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Pharmacology

Flight of the Phoenix

A fragment of protein linked to the control of arterial pressure could help weight loss and treat chemical dependence.

eduardo cesarProtein dosage plate: the stronger blue indicates greater concentrations of protein where antibodies will bind togethereduardo cesar

Emer Ferro tried to forget hemopressin, but could not. When he and biologist Vanessa Riola discovered it in 2003, in one of the laboratories on the fourth floor of the Biomedical Sciences Institute at the University of São Paulo (USP), he thought that it was only one more molecule capable of reducing arterial pressure. He then gauged his strength, and concluded that to continue he would have to get into a wearisome struggle that would lead him, at best, to one more anti-hypertension medication amongst dozens of others, and put the research aside. Like a phoenix, this molecule gained a new life when two biologists from the Butantan Institute, Camila Dale and Rosano Pagano, persisted in studying the biological properties of hemopressin and showed that it also served to placate pain in rats. At this point the fragment of protein gained another dimension and rekindled the attention of Emer, who did not yet imagine that this molecule might do much more – or that he himself, months later, would help to find a new method for speeding up the development of new pharmacological agents.

At the end of July 2006, as part of his work schedule at the Albert Einstein School of Medicine at Yeshiva University, in New York, Emer re-examined the results of experiments with hemopressin available up to then. This was when he suspected that to explain its effects on arterial pressure and pain, the molecule had to act on specific proteins on the cell’s surface – the receptors of cannabinoids called CB1, activated by compounds produced by the body itself or by components of narcotic plants such as marijuana. Intrigued, he crossed New York and asked for help from a Brazilian colleague, Andrea Heimann, a researcher at the Mount Sinai School of Medicine, a few blocks away from Central Park. As she had finished her work ahead of time and had the material available, in just three days Andrea did the experiments on the cells which rendered hemopressin a noteworthy candidate for becoming medication: because of the now confirmed nature of its action on CB1 receptors, it could also help people to lose weight, treat type 2 diabetes, reduce dependency on drugs and placate the need to smoke.

Hemopressin has great versatility because it really blocks cannabinoid receptors, which regulate hunger, mood and pleasure. It has the same effect as rimonabant, a drug already approved for the treatment of excess weight and obesity in Europe and Brazil, but still being analyzed by the US government’s regulatory authorities. Rimonabant has advanced slowly in the United States because of its side effects, such as the risk of severe depressions and suicide. According to a study published in November in The Lancet, Danish researchers tracked 4,105 people for one year and concluded that this drug could cause depression even in those who had never suffered from it before.

eduardo cesarLiving Factories: one of the rabbits used for antibody productioneduardo cesar

A good beginning
According to Emer, hemopressin could certainly overcome these limitations as it is a peptide (a fragment of protein) produced by the body itself, possibly from the recycling of hemoglobin, the molecule that transports oxygen to the body’s cells. Preliminary experiments on animals showed no relevant toxicity or apparent side effects, apart from indicating that hemopressin could also be taken orally, as Camila Dale and Rosana Pagano found in experiments carried out at Butantan.

“Hemopressin is beginning to look like an interesting molecule” says Emer. “This is the first natural peptide that works as an inverse agonist of CB1.” Put more simply, what he is saying is that hemopressin reduces action from the active forms of one type of cannabinoid. This molecule does a job previously carried out only by the hormones and neurotransmitters involved in the control of hunger and pleasure. The discovery was sufficiently significant to be accepted for publication by the scientific journal PNAS, edited by the National Academy of Sciences of the United States.

There is another reason why this fragment of protein, with only nine amino acids, will not quickly reach the same stage of development as rimonabant, which acts on the same surface proteins in the brain, liver and muscles. According to Andrea, the production of peptides, such as hemopressin, on an industrial scale tends to be more expensive and more refined than synthetic chemical compounds such as rimonabant. She and Emer believe that the best way to advance is to develop synthetic compounds with the same function as the active fragment of hemopressin, formed by four of the nine amino acids of this molecule; this is the same path used to develop many other drugs. “We now have to work with chemists who can find a synthetic version of hemopressin”, acknowledges Andrea.

This will not be first time that she has challenged fortune. Four years ago when she was about to finish her doctorate, Andrea was soundly scolded by her mother, when Andrea told her that not only would she leave the ten thousand square meters laboratory on the tenth floor of Heart Institute near Avenida Paulista [in the city of São Paulo], but that she planned to pursue an academic career, not an easy achievement, given the lack of vacancies for new researchers. At this moment some colleagues even told her she was foolish when they found out that she had become the principal partner in Proteimax, a small biotech business in the back of a house in a gated community in Cotia, Greater São Paulo.

Now Andrea gets even by showing that she produces quality science and has become fully conversant with a technique that could make it far easier to select compounds of pharmacological interest through antibodies produced by ten white, mottled and brown rabbits. These antibodies have a Y-shape, as do all antibodies that the body produces to fight tumors, viruses or bacteria. They are special, however, for preferentially joining up to surface cell receptors when activated by hormones such as adrenaline, neurotransmitters such as serotonin, peptides such as hemopressin and, in the retina, by light. Once activated, these receptors take on another shape and activate the so-called protein B. This is a strategic protein for the flow of information through the body, as it amplifies the signals from the exterior to the interior of the cells. The antibodies help to map these connections and to select the compounds that should or should not arouse protein G.

Of course, Andrea began slowly, with many doubts, fears and only seven antibodies, each one capable of attaching itself to specific molecules. She had almost no equipment or reagents when she met Lakshmi Devi, a pharmacologist of Indian origin, who studies protein G and the biochemical mechanisms of drug dependence. Three years after having met, when he came to Brazil for a pharmacological congress, Lakshmi swelcomed her to his laboratory at the Mount Sinai School of Medicine, in New York. It was there that Andrea worked, checking if the first seven antibodies did exactly what she wanted, while Emer re-examined the experiments with hemopressin at Yeshiva University, where he was working at the time, at the invitation of pharmacologist Lloyd Fricker.

eduardo cesarAt hand: antibodies for CB1eduardo cesar

Exports
In 2007, Andrea and Lakshmi presented the antibodies with specific configurations as a new tool to identify compounds capable of setting protein G in motion, in an article published in the Journal of Biological Chemistry. This work drew the attention of Michael Melnick, a biologist at Stanford University with a PhD from Harvard, who had founded and directed Cell Signalling Technology, a pioneering company in the development of antibodies for cancer research. “I believe that the Proteimax antibodies will be extremely useful in basic research and in many illnesses where receptors of protein B are involved” comments Melnick, who showed interest in some way associating himself with the Brazilian company. Almost half of the drugs currently in use set in motion superficial receptors linked to protein G.

The work published in the Journal of Biological Chemistry also brought the first requests for exports and facilitated an agreement with US company Assay Designs Inc. (ADI) for the distribution of antibodies produced and purified by Proteimax. Now duly equipped, Andrea can check, in Cotia, the efficiency of compounds through specific antibodies – in membranes of neurons – in the same three days that the hemopressin tests took her in New York.

The original seven antibodies have evolved to 35 today, but the Proteimax team, formed by the director herself and by biologists Laura Leticia de Souza and Bianca Alves Pauletti, believe that they could reach 50 with six more months of work. They have created what Emer calls a platform for molecular analysis, which could cut down by years the work of selecting chemical compounds with potential therapeutic use.

From each experiment, Fricker, who worked for two months as a visiting researcher in Emer’s laboratory, obtains hundreds of new peptides which could be studied further. The problem is to choose which ones really merit further studies.

“We need a rapid selection method compatible with large scale analysis”, comments Emer. He previously used another work method, based on luminescent genes from fireflies. This approach showed whether the peptide was biologically active, but did not identify the receptor it linked up to. Already making use of the specific configuration antibodies and while he plans stricter tests for hemopressin toxicity and action, Emer identified 14 new peptides which are linked to certain cannabinoid receptors (CB1 or CB2) and at times to both, as well as to enzymes that regulate arterial pressure. Although it is still a very distant possibility, one can envisage a single compound that could result from this and control arterial pressure, the urge to smoke and hunger at the same time.

The projects
1. Cellular molecular biology of oligopeptidases (nº 04/04933-2); Modality: Thematic Project; Coordinator: Emer Ferro – ICB/USP;
Investment: US$ 271,000.00 and R$ 270,000.00 (FAPESP).
2. Antibodies with specific conformation; Modality: Innovative Research at Small and Very Small Companies (Pipe); Coordinator: Andrea Heimann – Proteimax; Investment: US$ 147,158.16 and R$ 113,720.10 (FAPESP)

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