Kommersant: ‘Let’s remove the smell of rot, and your emotional state will improve’

Rotten fish smells disgusting to us — this is how our sense of smell protects our health. We unconsciously select the perfect life partner by smell. Thus the immune system protects the health of our future offspring. Both smell-related disgust and attraction are triggered by molecules of the same class, the so-called trace amines. We seem to perceive these molecules on a much deeper level than just by olfactory receptors in our nose. Recently, trace amines and their receptors have come to the forefront of advanced neuropharmacology. One of the most cited researchers in the world in this field, Raul Gainetdinov, Director of the Institute of Translational Biomedicine at St Petersburg University, explained the potential benefits of trace amines, how rats are better than mice, and how he was able to keep his laboratory going in the 1990s with money from MMM (Russia’s largest Ponzi scheme company founded in 1989 by Sergei Mavrodi).

Let’s start from the beginning. How did you become a biologist?

It runs in the family. I came from a family of physicians. My father was Chief Physician and my mother was Deputy Chief Physician of the Republican Children’s Clinical Hospital in Ufa. One of my aunts was a head of a hospital department and another aunt was Chief Paediatrician of the Republic of Bashkiria. One of my uncles was a head of a department in the Clinical Hospital, while another uncle was Head of Accidents and Emergency Services in Ufa. I grew up in this medical family. Every year, we celebrated the national Doctor’s Day. From my childhood, I knew that I was to go into medicine. But at the same time, I understood early on that treating patients in a hospital clinic is not enough for me. I wanted to do research. Indeed, I couldn’t verbalise my thoughts back then, because I had no idea there was such a thing as a research physician, but I felt exactly that — I was interested in medicine as a science.

Then, I got lucky. By chance, I entered the Faculty of Biomedicine of the 2nd Moscow State Medical University, where they were training research physicians. That is how I ended up graduating as a biochemist without the right to practice medicine. In fact, I got exactly what I wanted when I was 14. It was a very strong science education: five years of chemistry and biology; five years of mathematics; five years of physics and everything a clinical researcher would need besides medicine itself, which we studied, like all medical practitioners, for six years.

After graduation, I came to work at the Institute of Pharmacology of the Russian Academy of Medical Sciences. I became a junior research associate in the laboratory led by Kirill Rayevsky. He was a remarkable scientist. His primary research focus was antipsychotic drugs that block the D2 dopamine receptor. At the time, it was the best dopamine laboratory in the country! I became fascinated with dopamine. I began measuring dopamine levels in experimental animals at different states, I was reading research literature on the subject — that is how it all started. Dopamine has been my main subject since graduation thesis, which was about measurements of dopamine levels in various brain structures. I was lucky, indeed.

That laboratory was where you wanted to get, wasn’t it?

No, as a matter of fact, it wasn’t. I got into that laboratory by chance. The thing is, I was not very good at molecular biology, which was very popular. The top students went into genetics and molecular biology, while the Institute of Pharmacology was not really popular with graduates back then. I chose "boring" pharmacology because it is closer to medicine, like everyone in my family.

Why on earth is it "boring"?

It is commonly considered "boring" and it is still not very popular. It may appear as though all you do is routine pharmacological testing. It seems unlikely that you may be the one to find a pharmaceutical agent that will be used to treat millions of patients. The citation rate in pharmacology is also lower. But I have no regrets. I found my niche when I got into the dopamine field. It was great — when I was still young, I already knew personally all the leaders of my field, including Nobel Prize laureate Arvid Carlsson who had discovered the role of dopamine in the brain. I knew Oleh Hornykiewicz who had proposed the most effective anti-Parkinsonian medication titled levodopa. This drug has remained the gold standard in the medicinal treatment of Parkinson’s disease since 1958. I had known them all before I went to America thanks to Kirill Rayevsky, who took me to international conferences overseas. It was a tremendous source of insight and inspiration.

In 1992, I defended my candidate’s dissertation and spent another four years trying to fight for the opportunity to do science in Russia. The 1990s were "funny" times: for three years, I supported my laboratory with a "grant" from MMM.

You received a grant from Sergei Mavrodi, did you?

No, of course not. It wasn’t a research grant from Mavrodi. I invested in MMM myself, quite early on... I took a risk and managed to get the money out. It was that money that I used to feed my lab team and my family for three years.

In 1996, it became clear that I would not be able to do science in Russia. My lab team fell apart, and I left for the USA. I became a student of a man called Marc G Caron and joined his laboratory at Duke University (Durham, North Carolina). To our deepest regret, he passed away last year. Back then I believed — and I still believe — that Caron was number one in the dopamine field (after Nobel Prize winner Arvid Carlsson). He was an outstanding multidisciplinary researcher, equally strong in pharmacology, animal behaviour and molecular biology. There were no other experts like him back then. Nobody else was equally well-versed in all those things at the same high level. Caron would ask: have you done such and such a behavioural test on animals? And in a flash, he would discuss receptor mutations and their biochemical properties. I was utterly impressed when I met him at a conference. Then I decided that if I were to go to work overseas, I would only go to work with him.

You mean, you didn’t write dozens of postdoc applications, as people usually do now?

No, I didn’t. In fact, there are two anecdotes about my employment at Duke University Medical Center. I knew Caron was a Canadian but I didn’t know that he was working in the USA at the time. I wrote him that one letter saying that I would want to work with him, but could I work in Canada and not in the USA? I thought that he had two laboratories: in Canada and in the USA. I can imagine him heartily laughing about it. He replied: Come, but only to the USA, sorry!

The second anecdote is this. I wrote to Caron in February. Roughly at the same time, he published a paper in the journal Nature. That was a breakthrough. At this point, a digression is in order. Shortly before that, gene knockout animals came along. The first one was created in 1989 by Oliver Smithies, who later was awarded the Nobel Prize for the development of gene targeting strategies in mice. Scientists began to think about how knockout animals could be used in physiology and pharmacology research. The first to enter the knockout race were immunologists. It is less troublesome for them to do experiments — they take blood and carry out a laboratory blood test. So, immunologists were first to use the knockout mouse technology to create animal models of human disease. Experiments in neurosciences are different — we need to study behaviour and obtain statistics. Hence, initially the neurosciences were quite sceptical about knockouts... until that 1996 paper by Marc G Caron. The paper was about knockout mice lacking the dopamine transporter. This means they have high levels of dopamine — they are sort of genetically on cocaine. The mechanism of dopamine reuptake was removed — this is how cocaine and amphetamine work. So, Caron created those mice "on genetic cocaine", and then, all neurophysiologists and pharmacologists were amazed that such a technology exists. They began to ask Caron questions about how much the dopamine levels increased? And as you remember, the measurement of dopamine levels had been my specialisation since my graduation project. In short, Caron, having received my letter, thought that I was asking him because of these mice — in other words, because I thought that my skills could be in demand. The fact of the matter is, at that time, we were lagging behind in Russia. I didn’t even know about that paper of Caron! It was just a lucky coincidence. I came to Caron, we began to work together on this very hot topic, and we published over a hundred research papers on genetically modified rodents with knocked out genes.

What a remarkable coincidence!

Indeed. I became Assistant Professor at Duke University pretty soon. When I left the USA 12 years later, Marc G Caron had 47 lines of knockout animals in his laboratory. By comparison, in the Institute of Translational Biomedicine at St Petersburg University there are currently nine lines of knockout animals, and we are already overcrowded. And Caron had 47 lines! Just keeping and looking after genetically modified animals is costly and burdensome. Anyway, Caron’s lab was a real paradise for a researcher. You could go and test any ideas straight away. Imagine, you get an idea that a certain protein is responsible for something, and you just go and test it here and now.

You know, rising inflation does affect researchers. In recent years, the cost of maintaining large laboratory colonies has become prohibitive; even two or three grants may not be enough. Then, it wasn’t a collaborative research centre, it was Caron’s laboratory. He maintained lab animals at his own expense as a kind of research infrastructure. The cost of maintenance was up to $30,000 a month. Since then, I think, this figure may have doubled. That time was a golden age of the gene knockout strains in pharmacology research.

Are we talking about rats or mice?

Rats, of course, are always preferable, but they are more difficult to work with. Back then, genome-editing technology was available only for mice. Genetically modified rats came along later, only when the CRISPR-Cas gene-editing method became available. In fact even now, few laboratories have knockout rats, mostly due to inertia — everyone already has the equipment for mice. In the context of psychopharmacology, rats are better because they are closer to humans in many aspects. Rats are very intelligent animals, and since we are studying cognitive abilities this is important for us. You can study much more subtle nuances using rats; their behavioural repertoire is much more elaborate. In fact, when knockout mice appeared, many cognitive scientists said that you cannot study cognition on mice because they are too primitive. In the past, experiments in cognitive science were always conducted using rats.

We seem to be getting way ahead of ourselves without defining the terms. What is the proverbial knockout and why is it important?

Good question. A knockout refers to the use of genetic engineering to inactivate a certain gene. The knockout animals are viable and appear normal, in spite of having one gene eliminated. Since that gene is "turned off", there is no protein that is encoded by that gene. Hence, such animals can be used as very good models for some human diseases, for instance, to test new therapeutic methods. These days, knockout animals are created using CRISPR-Cas gene-editing method, whereas previously other methods were used. Anyway, the idea is the same — to turn off a certain gene.

I see, the gene knockout is a means to an end, but what is the end goal?

I need knockout animals for my specific task: I am trying to find new targets for pharmacological agents to change something in the brain. I use knockouts as models of various disorders and diseases: for example, high dopamine levels can contribute to schizophrenia or drug addiction. I am searching for drugs that can affect this pharmacological target in mice. If it works on mice, then we can try it on humans. I have different animal models. I have rats with high levels of dopamine. I have rats that have no serotonin. And my main focus now is trace amines.

I call them cousins of dopamine. Their structures are similar. Trace amines were discovered before norepinephrine and dopamine, but no one could figure out their functional role in the brain.

Things became a little clearer when trace amine-associated receptors (TAARs) were discovered. TAARs are a class of G-protein coupled receptors (GPCRs). This class of receptors was discovered at Duke University in Robert Lefkowitz’s laboratory. In 2012, Lefkowitz was awarded the Nobel Prize for his discovery of GPCRs. Robert Lefkowitz was Marc G Caron’s postdoc supervisor. Later, Caron opened his own laboratory next door to Lefkowitz. In fact, these two labs worked in conjunction. Lefkowitz and Caron sat in adjacent offices throughout their lives. I worked closely with both of them and consider them both my teachers. As a matter of fact, Caron was even better known for his work with dopamine receptors, which are also GPCRs. He cloned two of the five types of dopamine receptors, although he never won a Nobel Prize, alas. So, they discovered the first trace amine-associated receptor (it was the b2 adrenergic receptor activated by adrenaline) and boldly claimed in their paper in Nature that there would be many such receptors. There turned out to be over 800 of them! They could not have imagined that there would be so many of them. It turned out that 4% of the genome are GPCRs. In other words, humans have around 20,000 genes, and there are about 800 GPCRs encoded by the human genome, which play a role in an incredible array of functions in the human body.

GPCRs bind to a large class of neurotransmitters and neuromodulators such as: histamine, opioids, norepinephrine, serotonin, dopamine, and so on. They convert extracellular signals into intracellular responses. Thus, GPCRs have great clinical importance, which is reflected in pharmacology − up to 40% of all modern therapeutic treatments target GPCRs by either activating (agonists) or blocking (antagonists) some of these receptors. For example, all currently known antipsychotics (over 40 of them) block the D2 subtype of dopamine receptors.

Now, it is time we returned to trace amines, right?

Exactly right. A lot was done on the wave of interest in the subject; in particular, new trace amine-associated receptors (TAARs), the analogues of dopamine, were discovered. At first, they were considered some of the so-called orphan receptors, because the chemicals that could activate them were unknown at the time. In 2001, when TAARs were discovered, Caron and Lefkowirz literally pushed this topic on me, and I’ve been researching it for over two decades now. Anyway, humans have six subtypes of these receptors. I began to study them in detail and got some very interesting data. And since the ligands are future drugs, I started collaborating with several pharmaceutical companies. In 2007, I decided to go to Genoa to work at the Italian Institute of Technology (Instituto Italiano di Tecnologia, IIT), which is regarded as the best Italian scientific research centre. I brought the TAARs topic to Italy with me because Marc G Caron did not have anyone willing or able to carry out this research. At the Italian Institute of Technology, I started collaborating with the Swiss pharmaceutical company F Hoffmann-La Roche. For five-six years, they supported our research. During that time, we jointly published 20-30 papers on topics related to TAAR1, the first of the six trace amine-associated receptors. Then, the company launched clinical trials on a TAAR1 agonist for psychosis in schizophrenia. That collaboration was a success, but eventually my work was done. Once clinical trials had begun, they did not need me anymore. This is the way it always happens. We carry out basic research — in particular, we proved therapeutic potential of TAAR1 agonists in schizophrenia − and then, we pass the baton to clinicians who conduct clinical studies.

As for myself, I continued to study other trace amine-associated receptors. In 2007, however, when I had just moved to Italy, the journal Nature published a paper by Nobel Prize Laureate Linda B Buck. In this paper, she claimed that all TAARs, except for TAAR1, are purely olfactory receptors. Buck was categorical about it: although structurally TAARS are more or less the same, their analysis shows that five of the six TAARs are located only in the olfactory epithelium and there are no such receptors in brain tissue. By that time, I had already started to study these receptors in depth, I already had the knockouts for that purpose, and I strongly disagreed with that statement. Indeed, TAARs function as olfactory receptors that are responsible for innate odours, such as rot or pheromones, the mechanism of how the cat detects a mouse or an antelope senses a tiger. But what kind of smells are these?

There was an interesting story. Our colleagues at Harvard University collected the urine of 400 different animals — both herbivores and carnivores — and it turned out that the carnivores had high levels of b-phenylethylamine in their urine. It is a derivative of the essential a-amino acid phenylalanine. b-phenylethylamine is abundantly found in meat products; hence, whoever eats meat has a high level of it. It is one of the best known trace amines. In general, trace amines are the products of decarboxylation of amino acids. In other words, we are all made up of 20 building blocks of proteins — amino acids. If the acid part (the carboxyl group) is removed from an amino acid, we get an amine, or, to be precise, a trace amine. As a result of decarboxylation of amino acids, at least 20 trace amines should be formed, that is, each amino acid has its amine. The acid part is removed either endogenously by the body’s own enzymes, or exogenously, by certain bacteria. The trace amines are particularly abundant in foods produced by bacterial fermentation — wine, cheese, cured meats, and beer. This is how we came to the point when we decided to study what we eat. It seems like the right choice for Bashkir-Italian researchers!

It is a kind of recognition system, isn’t it?

Yes, among other things. Take, for instance, the immune system. We have different bacteria living on our skin that are determined by the type of immune system. These bacteria metabolise amino acids to trace amines. And there is a theory that we select our perfect partner based on smell. Essentially, we choose a matching immune system for our future offspring. At least this has been shown in bats. Also, innate aversion to odour is a signal of danger. I’ve already mentioned predators and herbivores, but there are also smells of a dead body and rotting fish — these are also trace amines. They warn us to stay away.

That is how I came to the subject of smell, although I had never thought I would pursue it. Anyway, I firmly believe that these smells are more than just smells. Linda B Buck stated that all TAARs, with the exception of TAAR1, are located only in the nasal olfactory epithelium, but not in brain tissue. How did she determine that? She homogenised the brain tissue for RNA extraction. What if she just diluted them in a large volume of the brain tissue and failed to see that TAARs could only be found in certain groups of neurons? All these years, I have been trying to prove that Buck was wrong: these receptors are expressed in the brain, and they are emerging therapeutic targets.

Has there been any progress on this?

Or course. I have been working exclusively in St Petersburg for seven years now. In 2015, the Institute of Translational Biomedicine at St Petersburg University was established under my leadership. We won an infrastructure development grant from the Russian Science Foundation and immediately opened five laboratories. At present, there are ten laboratories at the Institute. We renovated the vivarium so that it complies with the international standards. We have gene knockouts of all six trace amine-associated receptors. Some of the lines I brought from Italy, while other lines we bought or created ourselves at the Centre for Transgenesis and Genome Editing at St Petersburg University. Hence we are at the forefront of global research efforts in this area. We study changes in the behaviour and emotional state of genetically modified animal models, and gradually we will prove that TAARs are new pharmacological targets. In this area, we are ahead of everyone else.

Do you use artificial intelligence to select the ligands?

No, we don’t. We have partners among chemists who, in my opinion, can accomplish this task much better. I had a great colleague at the Laboratory of Chemical Pharmacology of St Petersburg University — Professor Mikhail Krasavin, who, unfortunately, died prematurely a few weeks ago. We worked together on a project supported by a grant from the Russian Science Foundation. He was a great medicinal chemist. We talked with him about it, and he agreed with me that human instinct is much stronger than AI. As a medicinal chemist, he could predict what would work, taking into accounts the patterns; and he knew how to pick the right compound. He liked to sit and draw to think about the task at hand. Then, he would send us the compounds, we would test them, discuss the test results, and he would optimise them. Sadly, he is no longer with us, but we continue to work with his students.

Which receptors have already been well investigated?

The first step is TAAR1 — it is the best investigated among TAARs. As I’ve said, I worked with F Hoffmann-La Roche on the ligand to TAAR1. They spent five years on clinical trials, obtained very promising data, but suddenly they found out that in 17% of African-Americans this drug caused a serious adverse reaction. There was a problem with the enzyme that metabolises this particular substance. They had to start all over again. The target is the same, but the pharmaceutical agent is completely different.

That set them back five years, while I continued my work. Then, one day I was at a conference in Canada, in Quebec. I made a presentation on trace amine-associated receptors, and some people believed me and some didn’t. It was a new subject area and not many people knew about it. After my presentation, I wanted to go to my room and catch up on some sleep — to get over my jetlag — the day overlapped the night. I was walking past conference halls and suddenly saw an open door. The speaker was talking about a new antipsychotic medicine, which he called a serotonin agonist. I could not believe my ears. All pharmacology textbooks say it is impossible. Additionally, he showed the structure of a substance very similar to trace amines. I was surprised, to say the least... but went to bed.

In the evening, I went to a bar with a friend of mine — a Canadian Martin Ballew. We were having a beer and a stranger happened to be sitting next to us. And as often happens at conferences (which is why they are great), it was not a random encounter. The man sitting next to us was the CEO of Sunovion, the US-Japanese pharmaceutical company worth $4 billion. We got to talking, and I asked him if it was their presentation on the new antipsychotic drug. He replied in the affirmative. Then, I told him directly that in my opinion it was not a serotonin agonist, but a TAAR1 agonist. I said that F Hoffmann-La Roche were stalling at the moment and that Sunovion might be able to win the race. He didn’t believe me then, but three or four years later, they returned to this topic, and my prediction was correct. I learnt about it from his colleagues in 2019. They then published a paper in the New England Journal of Medicine (which is one of the most influential journals in medicine) about a new TAAR1 agonist. Sunovion have conducted a phase 2 clinical trial in schizophrenia with excellent results, and most importantly, without side-effects.

They were so impressed by that story that they offered me to be their consultant. I helped them to make an educational animation film for doctors about trace amines and their receptors. I have been involved in production of information materials. At conferences where they have a stand, even in our challenging times, they show a video where I talk about this receptor for ten minutes. They play this video, despite the fact that I am from Russia. They are not afraid to take a stand. I am still their main consultant on TAAR1.

Thus, there are two drugs based on the TAAR1 target that are being tested at the moment, and I’ve had some hand in both of them. They are still in clinical trials carried out by two pharmaceutical companies: the US-Japanese company Sunovion are in phase 3, and the Swiss company F Hoffmann-La Roche are in phase 2. Sunovion are now trying to widen the range of indications for use. Since there are no side effects, they want to evaluate the use of their drug as an adjunctive treatment of depression and anxiety disorders. In total, Sunovion are conducting 25 different clinical studies. Last year, the official names of these drugs were announced: our Swiss colleagues named their drug "Ralmitaront", while the Americans used the name "Ulotaront". If you add up the first two letters of these names, you get "Raul". That was entirely coincidental and unintentional, but what a funny coincidence! I was the main consultant for both companies.

Ulotaront is expected to receive the FDA approval this year and ralmitaront — in three to four years. The sales potential of ulotaront is estimated at around $5 billion over a five-year-time frame.

We have talked about TAAR1, but what about other TAARs?

That is our main focus right now. We have all these knockouts now, and we can see a major change in the emotional patterns and behaviours of these model animals. Additionally, we have two strains of mice where we have deleted the gene in question and inserted a blue screenable marker at the site where this gene should be expressed. We began searching for the receptors corresponding to this gene in brain tissue and we found them in the olfactory cortex. It is part of the limbic system, which is among the oldest structures of the brain, responsible for emotions and survival instincts. Hence, TAARs are not just olfactory receptors after all. The response is projected into the structures involved in the processing of emotions. They translate instinctive olfactory information into an emotional state. These could be fundamentally new pharmacological agents, and this is what we are trying to prove.

Of the remaining five trace amine-associated receptors, TAAR5 is the best studied. It has been shown to be specifically activated by trimethylamine, an organic compound that smells like rotten fish. We found that TAAR5 is blocked by timberol, a synthetic terpene, commercially used in the perfume industry as a pleasant ambery cedarwood fragrance. I think, this pairing reflects the cultural characteristics of mankind. After all, woody coniferous fragrances have always been used to cleanse a space. Think of funerals, Christmas trees, frankincense, myrrh, or eucalyptus in saunas. These are terpenes, many of which are derived from conifer resins. We are very intrigued by this subject, and I am looking for other antagonists among the terpenes.

We have already tested a dozen terpenes. We have found two that work, but there are thousands! There is so much potential! I’d like the study to come on stream to understand them better. Again, my intuition tells me that many of terpenes are folk remedies for a reason. Indeed, taking walks in coniferous forests, or spending time under pine trees on the seashore has a soothing effect and is beneficial for our overall health. Wood tar soaps and resins — every plant is different. And different plants have different combinations of terpenes. All this information needs to be analysed systematically. All in all, the status of our work right now is as follows: we already have our own active TAAR1 agonist, and we are looking for an investor to develop our own drug in Russia. As for the other trace amine-associated receptors, we are still studying them at a basic level, confirming their role as potential targets for therapy.

Are you able to do research at the same level as before?

In general, yes, but of course, everything is more difficult and expensive. For instance, now we are trying to buy a test system so we can test an unlimited number of terpenes for activity in relation to TAAR5. It is incredibly expensive. In Europe, it costs 30,000 euros, while we have been asked to pay 90,000 euros for it.

What is to be the final outcome?

The hypothesis, in general, is very simple: let’s remove the smell of rot from your perception, and your emotional state will improve. It is potentially a new antidepressant, a cure for anxiety, and so much more.

Dо you know this chapayev joke?

— Vasily Ivanovich, go wash, It’s awful, you stink.

— Come on, Petka, I bathed last week.

— For pity’s sake, Vasily Ivanovich, go wash... It’s unbearable... Vasily Ivanovich went to the shop and bought a bottle of conifer cologne. He sprayed it on...

— Well, Petka, any better now?

— Well, how shall I put it, Vasily Ivanovich... It smells like someone has shit under a Christmas tree.

That is the essence of our discovery. Fir wood contains terpenes that block the smell of rot. That’s me preparing my future Nobel Prize acceptance speech when they award me for this discovery.

I still believe that trace amines and their receptors have huge potential in pharmacology and medicine. Remember, in Patrick Süskind’s novel and film Perfume, the perfumer was collecting scents of dead girls? It was the collection of trace amines that he was compiling. These are our biochemical signatures. This is what we leave behind. One day, each of us will become a leather bag full of trace amines — sometimes, I cheer up my students like that...