Scientists working on MAO – part 2



Kálmán Magyar
University Professor
Department of Pharmacodynamics
Semmelweis University of Medicine, Budapest, Hungary
1970 PhD
1978 D. Sc. (doctor of science)
1987 member of the Hungarian Academy of Sciences

My main scientific contribution is firmly connected to deprenyl (phenyl-isopropyl-methyl-propargylamine) research, which is an irreversible, B-type selective monoamine oxidase MAO inhibitor. During the late fifties and the early sixties many MAO inhibitors have been synthesized (iproniazid, phenelzine, tranylcypromine) in order to develop antidepressive agents. As the other MAO inhibitors, deprenyl was synthesised also as a psychic energizer (1964). Clinical experiences declared mostly good effectiveness of these compounds in depression, but after the intake of tyramine containing foods (cheese, red wine, salted herring) they induced serious side effects called “cheese effect”, which resulted in life dangerous hypertonic crises. Because of this toxicity all of the MAO inhibitors have fallen into disrepute and tricyclic antidepressants became the drugs of choice in the treatment of psychic depression. Deprenyl, as the other MAO inhibitors, has been dropped into the same basket. Nevertheless, we published that deprenyl has a peculiar spectrum of activity and it did not potentiate the effect of tyramine. We have shown, that the (-)-optical isomer of deprenyl is responsible for the inhibition of MAO (Magyar et al., 1967). Measuring enzyme activity 14C-labelled tyramine was used as a substrate and the inhibition profile, surprisingly seemed to have a double sigmoid shape. I was seriously criticised by my colleges because in their opinion the enzyme inhibition profile usually seems to be simple sigmoid one. Luckily, due to my obstinacy, I firmly stuck to my findings, which reflected on the heterogenous nature of the enzyme. Meanwhile, the outstanding studies of Johnston with clorgyline revealed in 1968, that MAO has different isoforms, such as MAO A and MAO B, according to its substrates specificity and inhibitor sensitivity. The preferred substrates for MAO A are serotonin (5-HT) and noradrenaline (NA), while for MAO B are beta-phenylethylamine and benzylamine. There are common substrates for both types of MAO such as tyramine and dopamine. In 1972 we published (Knoll and Magyar), that in contrast to clorgyline the selective inhibitor of MAO A, (-)-deprenyl (selegiline) is a selective irreversible inhibitor of MAO B. The paper became a “Science Citation Classic,” which has been cited nearly a thousand times in the literature. Since 1972 selegiline played an essential role in MAO research and it has become the “golden standard” of MAO B inhibitors. That was the only MAO inhibitor, which survived the shock, caused by the “cheese reaction”.

From these studies it became clear that selegiline in a dose required for selective MAO B inhibition, does not inhibit the metabolism of 5-HT and NA, which are playing the most important role in mood disorders. Due to the formers selegiline does not possess definitive antidepressive properties. The lack of its tyramine potentiation may partly explain that selegiline does not induce “cheese effect” frequently observed in patients treated with non-selective or MAO A selective inhibitors after ingestion of foods, rich in directly acting sympathomimetics, such as tyramine. Selegiline inhibits the metabolism of dopamine (common substrate) and it leads to a symptomatic improvement in parkinsonian movement disorders. The irreversible MAO A selective and the non-selective inhibitors proved to be good antidepressants, but because of their “cheese effects” they cannot be used in the treatment of depression, without serious food restrictions. Selegiline is not the only substance, which ultimately found the place of its therapeutic application in a totally different field, than its original clinical indication. Austrian, German and Hungarian clinicians played essential role to find its clinical effectiveness. Its name is so strongly incorporated into scientific knowledge, that the originators and the original country, Hungary, where it has been developed, almost totally forgotten.

Selegiline has a seesaw fortune. Its pharmacological activity is rather complex, which cannot be explained totally with its MAO B inhibitory action (Magyar et al., 2004). Its meaning went up-words, when in pre-treatment protected the toxicity of dopaminergic, noradrenergic and cholinergic exogenous neurotoxins (MPTP, DSP-4, AF64A). In many laboratories, including in ours (Magyar and Szende, 2000) it was proved that selegiline possesses antiapoptotic activity in tissue cultures, induced by neurotoxins or serum deprivation. It increases cell-cell adhesions, the physiological role of it is not known. The metabolit conversions of selegiline, including its distribution, elimination were widely analysed in my laboratories. An intensive “first pass” metabolism of selegiline was proved in the case of its oral application, which ought to be taken into consideration in the case of clinical application of the drug. To avoid “first pass” conversion of the drug the development of transdermal or lyposomal preparations should be considered, possessing better pharmacokinetic properties.

There is no MAO inhibitor in the palette, which has been more widely studied as selegiline was. Soon after I finished my university studies in the Semmelweis Medical University of Budapest, I went to London in 1961, where I studied GABA formation in the tricarboxylic-acid cycle of the brain. Coming home, my boss gave me the task to determine the enzyme inhibitory action of selegiline, the patent name of which was E-250 at that time. I thought this request is so simple, and I will be able to finish it during a month. Now I realised that it will never be really finished. Clinical observations indicated many beneficial and contradictory data, regarding the therapeutic action of selegiline. Among these it was declared, that selegiline improves the quality of life, prolongs life expectancy and slows dawn aging. The only thing I am not convinced about is its action on aging. I can show you my photos, one of them has been taken in 2005 and the other one 30 years ago. I leave the decision on the readers.

On the horizon, I can see nothing except just continuing to do research with the aim of improving the condition of neurodegenerative disorders. At the moment we can treat diseases, but not to cure them. Nerves are post-mitotic cells, which cannot divide or regenerate; because of this the field is not very promising for pharmacological research. Nevertheless, when the life expectancy of the population becomes longer, then we have to realize that more and more disabling neurodegenerative diseases will affect mankind. Scientists should think about how to fulfil the requirements of living longer and living a liveable life.

References:

Magyar,K., Vizi,E.Sz., Ecseri,Z., Knoll,J.: Comparative pharmacological analysis of the optical isomers of phenyl-isopropyl-methyl-propinylamine (E-250). Acta Physiol. Acad. Sci. Hung. 32 (4) 377-387, 1967.

Knoll,J., Magyar,K.: Some puzzling pharmacological effects of monoamine oxidase inhibitors. Adv. Biochem. Psychopharmacol.  5, 393-408, 1972.

Magyar,K., Pálfi,M., Tábi,T., Kalász,H., Szende,B., Szök_,É.: Pharmacological aspects of (-)-deprenyl. Curr.Med.Chem., 11 2017-2031, 2004.

Magyar K,  Szende B: The neuroprotective and neuronal rescue effect of (-)-deprenyl. in: Handbook Exp Pharm, (Eds: Cameron RG, Feuer G) Springer, Heidelberg, 142, 457-472, 2000.

 


A Japanese team of researchers:

Professor W. Maruyama, PhD
Department of Neurology
Nagoya University School of Medicine
Nagoya, Japan
&
Laboratory of Biochemistry and Metabolism
Department of Basic Gerontology
National Institute for Longevity Sciences
Obu, Japan
…………………………………………………………….
Professor Makoto Naoi, MD, PhD
Department Head
Department of Neuroscience
Gifu International Institute of Biotechnology
Kakamigahara, Gifu, Japan
received PhD in 1965

 

MAOI or NAOI?
When I was a postgraduate student, I wrote a paper as the first author, and by the publication, the name of the first author was printed as MAOI. Since then I have been called “a man of a MAO inhibitor”. Still now I am working on the intracellular function of MAO and MAOI. At last we found that MAO inhibitors show neuroprotective function. Please, check carefully your name in your paper before the publication, even though you may misspell the name of your professor!

Fig. 1: An endogenous neurotoxin, N-methyl(R)salsolinol, induces mitochondrial permeability transition and apoptosis in MAO A only expressing cells.

Fig. 2: Reactive oxygen species (SIN-1) induce apoptosis in neuronal cells, as stained with Hoechst 33314. An MAO B inhibitor, rasagiline, protects the cells from apoptosis.

 


Lars Oreland
Dr. of Pharmacology, Medical Faculty and Head of the Department of Neuroscience
Uppsala University, Uppsala, Sweden
received PhD Jan 1971

MY RESEARCH
My thesis dealt with purification and characterization of pig liver MAO. All the time thereafter I have kept an interest in studying the structure, properties, regulation and physiological functions of the MAO enzymes.

However, being a medical student, the question immediately arose after my Ph.D.: what might be interesting with this enzyme from a medical point of view. Thus, we started analyzing MAO activity in brain tissue from suicide cases of whom many had been alcoholics. Already in those first studies we noticed an increase in MAO B activity with age and ever since then, the role of the MAO enzymes in aging and neurodegenerative disorders, especially Alzheimer’s Disease, as well as disorders with an association with temperament, such as depressive disorders/suicidal behavior, substance abuse and various forms of antisocial behavior, have been favorite themes for my research.

Already at the time of my thesis it was clear that platelet MAO should provide an easily accessible source of human MAO and a substantial part of my research has been spent on the question to which extent human platelet MAO (which is highly genetically controlled and stable over life-time in the individual) reflects monoaminergic (serotonin in particular) capacity of the brain. Key findings within this area are associations between low platelet MAO activity and personality traits related to impulsiveness and sensation seeking. This line of research has brought an immense amount of fun with studies on MAO, personality and neuro-physiological tests of various groups of students, Himalaya mountaineers, boxers, air force pilots, heavy criminals etc. The associations between platelet MAO and personality have not always been possible to reproduce. My guess is that part of the explanation for some of the failures is that the variations in MAO activity are not that great and that the need for a very precise method for the analysis cannot be underestimated. The question about which mechanisms are underlying those associations have been residing in my head now for some 40 years. The existence of the associations have been beautifully confirmed in studies on free-living monkeys, in whom a lot of possible confounding factors in humans can be excluded. With regard to the molecular nature of the association, it is likely to be understood within a short time with the help of all new techniques in the area of molecular genetics. Thus, the answer is likely to be found in some transcriptional mechanisms shared between genes involved in the expression of platelet MAO and genes of importance for central monoaminergic function.

During this journey there have been a lot of opportunities to make excursions into related research areas such as other enzymes connected with personality or neuro-degeneration and especially around the sister enzyme, the semicarbazide-sensitive amine oxidase, usually named SSAO. During recent years, work involving molecular genetics has also initiated studies on a variety of genes expressing other proteins than MAO into the picture, especially genes expressing transcription factors of importance for monoaminergic genes, such as a family of transcription factors called AP-2.

FUN
Especially in the early times, I was rewarded by all the opportunities of traveling to places I would otherwise never have seen if I had chosen another career. Even before my PhD I had guest researchers coming from Russia and Japan and such visits, of course, generate long-standing friendships with a lot of visits in either direction. Certainly the story of everyone is unique and my early story, as well as that of most others, contains both frustration and reward. I started with the MAO purification project in 1962 and was allowed to work almost completely on my own from the very start. However, in 1969, that is, after seven years, nothing at all of any interest had come up, in spite of my (not always very ingenious) efforts. It was frustrating to see my previous fellow medical students entering promising careers as doctors. However, a few months before one of my own self-imposed deadlines, things started to go right and a kind of catch-up effect brought guests to my lab. In June 1971 it also brought an invitation to Caliari, Sardinia, the first–and probably still most exciting–of a long row of future Amine Oxidase meetings in different places around the world. But what if this break-through had not happened? My message is that choosing a research career involves a component of gambling. For sure, lack of luck can be partially compensated by hard work, but an extra piece of luck can add a lot to it…. Looking back at the list of contributors to the 1971 meeting shows how a kind of MAO family was formed, since several of those people still, 35 years later, regularly meet at different events. For anyone interested, I have outlined my Amine Oxidase story with some more details in a book chapter. (Oreland L. Amine oxidases; from purification to transcription – a very personal view. In MAO inhibitors and their role in neurotransmission (drug development). Eds Török TL and Klebovich I. Medicina Publ House Co Budapest 2004, p. 115-132.).

 


Dr. Rona R. Ramsay
Reader in Biochemistry, University of St Andrews, UK
received PhD (1977) University of Cambridge, UK

DATA ON MAO:
It was after I established that accumulation of the neurotoxin MPP+ by respiring mitochondria was a critical step in its toxicity that I got involved in the metabolism of MPTP to MPP+ by MAO. It proved a career move because I have studied it as funding allows for the past 15 years.
a. My favorite part is rather deep into scientific specialism since it was working out the kinetic mechanism of the enzyme. MAO has two forms, MAO A and MAO B. The A form in neurons is stimulated to go faster by the presence of high levels of its substrate thus protecting the mitochondria (critical sources of energy for the neurons) from inhibition. The B form found in the glial cells surrounding the neurons is less stimulated by its substrates but is critically dependent on oxygen. When the brain is low on oxygen it will not waste it on this reaction.

Recently, I have worked with Astra-Zeneca to make its novel oxazolidinone antibiotics safe. The lead compound for these drugs was known to inhibit MAO. MAO is a promiscuous enzyme that will metabolize almost anything with an amine group that is either relatively flat or flexible and hydrophobic. MAO in the intestine provides protection from ingested amines in drugs or food such as cheese. By exploring many derivatives of the oxazolidinones we were able to identify features that favored the antibiotic activity but prevented binding to MAO. The Astra-Zeneca chemists synthesized the compounds, we measured the inhibition of MAO A (they are poor inhibitors of MAO B) and together we modeled the structures into the crystal structure of MAO (illustrated on right) to show how the compounds interacted.

My best moment as a graduate student came only three months after I have started at Cambridge in the laboratory of Dr Philip K. Tubbs. I had to persuade my supervisor that I really had reliable data indicating that I had discovered a new mitochondrial transport system. The mitochondrial carnitine exchange system is now in all the textbooks.

The reality that research is 99% perspiration set in during my post doc at UCSF when I made no headway (working on the huge 46 component Complex I that is the start of the mitochondrial energy production pathway) for the first 6 months. A cartoon in the San Francisco Chronicle consoled me: it showed an elephant with a mortarboard and a PhD diploma with the caption, “The worst is yet to come”. So true at that point but I have continued to have fun, to be the prepared mind at the right time to make discoveries. (For example the role of MAO in producing from MPTP the neurotoxin that induces Parkinson’s Disease.) Research is ALWAYS fun.

Finding a balance in life is very important. I have great joy from both sides of my life – both research (and teaching) and my family of four children.

 


Dr. Peter Yu is a Professor of Neuropsychiatry at the University of Saskatchewan, Saskatoon, Canada

His chief areas of clinical practice include:
neurochemistry, oxidative deamination, vascular disorders, Alzheimer’s Disease, and protein mis-folding. His recent research interests include Parkinson’s Disease, regulation of glucose transport and weight gain, diabetic complications, atherosclerosis, and oxidative stress.

 


Dr. Keith Tipton, BSc, MA, PhD
Professor of Neurochemistry, Enzymology and Systems Biology
School of Biochemistry and Immunology
The University of Dublin, Trinity College

Research Interests of the group include:

1. Neurochemistry: The Metabolism of neurotransmitters in the brain and the interactions of drugs with these processes. Amine metabolism and drug interactions in the central nervous system and periphery. The behaviour of the enzymes of amine metabolism, their interactions with drugs that behave as antidepressants or neuroprotective agents. Optimal structure-function relationships of monoamine oxidase inhibitors. The functions and interactions of enzyme inhibitory drugs of potential value in the treatment of depression and Parkinson’s Disease. The interactions of ethanol and amine metabolism, with particular reference to the formation of alkaloids which may be involved in the addiction or tolerance processes.

2. Enzyme behaviour, enzyme inhibition and enzymes with alternative functions:
Mechanisms through which the copper-containing amine oxidase (semicarbazide-sensitive amine oxidase; EC 1.4.3.6) also functions as a vascular-adhesion protein and modulates cellular glucose uptake.

Mechanisms of the complex behaviour shown by the oxidase reaction catalysed by peroxidase (EC 1.11.1.7).

The multiple functions of ceruloplasmin (ferroxidase; EC 1.16.3.1).

The behaviour and applications of suicide inhibitors and suicide substrates.

3. Systems biology: Systems biology is the application of computational approaches to the understanding of the normal and dysfunctional behaviour of cellular systems.
Current projects include:
(i) Pathway tracing and metabolite connections.
(ii) Regulation of glucose transport in adipocytes and metabolism in normal and diabetic states.
(iii) Regulatory behaviour of branching processes.
(iv) Function and dysfunction of the aminergic nerve terminal.
(v) Complex dynamics and their biological significance.

The systems biology website is at http://www.systemsbiologyireland.org/

4. Enzymes, metabolic systems and Enzyme Bioinformatics:
We are responsible for curation of the International Union of Biochemistry and Molecular Biology’s (IUBMB’s) Enzyme List. This is a functional classification system in which novel enzymes are classified according to the reactions they catalyse, with each new enzyme being assigned an EC number. The work also involves updating entries for previously classified enzymes in the light of current knowledge. As the EC system is used extensively in many bioinformatics efforts, our group collaborates with many of the major world-wide bioinformatics centres, including the European Bioinformatics Institute, Cambridge (IntEnz), Kyoto University Bioinformatics Center (KEGG), University of Geneva (ExPASy and SWISS-PROT databases), University of Cologne (BRENDA database), University of Missouri at Columbia (KLOTHO database) as well as with IUBMB and IUPAC in this work.