of docking of test compound 6373721 into LSD1 enzyme.

108

Figure (53): 3D demonstration of docking of test compound 6373721 in the active site of crystalized LSD1 structure. The ligand is in green. The image is created by Pymol v 1.3. The hydrogen bonds are in red.

109

Figure (54): 2D demonstration of docking of test compound 6636424 into LSD1 enzyme.

110

Figure (55): 3D demonstration of docking of test compound 6636424 in the active site of crystalized LSD1 structure. The ligand is in green. The image is created by Pymol v 1.3. The hydrogen bonds are in red.

111

Figure (56): 2D demonstration of docking of test compound 7138125 into LSD1 enzyme.

112

Figure (57): 3D demonstration of docking of test compound 7138125 in the active site of crystalized LSD1 structure. The ligand is in green. The image is created by Pymol v 1.3.

The hydrogen bonds are in red lines.

113

Figure (58): 2D demonstration of docking of test compound 7320244 into LSD1 enzyme.

114

Figure (59): 3Ddemonstration of docking of test compound 7320244 in the active site of crystalized LSD1 structure. The ligand is in green. The image is created by Pymol v 1.3. The hydrogen bonds are in red lines.

115

3.5. Bovine serum albumin binding assay (BSA):

This assay is used to assess binding affinity of the test compounds to human serum albumin (structurally similar to bovine serum albumin) that is essential for their tissue distribution including central nervous system. The BSA assay principle depends on the quenching ability of test compound on the intrinsic flourscence of the bovine serum albumin that is attributed to its tryptophan residues. Table (9) shows the EC50

(concentration at which the compound is 50% bound to the bovine serum albumin) in addition to the BSA binding curves for test compounds 6366286, 6373721, 7138125,

6636424 and 7320244.

Compound 6636424 has the highest binding affinity to the bovine serum albumin where it reduces the percentage of free BSA into (33.26 ± 0.3520) of the control with lowest EC50 (8.964μM). Compound 6366286 has the lowest binding affinity to bovine serum albumin where it slightly decreases the free BSA percentage into (91.11 ± 1.964) of the control with highest EC50 of (104.9μM). Compound 6373721, 7138125 and

7320244 lower the percentage of free BSA into (49.39 ± 4.637), (80.29 ± 3.004) and

(41.31 ± 0.09183) of the control respectively. Figure (60) show the percentage of the binding affinity of test compounds to BSA.

116

Figure (60): Effect of most active test compounds at 11.11μM concentration on

Percentages of free BSA. The flourscence intensity values are normalized by the control values and expressed as Mean ± SEM.

117

Table (9): EC50 and BSA binding curves for test compounds 6366286, 6373721,

7138125, 6636424 and 7320244

Compound ID Mean ± SEM BSA binding curve and EC50 (μM)

1 6366286 91.11 ± 1.96

(11.11μM)

2 6373721 49.39 ± 4.64

(11.11μM)

3 7138125 80.29 ± 3.004

(11.11μM)

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4 6636424 33.26 ± 0.35

(11.11μM)

5 7320244 41.31 ± 0.092

(11.11μM)

3.6. Parallel artificial membrane permeability assay (PAMPA):

This non-cell based, high through output screening assay is designed to calculate the passive permeability parameters (Pe) through an artificial membrane (polycarbonate membrane). The permeability parameter (Pe) can be used to estimate the ability of test compounds to pass through lipid membranes such as BBB. Distribution of test compounds into the central nervous system through BBB is essential for their actions as

MAO-B inhibitors in patients with Parkinson disease and other degenerative disorders.

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All five compounds are highly permeable that can pass through the lipophilic membrane of the BBB with ease to ensure high neuronal tissue distribution required for their therapeutic effect in the treatment of Parkinson disease and other neurodegenerative disorders. Only two test compounds concentrations at the acceptor compartments can be determined by standard curve interpolation created by serial dilution of test compounds at

500μM. The concentrations are illustrated in table (9).

176 Pe can be calculated by an equation reported by Faller, B . High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. As follow:

[ ]

Where:

VD: volume of the donor compartment (0.15cm3).

VA: volume of the acceptor compartment (0.3 cm3).

Area: active surface area of the artificial membrane (0.048cm3).

Time: incubation time required for the assay in seconds (4 hours = 14400sec.).

By applying all the variables with our experiment results; table (10) show the permeability Pe and log Pe values for test compounds 6366286, 6373721, 7138125,

6636424 and 7320244 in addition to concentrations of three compounds in the acceptor compartments.

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Table (10): Permeability Pe and log Pe values for test compounds 6366286, 6373721,

7138125, 6636424 and 7320244 in addition to concentrations of two compounds in the acceptor compartments

-6 -1 Compound ID Pe (10 cm ) Log Pe Permeability Concentration

in the acceptor

compartment

1 6366286 423.207 - 3.373 Highly permeable N/A

2 6373721 97.145 - 4.013 Highly permeable 71.71249 μM

3 7138125 53.972 - 4.268 Highly permeable 59.23801 μM

4 6636424 203.284 - 3.69 Highly permeable N/A

5 7320244 179.802 - 3.745 Highly permeable N/A

The results are compared to the PAMPA- BBB Pe ranges established by a previous literature 177. Predicting blood-brain barrier permeation from three-dimensional molecular

-6 -1 structure where compounds with Pe (10 cm ) > 4.0 are considered as highly BBB

-6 -1 permeable while compounds with Pe (10 cm ) < 2.0 are considered as low BBB

-6 -1 permeable. Test compounds with Pe (10 cm ) values in the range (2.0-4.0) are considered to have uncertain BBB permeability.

Chapter four: discussion

4.1. Discussion:

Parkinson’s disease is the second most common neurodegenerative disorder after

Alzheimer’s disease that affects 1% of elderly people older than 65 years of age and 4% of elderly people older than 80 years of age1,2. Clinically it is characterized by motor symptoms such as; tremor, dyskinesia, rigidity and postural instability, and non-motor symptoms such as cognitive, behavioral and psychiatric symptoms3,4. It is mainly idiopathic but Parkinson’s disease may be caused by environmental factors such as pesticides and head injuries or genetic factors such as synuclein alpha (non-A4 component of amyloid precursor) (SNCA) (gene codes for α-synuclein), leucine rich repeat kinase 2 (LRRK2)26, E3 ubiquitin ligase Parkin28, PETN-induced novel kinase 1

(PINK1)29. Parkinson’s disease is a progressive disorder that is pathologically caused by dopaminergic neuronal cell death of the pars compacta of substantia nigra at the mid brain. About 70% of the dopaminergic neurons are lost at the early symptoms of the disease. Since there are no pharmacological treatments that permanently stop the disease progression so there is no permanent cure for Parkinson’s disease. Pharmacological interventions are only intended to alleviate motor and non-motor symptoms of the disease31. These medications act either by increasing the dopamine level at the substantia nigra or mimic its effects.

121

122

MAO inhibitors can be used as mono or adjuvant therapy with levodopa in treatment of PD; they inhibit main catabolic pathway (oxidative deamination) of the dopamine at the synaptic cleft; thus elevate dopamine level and reduce the required dose of levodopa. There are selective and non-selective MAO inhibitors; Selective MAO-B inhibitors are preferred in the treatment of Parkinson’s disease since they are associated with low risk of side effects such as hypertensive crisis and other psychiatric adverse reactions. In addition selective MAO-B inhibitors have a neuroprotective effect that result from combating neurotoxic effects of MAO-B enzyme and inactivation of MPTP-induced neurotoxicity116-118.

Using similarity analysis approach161 and matching method (docking)162; sixteen compounds are identified as alternative models derived from two lead compounds;

Pioglitazone (antidiabetic drug) and Zonisamide (antiepileptic drug) that have shown inhibitory effects on MAO enzymatic activity at very low concentrations178 179. Initially these compounds are virtually screened by docking studies that are structure based techniques involve automated docking of each test compound in the active site of the target proteins (MAO-B or LSD1) in different conformations with various binding energies and dissociation constants (Ki). These compounds are then tested in the laboratory for their actual (Invitro) inhibitory effect on MAO-B enzymes and LSD1.

Some of these compounds are more active than Pioglitazone where they inhibit

MAO-B enzyme at nanomolar ranges, others are less active as compared to Pioglitazone

(IC50 ~300nM). Compound 9192036 has shown no inhibitory effect on MAO-A or

MAO-B enzymes. All test compounds have shown higher inhibitory effect toward MAO-

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B enzyme than MAO-A except test compound 5114860 that has approximately equal inhibition effect on both enzymes (see table 4). Of sixteen (16) compounds, five thiazolidinedione based test compounds have shown a pronounced inhibitory effect on

MAO-B enzyme activity with IC50 in nanomolar ranges and higher affinity toward

MAO-B enzyme as compared to MAO-A. Selectivity toward MAO-B enzyme is an important goal in identification of novel models in treatment of Parkinson’s disease since they are associated with low risks of adverse reactions (that may developed with non- selective and MAO-A inhibitors such as hypertensive crisis) and enhanced neuroprotective effect.

Test compound 6366286 and 6373721 are the most active test compounds in our group. They markedly decrease MAO-B activity into ~ 30% of the control (100% MAO-

B activity) at very low micromolar ranges (0.056-0.11μM) with IC50 of ~ (30nM-50nM) respectively as compared to that of pioglitazone (IC50 of ~300nM). Test compound

7320244 is the weakest MAO-B inhibitor among these compounds where it decreases

MAO-B activity into ~30% of the control at micromolar concentration of 1μM (higher than that used for previous test compounds). It is less active than Pioglitazone with an

IC50 of 385nM. Test compounds 7138125 and 6636424 with micromolar ranges of (0.5-

1μM) decrease MAO-B activity into about (20%-40%) of the control activity (100% activity) with IC50 of 0.08108μM and 0.206μM respectively.

All these five test compounds have higher affinity toward MAO-B enzymes as compared to MAO-A where 100 (as for compounds 6373721 and 6366286) or 10 times

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(as for 7320244, 7138125 and 6636424) more micromolar concentrations are required to decrease MAO-A enzymatic activity into 50-95% of the control (100% MAO-A activity).

From docking studies; these five active compounds have conformations with very low MAO-B enzyme binding free energy of ~ (-8) to (-10) except compound 7138125 that have binding free energy of ~ (-6) that suggest their strong hydrophobic interactions within enzyme active site.

For inhibitors (or substrate) to reach the MAO-B active site (flavin ring), loop 99-

112 must be transiently displaced to allow inhibitor entry into the entrance cavity then test compound may moves four gating residues (mainly Ile199 and Tyr326) that separate entrance cavity from substrate cavity of MAO-B enzyme to reach active site at the flavin ring. Test compounds molecules must move a distance of 20Aº from the protein surface through hydrophobic channel to reach flavin active site83. Ile199 side chain plays an important role in substrate and/or inhibitors specifity and recognition where it act as gating residue that gate the substrate cavity and adopt one of two conformations ‘’open’’ or ‘’closed’’ depending on the chemical charges and size of the substrates or inhibitors.

When small inhibitors enter MAO-B substrate cavity, Ile199 side chain adopt closed conformation that result in bipartite cavity with entrance and substrate cavity. The large chemical structure of all five test compound 6366286, 6373721, 7138125, 6636424 and 7320244 occupy both entrance and substrate cavity forcing the Ile199 side chain to adopt an open conformation that result in converting bipartite cavity (entrance cavity with volume of 290Aº and substrate cavity of 420Aº) of MAO-B enzyme into monopartite cavity with a volume of 700Aº. This substrate cavity plasticity has important

125

pharmacological implications where small inhibitors (such as compound 5114860 in table

4) have similar affinity to both MAO-A and MAO-B enzymes while large inhibitors

(such as our top five compounds) have shown higher affinity to MAO-B enzyme83.

There is a steric effect between Ile199 side chain and Phe103 residues on loop 99-

112 that may contribute in gating control of entrance cavity. When MAO-B enzyme in ligand free state the phenolic side chain of the Tyr326 reduce the steric effect of the

Ile199 side chain on Phe103 side chain on loop99-112 that keep opening of the entrance cavity. When Ile199 side chain adopt ‘’close’’ conformation, the Phe103 side chain on loop 99-112 will experience no steric effect that lead to opening of the entrance cavity100.

When large inhibitor such as our test compounds enter substrate cavity; the Ile199 side chain is rotated into the open conformation and Phe103 side chain on loop 99-112 experienced a steric effect and closes entrance cavity. Tyr326 is a component of the hydrophobic residues that line the substrate cavity near the junction between the substrate and entrance cavity90. When large test compound binds to the substrate, the effect of phenolic side chain of Tyr326 is reduced and steric effect of substrate cavity on Phe103 is increased. Figures (61, 62,63,64,65 and 66) show the Ile199, Tyr326 and Phe103 side chains in MAO-B enzyme and orientations for most active test compounds.

Elongated substrate binding site of MAO-B enzyme and aromatic cage tyrosyl residues are thought to affect orientations of the test compounds molecules in the enzyme active site. All top five compounds are oriented in extended conformations with their tail aromatic or thiazolidinedione rings situated between tyrosyl residues of the aromatic cage.

126

FAD dependent MAO-B enzyme mainly deaminates primary and secondary amines. In addition to hydrophobic interactions, test compounds orientation in active site of MAO-B enzyme facing the FAD cofactor is also regulated or controlled by two phenyl rings of tyrosyl residues (Tyr398 and Tyr435) that are in perpendicular position to the flavin ring and form an ‘’aromatic cage’’ that exert steric effect on substrates or inhibitors. The dichlorobenzyl ring of test compounds 6366286, 4-ethylphenoxy ring moiety of 6636424 and 4-fluorobenzyl of 7320244 are facing isoalloxazine ring unlike their lead Pioglitazone in which thiazolidinedione ring is facing the flavin ring. This suggests that different orientations of molecules at the MAO-B active site of these compounds may affect their inhibitory effects on MAO-B enzymatic activity. The thiazolidinedione rings of the test compound 6373721 and 7138125 are facing the flavin ring of the FAD cofactor as shown in figures (61, 62,63,64,65 and 66).

Most active test compounds are also screened for their potential inhibitory effect on another flavin dependent enzyme (LSD1). LSD1 is flavin dependent enzyme share a

20% sequence identity with MAO enzymes and found to be expressed in certain cancers94. Inhibition of LSD1 has important therapeutic implications in cancers and neurodegenerative diseases where its inhibition may reactivate gene expression that inactivated or silenced by LSD1 in those diseases146,147. Because MAOs and LSD1 share similar oxidative deamination reaction of the amine substrates so many MAOs inhibitors are screened for their potential inhibitory effect on LSD1147. MAO enzymes catalyze the oxidative deamination of amine substrates through formation of covalent flavin adduct at

127

C4a position of the flavin ring while LSD1 enzymes catalyze the oxidative demethylation of Lys4 of histone by forming covalent adduct at N5 position of the flavin ring.

There are several established functional groups (Tranylcypromine159,

Propargyline158, Hydrazine147 and polyamine180) that reversibly and irreversibly inhibit the LSD1 enzymatic activity by forming flavin adduct on N5 position of isoalloxazine ring. All five thiazolidinedione based test compounds 6366286, 6373721, 7138125,

6636424 and 7320244 do not have a significant inhibitory effect on the enzymatic activity of LSD1. They slightly decrease LSD1 enzymatic activity into 90% - 96% of the control (100% activity).

Docking studies as shown in 3D figure (51, 53, 55, 57 and 59) and 2D (50, 52, 54,

56 and 58) representations of the five test compounds at the active site of LSD1 crystal structure; have shown that required LSD1 binding free energy of these compounds are ranges between (-5) to (-7) that are higher than that required for MAO-B ((-8) to (-10)).

In addition the inability of these compounds to inhibit LSD1 enzyme may partly be attributed to their folded (bent) conformation that force thiazolidinedione ring of the compounds to be in distant locations away from the flavin active site. In our docking studies all test compounds adopt an extended conformation in substrate binding site of

MAO-B that result from the effect of narrow substrate active site with volume of (700Aº) and aromatic cage steric effect. Lack of aromatic cage in LSD1 enzyme and wide substrate binding site with volume of (1245Aº) allow these compounds to adopt folded conformations as shown in figures (66, 67, 68, 69 and 70). Only test compound 7138125 formed H-bond with a length of 3.04 on N5 of the flavin ring of LSD1 enzyme. Previous

128

studies have shown that the strength of H-bond with distance of 2.5-3.2Aº is considered as moderate and mostly electrostatic181.

Most active compounds are also screened for their potential distribution in the brain by measuring their ability to bind bovine serum albumin in addition to their potential permeability through blood brain barriers. Human serum albumins (HSA) are soluble constituents of the circulatory system that can bind and transport organic and inorganic molecules into various tissues including C.N.S. Binding of the compounds with human serum albumin can affect pharmacokinetics and pharmacodynamics of the test compounds where it can alter their volume of distribution, decrease rate of clearance, and increase its half-life 182,183.

Since there is a structural homology between HSA and bovine serum albumin

(BSA), so BSA is used to assess or screen ability of these compound to bind HSA.

Moderate human serum albumin binding is an important feature for our test compounds

(50%-60% of protein binding). Highly protein bound drugs are associated with very low amount of free fraction which require administration of large doses to achieve desired tissue distribution that may be associated with high risk of adverse reactions. Low protein bound drugs (free unbound fraction is very high) cannot reach active site due to its inability to bind albumin for transport into the target tissues.

Test compounds 6366286 and 7138125 are low protein bound compounds. They have low binding affinity to BSA with EC50 of ~100μM and 15 μM respectively. Only small fraction of the compound will be transported into the target tissues for therapeutic effect with low biological half-life and increased rate of clearance. Test compounds

129

6373721 and 7320244 have moderate serum albumin binding affinity (between 50%-

60%) with EC50 of 12μM and 9μM respectively so that low doses of the drug are required to achieve therapeutic concentration. Test compound 6636424 show high protein binding affinity that lead to increase its volume of distribution and minimize its elimination half-life. It reduce the percentage of free serum albumin into about 30% of the control (100% free BSA).

All five test compounds can cross the blood brain barrier and reach brain tissues with reasonable concentrations where they have been shown high blood brain barrier

(BBB) permeability with Pe between ~ 100-400 and logP between -3.3 and -4.3 according to the BBB permeation predictions177. The PAMPA BBB-permeation ranges

-6 -1 suggest that compounds with Pe (10 cm ) > 4.0 are considered as highly BBB permeable177.

130

Figure (61) show a 3D representation of docking of test compound 6366286 in MAO-B crystallized structure. The Ile199 residues and side chains, Tyr326, and Phe103 side chains are in blue, the aromatic cages residues (tyrosyl residues; Tyr398 and Tyr435) are in red. The test compound is in green and FAD is in yellow. The figure is created by Chimera v.1.8.1.

131

Figure (62) show a 3D representation of docking of test compound 6373721 in MAO-B crystallized structure. The Ile199 residues and side chains, Tyr326, and Phe103 side chains are in blue, the aromatic cages residues (tyrosyl residues; Tyr398 and Tyr435) are in red. The test compound is in green and FAD is in yellow. The figure is created by Chimera v.1.8.1

132

Figure (63) show a 3D representation of docking of test compound 6636424 in MAO-B crystallized structure. The Ile199 residues and side chains, Tyr326, and Phe103 side chains are in blue, the aromatic cages residues (tyrosyl residues; Tyr398 and Tyr435) are in red. The test compound is in green and FAD is in yellow. The figure is created by Chimera v.1.8.1.

133

Figure (64) show a 3D representation of docking of test compound 7138125 in MAO-B crystallized structure. The Ile199 residues and side chains, Tyr326, and Phe103 side chains are in blue, the aromatic cages residues (tyrosyl residues; Tyr398 and Tyr435) are in red. The test compound is in green and FAD is in yellow. The figure is created by Chimera v.1.8.1

134

Figure (65) show a 3D representation of docking of test compound 7320244 in MAO-B crystallized structure. The Ile199 residues and side chains, Tyr326, and Phe103 side chains are in blue, the aromatic cages residues (tyrosyl residues; Tyr398 and Tyr435) are in red. The test compound is in green and FAD is in yellow. The figure is created by Chimera v.1.8.1.

135

Figure (66): 3D representation that shows the orientation of test compound 6366286 inside substrate binding site in LSD1 enzyme. The image is created by Chimera v.1.8.1.

136

Figure (67): 3D representation that shows the orientation of test compound 6373721 inside substrate binding site in LSD1 enzyme. The test compound is in blue and FAD cofactor is in red. The image is created by Chimera v.1.8.1. The H-bonds are not shown.

137

Figure (68): 3D representation that shows the orientation of test compound 6636424 inside substrate binding site in LSD1 enzyme. The test compound is in blue and FAD cofactor is in red. The image is created by Chimera v.1.8.1. The H-bonds are not shown.

138

Figure (69): 3D representation that shows the orientation of test compound 7138125 inside substrate binding site in LSD1 enzyme. The test compound is in blue and FAD cofactor is in red. The image is created by Chimera v.1.8.1. The H-bonds are not shown.

139

Figure (70): 3D representation that shows the orientation of test compound 7320244 inside substrate binding site in LSD1 enzyme. The test compound is in blue and FAD cofactor is in red. The image is created by Chimera v.1.8.1. The H-bonds are not shown.

140

4.1.1. Structure activity relationships:

The most active test compound (6366286) (derived from the lead (Pioglitazone)) is associated with 10 times (IC50 = ~30nM) more MAO-B inhibitory effect than that of

Pioglitazone (IC50=~300nM). several structural differences exist between these two compounds such as; methyl substitution of the nitrogen of Pioglitazone thiazolidinedione ring, double bond connect thiazolidinedione to the rest of the test compound, one carbon instead of two in the linker between the two aromatic rings of the test compounds and dichlorobenzyl ring of test compounds instead of ethylpyridine ring of Pioglitazone as shown in figure (71).

Docking studies (figure 72) have shown a flip in the orientation of test compounds 6366286 and 6838234 as compared to that of Pioglitazone inside MAO-B substrate binding site with dichlorobenzyl ring of compound 6366286 and 6836234 facing the flavin ring. Upon reversing the binding direction, the presence of electron withdrawal group like chlorine atoms at para- position seems to be able to reduce the

IC50 values greatly by collaboration with aromatic cage dipole moment. Methyl substitution of the nitrogen atom of thiazolidinedione ring seems to be favored since corresponding propargyl substitution in test compound 6836234 lowers MAO-B inhibitory effect (IC50 = 1.044μM) as compared to that of Pioglitazone.

The elongation of the linker (figure 73) between the two aromatic rings of test compounds 6636424 (IC50 = ~200nM) as compared to that of pioglitazone (IC50 =

300nM) seems to increase the MAO-B inhibitory effect. Chlorine substitution at ortho position instead of para substituted ethyl group of the second aromatic ring as in test

141

compound 6634507 (IC50 = 3.9μM) is unfavorable because it may reduce MAO-B inhibitory effect where ortho chlorine substitution prevent favorable interaction between thiazolidinedione ketones and water molecules within substrate binding site as illustrated in recently published study179.

Although the chemical structures of test compounds 6373721 (0.04μM) and

6634507 (IC50 ~ 4μM) are identical (see figure 74); docking studies (figure 75) have shown that their hydrophobic interactions and orientations inside substrate binding site of

MAO-B crystals are different and associated with different binding energy and inhibition constants. This orientations result in different actual MAO-B inhibitory effect where test compound 6373721 is 100 times more potent than compound 6634507.

Increasing hydrophobicity of the test compound 5472855 (IC50 = ~1.5μM) by para-methyl substitution enhance MAO-B inhibitory as compared to that test compound

6209863 (IC50 = ~8μM). See figure (76).

142

Figure (71): chemical structures of lead compound Pioglitazone and its derivatives

6366286 and 6838234. The chemical structures were obtained from hit2lead

(www.hit2lead.com).

143

Figure (72): Three dimensional image of Pioglitazone, test compound 6366286, and

6836234 orientations in MAO-B crystal structure. The ligand is green, the gating residues

(Ile199 and Tyr326) are blue, the aromatic cage residues (Tyr398 and Tyr435) are orange, carbon atoms are green, sulfur atom is yellow, chlorine atom is cyan oxygen atom is red and nitrogen atom is blue. The image is created by Pymol v 1.3.

144

Figure (73): Chemical structures of lead compound Pioglitazone and its derivatives 6636424 and

6634507. The chemical structures were obtained from hit2lead (www.hit2lead.com).

Figure (74): Chemical structures of Pioglitazone derivatives 6373721 and 6634507. The chemical structures were obtained from hit2lead (www.hit2lead.com).

145

Figure (75): Three dimensional docking of test compound 6373721 and 6634507 orientations in MAO-B crystal structure. The ligand is green, the gating residues (Ile199 and Tyr326) are blue, the aromatic cage residues (Tyr398 and Tyr435) are orange, carbon atoms are green, sulfur atom is yellow, chlorine atom is cyan oxygen atom is red and nitrogen atom is blue. The image is created by Pymol v 1.3.

146

Figure (76): chemical structures of 5472855 and 6209863. The chemical structures were obtained from hit2lead (www.hit2lead.com).

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