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Structural biology

The essence of molecules

Crystallography facilitates drug development

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SCIENCE PHOTO LIBRARY The above image represents oxygen carrying myoglobin, one of the first proteins to be examined through crystallography; the colored spheres represent amino acids of the myoglobin, which helps store oxygen in the musclesSCIENCE PHOTO LIBRARY

Twenty years ago, few laboratories in Brazil used crystallography, but today dozens of research centers have mastered this technique used to reveal the three-dimensional structure of proteins. In São Paulo, the use of crystallography saw a step change in 2000 and 2001 when the Structural Genome project was launched through a partnership between FAPESP and the Brazilian Synchrotron Light Laboratory (LNLS), which is operated by the Ministry of Science, Technology and Innovation. The Structural Genome project provided US$3.5 million in funding to dozens of laboratories, enabling research groups to finance the purchase of equipment for protein expression, purification and crystallization.

The Center for Structural Molecular Biology at the University of São Paulo’s São Carlos Institute of Physics (IFSC/USP) rapidly distinguished itself as a key player in the technology. The group had already explained the molecular structure of about 20 proteins through the use of X-ray diffraction crystallography. The macromolecules analyzed were potential targets of inhibitors for diseases such as hepatitis B, malaria and some types of cancer.

The first strides made at the crystallography center in São Carlos came in 1989. “The field was experiencing a boom all over the world, and many such centers were being created. We decided to do something of our own. The lab had one tiled sink and 16 square meters of space, compared to about a thousand today,” recalls Glaucius Oliva, coordinator of the center at USP São Carlos. Now president of the National Council on Scientific and Technological Development (CNPq), he began working in this field thirty years ago as an undergraduate in physics under the guidance of professor Yvonne Mascarenhas, who initiated the crystallography work in São Carlos. In 2000, his group achieved the status of a Research, Innovation and Dissemination Center (RIDC), and more recently it was named a National Institute of Science and Technology (INCT), with support from the federal government and FAPESP.

In 1992, a protein—glucosamine-6-phosphate deaminase from the Escherichia coli bacterium—was fully characterized for the first time in Brazil by means of crystallography. In 1997, the same year in which the group from USP’s São Carlos Institute of Physics grew crystals in zero gravity onboard U.S. space shuttles, a specific beam line dedicated to crystallography studies came into operation at the LNLS in Campinas. According to Oliva, in addition to the huge increase in the number of people trained in this field since the 1990s—many of whom now head research groups in a number of Brazilian states—crystallography has undergone an “enormous methodological and technological evolution during that period.”

Previously, all the solutions that were used to grow a crystal from a protein needed to be made manually, but today there are robots that practically put together the entire experiment. Specialists say that the speed and yield are much greater. “Despite all of this, the technique is still pretty experimental. There is no theory for crystallography. You can’t predict whether or not it will work,” Oliva says.

As the research center in São Carlos was coming of age in the early 2000s, the Structural Molecular Biology Network (SMOLBnet), also in coordination with the LNLS, became operational. The network served to facilitate the work of teams from dozens of laboratories, who identified the three-dimensional structure of 52 proteins in two years (see article). During that time Oliva’s team did another pioneering experiment that described the biochemical pathways for glucose production by Trypanosoma cruzi, the protozoan that causes Chagas disease (see article).

FAPESP’s Virtual Library, which keeps records of the projects supported by the institution since 1992, cites 93 research projects, of which 17 are ongoing and 76 have been completed. Researchers use crystallography to study proteins that could lead to new drugs or shed light on the development of some types of cancer.

The importance of modern crystallography techniques is easily apparent if we simply reverse the logistics of producing a drug. In order to be efficient, a drug must act on the relationship between parasite and host, as in the case of Chagas disease. The medication must work, preferably killing the parasite without interfering with the host.

But how can we see all of these biological interactions at the molecular level? Only by conducting an actual molecular examination of the entire process. This is the role of crystallography, a set of techniques that reveal the detailed structure of proteins, for example, through X-ray diffraction (scattering) in a crystal formed out of the proteins to be studied.

The further the research progresses, the more proteins are identified and validated by crystallography, thus expanding the biological database on a given problem. The number of options available to solve certain challenges at the molecular level tends to increase accordingly.

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This work of assembly and disassembly at the molecular level exists not only for Chagas disease, but for several maladies, and it allows for other applications within the field of biology in general. The objective is always to identify macromolecules, and then attempt to synthesize other compounds in the laboratory that are linked to the so-called biological targets. The idea is to block that pathway so that a few normally desirable developments, such as the death of T. cruzi, will occur.

Additional evidence that crystallography techniques are vital to scientific development comes from the world of snakes. Several ongoing projects in the state of São Paulo in recent years are using precisely this tool to decipher the venoms of these animals.

The applicable analogy is easily understood. As the discoveries under several research projects have recently shown, the components of venom are not suitable for clinical use. But specialists believe that, with a better understanding of the molecular structure of the venom, they could alter part of the molecule in order to change the way it acts upon the organism. It is believed that this planned modification of the original structure of a molecule could have therapeutic use in many diseases.

A research group from the Hermínio Ometto Foundation, in the city of Araras in São Paulo state, is moving in that very direction. The researchers are interested in isolating a group of enzymes from the venom of Crotalus durissus terrificus, the well-known rattlesnake. X-ray diffraction crystallography is among the tools being used to explain the three-dimensional structures of selected proteins.

According to the group, the L-amino acid oxidase (LAAO) enzymes that give the snake venom its amber-yellow color showed in vitro cytotoxic, bactericidal and antiparasitic potential. They have been described as inducers of a number of toxic effects in biological systems, including platelet aggregation, hemorrhage, edema and apoptosis.

Developing these techniques and the databases of protein structures, with their known molecular information, is only part of the problem. There are also gaps in the applied research.

When myoglobin—one of the first structures of a protein that was fully understood through crystallography—was validated in the United States in 1960, a parallel evolution began in the business sector. Few companies in Brazil, however, have invested in new-drug research, the principal area in which crystallography has been applied.

These advances in structural biology will yield the anticipated consequences in terms of health only if the processes of technological innovation and drug development are fully facilitated, the researchers warn. They believe that the development of molecules that can actually achieve the biological targets for which they were designed will occur only if universities and businesses are able to work together. Only a product viewed as profitable by companies will be able to receive financial support and be manufactured on a commercial scale.

Projects
1.
National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases (INBEQMeDI) (nº 2008/57910-0) (2009-2014); Grant mechanism Thematic; Coordinator Glaucius Oliva (IFSC/USP); Investment R$ 1,340,213.83.
2. Crystallography, molecular modeling and planning for substances of biological interest II (nº 1994/00587-9) (1995-1998); Grant mechanism Thematic; Coordinator Glaucius Oliva (IFSC/USP); Investment R$ 257,249.99.
3. Center for Structural Biotechnology (nº 1998/14138-2) (2000-2012); Grant mechanism Research Centers Program (Cepid); Coordinator Glaucius Oliva (IFSC/USP); Investment R$ 28,449,954.27.
4. Biochemical, structural and functional characterization of L-amino acid oxidase isolated from the yellow venom of the Crotalus durissus terrificus snake (nº 2011/12267-6) (2012-2013); Grant mechanism Regular Line of Research Project Award; Coordinator Maurício Ventura Mazzi (Hermínio Ometto University Center – UniAraras); Investment R$ 301,426.83.

From our archives
Jigsaw puzzle of life  – Special Cepids Issue – May 2007
The key to new medicines Issue 57 – September 2000
Identified genes may be related to CVC – Issue 38 – December 1998
Cooperation in space – Issue 21 – June 1997
Reconnaissance voyage –  Issue 19 – April 1997

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