Finding the key that opens a known lock, like the one of the door to the house, is relatively easy. With time, its main characteristics the make, the size, the shape and the color become familiar. When looking for the key to the house, it is simple to discard, with just a quick mental comparison, the objects whose outline differs radically from the looks of the keyhole. This strategy cuts short the search for keys that show some chance of opening the door. Identifying the ideal key in the midst of other keys without ever having seen the keyhole in the door can be torture, above all if these number of options to be tested is high. One thing is certain: the chances of finding the key increase as the fundamental characteristics of the lock are revealed.
It was more or less this revealing the outline of a chemical lock that a team of researchers from Rio Grande do Sul and São Paulo did. They produced the most detailed image of the three-dimensional structure of the crystal of a protein, the enzyme called PNP, short for purine nucleoside phosphorylase, whose action, when uncontrolled, seems to lead to the occurrence of some diseases of an immunological origin and to the rejection of transplanted organs. “It is as if we had zoomed in, in a photo”, compares Diógenes Santiago Santos, from the Pontifical Catholic University of Rio Grande do Sul (PUC/ RS), the coordinator of the research about the enzyme. These studies are being carried out in the ambit of the Millennium Institute for Integrated Strategies for Studying and Controlling Tuberculosis in Brazil, a project financed by the Ministry of Science and Technology (MCT).
“Now we can see particulars of the structure of PNP that were not clear before.” The new image of PNP shows a quality some 20% superior than that of the previous version. Its resolution is 2.3 ångströms, while that of the old representation of the molecule is 2.8 ångströms (1 ångström corresponds to 10 millionths of a millimeter). The lower the value of the resolution in ångströms, the clearer are the details of the structure disclosed. The image captured by the Brazilians throws light on tiny portions of the PNP that can be fundamental for finding molecular keys the medicines capable of linking to and acting on this enzyme.
Mutant PNPs Previous studies suggest that controlling the action of PNP can be vital for the treatment of autoimmune diseases, such as lupus, rheumatoid arthritis, psoriasis and some types of cancer, like the lymphomas. This is believed in, because anomalous forms of this enzyme, probably deriving from mutations in the gene that carries the recipe for the production of PNP, alter the pattern of activation of one of the pillars of the immune system, kind of defense cell known as T lymphocytes. These cells recognize and combat substances that are alien and potentially dangerous to the organism, the antigens. The threat, real or potential, can be represented by viruses, bacteria, parasites or any alien protein entering the organism.
Under normal conditions, the activity of the T lymphocytes is beneficial and indispensable for the production of antibodies against the antigens. However, dysfunctions in the production of these guardian cells of the organism, caused probably by an excess of stimuli sent by the PNP, may cause autoimmune diseases. In this kind of disturbance, a kind of rebellion against the immune system breaks out, a mutiny of the defense cells, which instead of turning on external enemies, start attacking its own tissues. In the light of the evidence that PNP has a direct influence on the behavior of the T lymphocytes, science started to look for inhibitors of this enzyme drugs that could treat or at least reduce the effects of the diseases with an immunological background. Achieving an excellent representation of the structure of PNP, in which these inhibitors have to slot themselves in so as to produce some effect, has become a priority in this line of research.
To produce a high resolution of PNP, the researchers had to get around difficulties inherent to the production of images in three dimensions of the structure of proteins (enzymes are a kind of protein). The first challenge was to get the protein in sufficient quantities and with a purity of almost 100%. “This enzyme is very difficult to be cloned”, comments biochemist Mário Sérgio Palma, of the São Paulo State University (Unesp), in Rio Claro, co-author of the work with PNP. Instead of working with the enzyme in its natural form, extracted from human blood, as other groups did without much success, the Brazilian team opted for a recombinant version: they made the Escherichia coli bacterium produce the human PNP in a large quantity. The strategy turned out so successful that the Brazilians are today selling the PNP to companies from abroad.
The next step was to generate a stable version of the enzyme. Like a child who never stays still when it is time to be photographed, the structure of the PNP in its original physical state, the liquid form, is very unstable and complicated to be caught, the way out was to mix the enzyme with a solution and to cool it down until it formed a protein crystal. The solidified enzyme was then exposed to a high intensity radiation beam at the National Synchrotron Light Laboratory, in Campinas which revealed its structure. That is how the image of the PNP with a resolution of 2.3 ångströms arose.
The researchers produced unprecedented images of the structure of the enzyme interacting with the two main drugs that are candidates for being PNP inhibitors, acyclovir and immucillin H, in possible new treatments against autoimmune diseases. “We are trying to understand why some drugs work better than others on the enzyme”, explains physicist Walter Filgueira de Azevedo Jr., from Unesp in São José do Rio Preto, who participated intensively in this part of the studies. The greater or lesser affinity between PNP and its potential inhibitors depends on a series of technical parameters, such as interactions between their atoms and the complementary nature of their respective contact surfaces. The next challenge for the Brazilians, who this year published nine scientific about PNP in international magazines will be to look for natural inhibitors of the enzyme. In collaboration with João Batista Calixto, from the Federal University of Santa Catarina (UFSC), a specialist in phytotherapic, the team led by Diógenes Santos is trying to isolate molecules of plant extracts with potential for acting on PNP.
Enzymes of Metabolic Pathways Defined as Targets for the Development of Drugs against Neglected Diseases (Malaria and Tuberculosis) and Chronic- Degenerative Diseases; Coordinator Diógenes Santiago Santos PUC/RS; Investment R$ 2,140,000.00 (MCT)