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Immunology

First movement

Enzyme that may cause arthrititic inflammation and its pain discovered

Reproductions from the book "Rodin" by Sommerville StoryDetail of the “The Gates of Hell”, by RodinReproductions from the book "Rodin" by Sommerville Story

While everybody was looking one way, they were looking in the other direction. They asked different questions and discovered many interesting things. Two scientists from the Butantan Institute, Catarina Teixeira and Cristina Fernandes, noticed that the same protein found in abundance during the most advanced stages of arthritis (something that everybody in this area already knew) could also be one of the causes of this illness (which is what nobody else seemed to have realized or demonstrated). What they discovered in conjunction with researchers from the Federal University of Ceará and from the University of Costa Rica, does more than merely shed light on the sinuous labyrinths of inflammation and pain that gradually consume joints such as knees, elbows, wrists and hands. It also helps scientists to gain a better understanding of how and why current treatments may or may not work in dealing with this disease, which predominantly affects women, at a rate three times greater than men, and which first appears around the age of 35. In Brazil, some two million people suffer from arthritis and, in extremely severe cases, avoid moving to avoid pain, almost as if they were prisoners within their own bodies.

“Nature is economic,” comments Catarina, explaining how, based on this simple fact, she and Cristina asked themselves whether the same protein might not have broader and more relevant functions. The article published last year in the British Journal of Pharmacology clearly illustrates how the molecule they studied (BaP1, extracted from snake venom and very similar to a molecule found in humans) activates and feeds the inflammatory processes common in arthritis. The protein also aids the release of inflammatory substances known as prostaglandins and cytokines, which cause pain in joints and also eat away at the cartilage at more advanced stages of the disease. In combination with results obtained by other research groups, this study helps to identify these proteins (enzymes called metalloproteases because they transport a metal, usually zinc) as a potential target in the struggle not only against arthritis but also against tumors and other diseases in the development of which they play a role.

Usually, different types of metalloproteases act as a maintenance team, removing what is no longer used in the space between cells, in addition to assisting in the formation and recomposition of the skin. They only rebel when standard body control for some reason no longer works. This is when they may foster the development of cardiovascular diseases, the spreading of tumors or the intense production of much smaller molecules such as the tumor necrosis factor (TNF-alpha). When activated, TNF-alpha recruits blood cells involved in inflammation, one of the body’s defenses in fighting against viruses, bacteria and tumors; however, if they are abundant they may intensify the arthritis pain and inflammation.

The current drugs used to fight arthritis pain aim at blocking the action of TNF-alpha, while the other approach blocks the action of the body’s entire defense system that, for reasons not yet known, begins to acknowledge joint cartilage as a foreign body to be eliminated. Putting the reins on metalloproteases, an alternative identified by more recent studies, would imply controlling the production of TNF, the small molecules that cause inflammation. Easy to imagine, but hard to do, and, according to the cautious researchers from Butantan, perhaps not very safe. In view of the versatility of these enzymes, arthritis might disappear, but relevant physiological processes such as scaring and fertilization might be jeopardized.

Targets
That is another vast labyrinth. In a review article published in Expert Opinion on Therapeutics Targets, professor of molecular rheumatology Andrew Rowan along with other researchers from the University of Newcastle in the UK describe 23 mammal metalloproteases, defects which could lead to a variety of problems, ranging from male sterility and nanism to obesity. They also analyze the compounds that could detain the said enzymes, called metalloproteases inhibitors: of the 50 plus inhibitors identified as potential alternatives to treat arthritis and cancer, further research for the majority was discontinued midway, due to side effects or low efficacy, despite the initially promising results obtained from lab cells and animals. Research continued only on seven inhibitors and so far only one has been approved – and for a periodontal disease, characterized by the inflammation of the gums and bones that support teeth.

In an article also published in Expert Opinion, Paraic Kenny, from the University of California at Berkeley, USA, puts a stop to what he calls the era of litany of deceptions, in order to discuss the progress and restrictions of proteins that inhibit an enzyme of this kind (referred to by its acronym, Tace) as a way to stop the development of lung, pancreas, breast and ovarian cancer, among others, in addition to fighting arthritis itself. According to Dr. Kenny, Tace may already be considered a therapeutic target, given that its antagonists have toxic effects that are tolerable and show reasonable efficacy in preliminary tests in cancer in human beings; however, the results obtained to date in relation to arthritis have not been very successful, possibly because other enzymes of that type continue to feed the inflammation and pain.

“The greatest development to date was to add one more mechanism of action produced by these enzymes, which have now been added to the group of mediators that spur the inflammatory reaction, possibly aiding the perpetuation of inflammation and structural damage to the joints,” comments Francisco Aírton Rocha, a rheumatologist from the Federal University of Ceará who worked together with the Butantan team. Researchers reached these conclusions by manipulating enzymes extracted from snake venom. Twenty years ago, Catarina, at that time a newly hired employee at the institute, began to study the effects of the venom of the jararaca snake (Bothrops jararaca), a common species and responsible for the largest number of snake bites in Brazil. She was particularly interested in understanding the mechanisms involved in inflammatory reactions found near the bite mark and barely neutralized by the anti-ophidian serum.

Reproductions from the book "Rodin" by Sommerville StoryDetail of “The Burghers of Calais”, by RodinReproductions from the book "Rodin" by Sommerville Story

After characterizing the local inflammatory reaction caused by Bothrops jararaca bites and that of a closely related species, the Bothrops asper, Catarina began looking for the components of the venom responsible for the said effects. Then, encouraged and with the support of José Maria Gutierrez, a researcher from the University of Costa Rica and the Clodomiro Picado Institute, the Costa-Rican equivalent of Brazil’s Butantan Institute, Catarina worked to characterize the inflammatory properties of an enzyme from the metalloproteases group found in the Bothrops asper venom; this species known as terciopelo (velvet in Spanish), is responsible for most of the snake bites in Central America.

Both Catarina and Cristina, as from 1999, were amazed by the impressive effects of one of these enzymes, BaP1, whose structure is very similar to metalloproteases produced by the human body. Cristina initially demonstrated the inflammatory effect of this enzyme, detailed in 2006 in Toxicon. Subsequently, combining their research results with reports from other research groups, which showed that the joints of individuals with rheumatoid arthritis accumulated metalloproteases, they concluded that these enzymes might have even broader functions that nobody had imagined before, triggering or furthering the onset of arthritis inflammation. They tested the hypothesis by applying the enzyme to rats, using the experimental model that Rocha has used to study the mechanisms of pain and injuries caused by arthritis for 15 years.

Toxins
Rocha taught Cristina to inject the snake enzyme in the tiny cavity between the likewise minuscule bones in rats’ knees. Just one hour after the BaP1 injection, a group of animals already had a significant number of inflammatory cells, specially neutrophils, in addition to substances associated with pain such as prostaglandins and TNF-alpha, characterizing the many defense cells and substances typically associated with arthritis; other groups of animals, which had previously been treated with compounds that blocked the TNF-alpha action or the formation of prostaglandins, did not develop inflammation or pain when they received BaP1.

The results yielded two simultaneous achievements. The first and more obvious one: confirming the initial hypothesis that this enzyme no longer plays a supporting role, but is in fact the main character in the painful drama of arthritis, given that it initiates and fosters the inflammation and the pain that consumes the joints; naturally the next step is to confirm whether the human equivalent has the same effect. The other achievement that Catarina pointed out is more subtle: the venomous toxins may also be used as a tool to better understand the mechanisms of body functioning and the development of diseases such as arthritis, clarifying by analogy the functions of many molecules in the human body, which, to date, remain a mystery to us.

The project
Study of the effects of metalloproteinase BaP1, extracted from the Bothrops asper snake venom, in the joints of rats
Type
Research Project Grant
Coordinator
Catarina de Fátima Pereira Teixeira
Investment
R$ 209,741.63 (FAPESP)

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