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Long range vision

Discoveries on protein essential in forming the brain make agreement with multinational feasible

Two weeks before Christmas last year, as a reward for the ceaseless efforts of the research group that he coordinates, Antonio Carlos Martins de Camargo – a physician by training who found his way into research in biochemistry and eight years ago set himself up in the Butantan Institute – signed a contract with the multinational pharmaceutical industry Merck, Sharp & Dohme. The agreement, which has only two pages, establishes a common objective for the teams from Butantan and from the company headquartered in Essex, in the United Kingdom: to develop medicines using a protein that represents one of the ramifications of work that was started back in the 40s by one of Brazil’s greatest scientists, Maurício Rocha e Silva.

From a discrete birth, it soon proved to be promising, and even, in recent years, essential, as a succession of scientific articles signed by researchers not only from Brazil, but also from the United States, Europe and Japan, proved the crucial role of this protein in regulating a series of functions, from the transmission of pain to the formation of the brain during gestation.There is still no forecast of what medicines may arise, nor when, but the prospects for this joint work are opening up a new chapter in the story that was born at the end of the 60s, as developments of Rocha e Silva’s discoveries, and is far from ending.

In one of the next episodes, there should be a clash between the big names of world science, in search of recognition of the authorship of the discovery of the protein to which Camargo dedicated almost half of his life. This month, if possible, the researcher from Butantan intends to demonstrate, once and for all, that the protein that he himself discovered and called endooligopeptidase A or endoA – presented in two articles, one published in 1969 in Biochemical Pharmacology , and another, two years later, in the Journal of Neurochemistry – is the same that the Americans, Germans and Japanese rediscovered in 2000 and baptized as Nudel, nuclear distributing-like protein, describing its activity in forming the cerebral cortex in independent articles published in December of that year in Neuron . “It is not enough for us to be and to be seen to be honest, like Caesar’s wife”, says he. “We Brazilians have to prove our honesty at every moment.”

But Camargo learned how to wait, as the agreement with the pharmaceutical industry demonstrates; it was stitched together with the same diplomatic sense that, coupled with diligence in work, made it possible for him to be hired by the Faculty of Medicine of the University of São Paulo (USP) at the end of the 60s, transferred to the Biomedical Sciences Institute at USP in São Paulo in 1984, to form a research nucleus at Butantan, and to become the coordinator of the Center for Applied Toxinology (CAT), one ofthe Cepids (Research, Innovation and Diffusion Centers) that make up FAPESP’s special program.

“We only accepted Merck’s proposal after the patent for the protein had been lodged”, he says. “We cannot be in a hurry, so as not to lose everything we have already done.” And what they have achieved is by no means little. Studying the properties of this protein has made it possible to educate almost 30 masters and doctors, besides being one of the oldest research lines supported continuously by FAPESP. The first project in Camargo’s name, without counting earlier ones, signed by his supervisors, date back to 1970. There followed another 45 projects supported by research grants, for laboratory infrastructure and for trips abroad, or by means of scholarships for the pupils.

Now at the age of 66, the physician born in São Carlos is part of a generation of researchers that show engagement with their own scientific problems, without expecting immediate recognition. “It is difficult to resist the temptation of the new technologies, which give immediate results, but we cannot neglect real science, which takes time”, says one disciple of Rocha e Silva. The scientists of this lineage, nowadays in the region of 60, 70 or even 80 years of age, adopt Darwin’s style of doing science: they value the formulation of hypotheses that can explain what has been seen and the search for evidence that sustains or overturns the original hypothesis.

Some time ago, however, there was a serious risk of the Darwinian style even being mistaken for arrogance. “I was so involved with a research program that I wasn’t worried about what other people thought”, he recognizes. “I didn’t want to please, but to explore my ideas and to do a good job, which I knew would be important sooner or later.”In 1962, when he set foot in a laboratory for the first time, Camargo was in his second year of studies in Medicine. He had to assist at child births and surgical operations, but research was what he really wanted to do.

Aware of his real vocation, he knocked at the door of one of Rocha e Silva’s assistants, Sêrgio Steiner Cardoso, who gave him the chance of taking part in a study on controlling the regeneration of tissues of the human body. Camargo worked in this line until 1966, when he went on the track of the protein that has called the attention of Merck. He himself was at the time an assistant of Maurício Rocha e Silva, known for being as spontaneous as he was brilliant, to the point of not measuring his words when addressing his colleagues.

Rocha e Silva convinced him in seconds to forget the regeneration of tissues and to take part in the search for the working mechanisms of bradykinin, a molecule that regulates blood pressure, discovered in 1948 by Rocha e Silva himself and by Wilson Beraldo (bradykinin reduces the pressure in the bloodstream and raises it in the central nervous system). At that time, the priority was to identify a group of special proteins, or enzymes, responsible for the transient effect of bradykinin. Applied in the brain of a rabbit, bradykinin would leave the animal hypertensive, restless and panting for a couple of minutes. Afterwards, the rabbit would become catatonic, spread out on the table, incapable of moving.

The evidence suggested the existence of enzymes capable of deactivating bradykinin – if kept in check, they could allow bradykinin to act for a longer time. Not even today would it be easy to prove this idea, since the proteins mingle or act on their own, or in conjunction, when they activate or deactivate other proteins. Camargo asked for a loan of the peptides (protein fragments) that another of Rocha e Silva’s assistants, Sêrgio Henrique Ferreira, to this date with the School of Medicine of USP in Ribeirão, had just discovered in the venom of jararaca.

Known as the bradykinin potentiating factor (BPF), this molecule blocks the action of the enzyme that destroys bradykinin. Years later, in one of the most notable results of this group, BPF inspired the creation of captopril, one of the best selling antihypertensive agents in the world, which earns some US$ 5 billion a year for the American laboratory Bristol-Myers Squibb, which since 1977 has held the patent for the synthetic version of this peptide.

The refusal
One of the enzymes that annul the action of bradykinin was endoA, as was to be seen later. At that point of time, it was only seen that Ferreira’s peptide left rabbits with hypertension, restless and panting for up to one hour. But it still remained to be discovered which enzyme was cutting – or degrading – the bradykinin. The years went by, until Camargo’s team purified endoA, at times running the risk of being electrocuted. “Twenty years ago, one of the ways of separating peptides was to use high voltage electrophoresis, applying a tension of 3000 volts to a sheet of paper with the proteins”, says he, without any nostalgia for these methods, which used to take four or five hours of work. Today, it is possible to separate peptides in moments, without any danger of unexpected shocks.

Even knowing the difficulties he would have to overcome, Camargo avoids the easy ways. In 1972, he was finishing his postgraduate studies with Lewis Greene, at the Brookhaven National Laboratory, in New York, when he met Sêrgio Cardoso again, the same one who had opened up the gates of the world of science to him. Cardoso, who had transferred himself to the United States, gave him another chance: to follow him, as his right hand man, in the task of setting up a protein laboratory at the University of Tennessee. His former student would receive a salary five times higher than in Brazil, besides winning a house ready for installing himself with his wife and three small children. Camargo said no. He preferred to go back, having decided to create a protein center in Brazil.

He left Brookhaven without forgetting a comment that he had had to swallow weeks after having arrived there, two years before, in 1970. He was unwrapping some packages of equipment, when a technician commented to Greene, the head of the laboratory: “This is the work that a Latin American doctor can do in the United States”. Months later, in Ribeirão, he formed a team that accepted the challenge of assembling the equipment, instead of buying it all ready. Not just for the savings that they actually achieved, cutting costs by half, but also because they wanted to discover how it worked, and particularly how to fix the protein analyzer, the most important apparatus of them all. It was a challenge only overcome with the help of two technicians from Brookhaven, Nicholas Alonso and Rosalyn Shapanka, who had already taken part in the initial studies of endoA – both appear as co-authors of the first articles on the characterization of the protein.

If there is one piece of advice that Camargo willingly offers his team, it is: do not be afraid of pursuing your own ideas. “I never believed that the biological role of endoA was to destroy bradykinin”, he confesses. With time, the group demonstrated that there was, in actual fact, a family of similar proteins – and that is how endoB arose, a few years after endoA. The two sufficed to give weight to the suspicion that they could have a broader action, acting in the formation or destruction of other proteins and in the regulation, not only of pain and blood pressure, as was thought at first, but also in hormonal control, cell growth, interaction between nerve cells, or the equilibrium of water in the organism, as was ascertained, over the years, as these molecules started to be studied all over the world.

EndoA and endoB are part of a group of enzymes called oligopeptidases, which destroy only peptides, and not entire proteins, as is usual. Camargo put forward this concept in 1976, in Biochemistry and in the Journal of Biological Chemistry , describing endoB, but nobody paid any attention right away. Recognition was to come 19 years later, when a Pole called Vilmos Fulop, from the University of Warwick, in Britain, cited his studies when reporting the crystallization of endoB, in the Cell magazine. In another polemical article, published in 1996 in the Biochemical Journal, Camargo demonstrated that the protein isolated in the testicle of a rat by a team from the Mount Sinai School of Medicine, United States, was not the same one he had extracted from the brain of a rabbit, and now suspected that the same molecule could have more than one function in the organism.

The heritage
Today, it is Miriam Hayashi, whom Camargo got to know in Tokyo in 1990, who is dedicating herself body and soul to proteins. A Pharmacy graduate, she was working on research into anticancer drugs at the Japanese branch of Roche. After one year over there, she was already thinking about going back to Brazil. In Butantan since 1993, she is dealing with endoA and endoB using molecular biology, complementing what had been done before.

It was Miriam who sequenced endoA, made up of 345 units – or amino acids. It also she who attested to the importance of this protein, by means of an experiment carried out with an embryo of an African frog (Xenopus laevis ). In four of the embryo’s eight cells – those that are going to form the right side or the left, while the other four are going to form the opposite side of the body -, she applied a high dose of the gene that induces the production of this protein. There was deformation of the eye and of the brain of the tadpoles, but only on the side in which the protein appeared in excess, indicating that any imbalance in its quantity could be harmful.

It was important evidence, but it came along with a disappointment. As soon as the experiment was concluded, at the end of 2000, Miriam read inNeuron an article by American researchers who took the merit for the discovery, for publishing first the results that she had arrived at months before. It was not enough, though, to take her wind away. Besides the object of her study, she seems to have inherited from Camargo a taste for betting on her own paths.