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Toxin, Reveal Thyself! – Clues to Deadliest Diseases Being Unlocked

Imagine drawing a face you’ve never seen by estimating the shape and size of every feature. Then, someone shows you a photograph and takes all the guesswork away.

Similarly, Wayne Anderson, PhD, professor of molecular pharmacology and biological chemistry at Northwestern University Feinberg School of Medicine, uncovers the atomic structure of proteins in order to move a new generation of drug development forward.

Throughout the world, bacteria are increasingly resistant to old antibiotics including penicillins, cephalosporins, and carbapenems (the main defense against “Superbugs”). Utilizing pharmacology and chemistry on cellular levels, pharmaceutical companies have tried diligently―but failed―to create alternative broad-spectrum antibiotics.

With that discouraging outlook and some potentially nightmarish scenarios ahead, the idea snowballed for the academic community to reset the stage for drug discovery. Anderson feels the pressure: “We can―and have to―do better.”

His team has taken the lead by administering the Center for Structural Genomics of Infectious Disease (CSGID) based at the Feinberg School of Medicine. CSGID finds the position of the thousands of atoms that make up a protein, which give scientists throughout the world unprecedented insight into their molecular structure and behavior to re-invigorate the quest for new drugs and diagnostics.

The Attacker Unmasked

Funded by the National Institute of Allergy and Infectious Diseases (NIAID), CSGID uses high technology and computation to determine 3-D structures of proteins using X-ray crystallography. Images are produced at the Advanced Photon Source at the U.S. Department of Energy’s Argonne National Laboratory and reveal convoluted shapes that scientists will use to design new drugs.

They are not just pretty pictures. Images pinpoint, down to a ten-billionth of a meter, the average position of atoms, how they interact, and the distance between them. This data tells researchers how to refine a compound so it can link atoms or produce desired reactions between them that reverse disease.

“We can do things that were nearly impossible before,” Anderson says. “Knowing where the atoms are that you want your drug to interact with makes it much easier to design compounds that fit in the space between atoms and create favorable interactions.”

Wayne Anderson, PhD, professor of molecular pharmacology and biological chemistry

With Anderson as CSGID’s director, Northwestern heads the effort. Other institutions that are also principal investigators with the center include: University of Chicago, University of Toronto, University of Virginia, Washington University, University College London, Southwestern Medical Center, Sanford-Burnham Medical Research Institute, and J. Craig Venter Institute.

Working together, these institutions function like an assembly line. Each performs one or more of the steps needed to process the protein so it can be imaged. PIs also collaborate on analysis of results, co-author publications, and can partner with organizations for further investigation of structures after they are revealed.

Closely related to CSGID, Anderson has served as director for the Life Sciences Collaborative Access Team for 11 years. Comprised of eight institutions, including Big Ten universities, members oversee operations for the structural biology sector at Argonne.

A sister center also funded by NIAID, the Seattle Structural Genomics Center for Infectious Disease, serves a similar function as CSGID and is headquartered at the non-profit Seattle Biomedical Research Institute. Both centers work closely to divide the protein mapping work.

CSGID researchers already hit milestones during the first five-year, $36- million grant period launched in 2007, when CSGID solved the structures for lethal Bacillus anthracis (anthrax), Salmonella enterica (salmonellosis food poisoning), Vibrio cholerae (cholera), and Yersinia pestis (plague). The Department of Defense funded these investigations to develop new treatments in case of bio-terrorism attacks.

Now in the second five-year grant period for $25 million, CSGID’s efforts branched out to decipher additional organisms from NIAID’s A-C priority pathogen list, including proteins in Staphylococcus aureas (staph infections) and Clostridium difficile (C-Diff).

Extra Innings at the Ballgame

To carry out crystallography, Anderson’s teams are entrusted to use Argonne’s ultra-powerful synchrotron at the Advanced Photon Source in Lemont, Ill.The size of a major league baseball stadium, the circular synchrotron is a particle accelerator where electromagnetic radiation is generated and shared by scientists throughout the world. Photons are accelerated to more than 99 percent of the speed of light, producing the brightest X-rays in the Western Hemisphere.

Protein ribbons (left to right): Staphylococcus aureus, Bacillus anthracis, Listeria monocytogenes, and Coxiella burnetii

In typical X-rays, the picture is a shadow of an object. In contrast, X-ray crystallography generates images through diffraction patterns. The synchrotron sends X-ray beams through crystallized proteins. Beam patterns are recorded when the intense light bounces off atoms and other elements in molecules.

Retracing those patterns, scientists use computerized computation to determine where atoms and other structures are located inside proteins. Scientists use computer graphics to build atomic models.

Another method, nuclear magnetic resonance spectroscopy, is used much less often on extremely small proteins. Images are generated by measuring the protein’s response when placed in a magnetic field.

All of these advanced capabilities catapulted Anderson light years ahead in his research. He has been keen on crystallography since the 1970s, when he was inspired as a Yale graduate student working for Thomas Steitz, another structural biologist and 2009 Nobel Prize winner for Chemistry for his work on the structure and function of the ribosome.

In those early days of crystallography, it took up to four years to map a protein, sometimes using plastic parts and weak computers. Now, Anderson’s team is averaging two to three maps a week.

Let’s Start Talking

CSGID services are free of charge to the global scientific community, including non-profit and for-profit organizations such as pharmaceutical companies and educational institutions. If requests are approved by NIAID, the service is an unbelievable bargain, offered by the Federal government to dramatically speed up drug discovery.

So far, CSGID has received approximately 6,700 requests: about 4,600 of them have come from the center’s member institutions and the rest from other scientific organizations throughout the world. Less than 10 percent of requests have been successfully imaged―a low rate because there are many opportunities for things to go wrong. To map a protein, it must be processed through multiple steps, and many don’t make it through all of them.

Some proteins are very dynamic, twitching and gyrating, refusing to “stand still” so they can be studied. Other wicked ones are so toxic that purifying them for experiments has been impossible.

When it comes to crystallography, a low success rate is the norm. However, when proteins are successfully imaged, it is a revelation to scientists who have studied them for years without ever seeing their faces. It catapults their investigations.

Karla Satchell, PhD, associate professor in microbiology-immunology, marveled when she actually saw what the toxin Vibrio vulnificus looked like after working with it for nearly five years. A pathogen found in seafood, the toxin is evolving into a huge problem as it spreads with global warming. Satchell is devising better surveillance methods for the FDA.

Argonne National Laboratory, Argonne, Illinois, USA

Anderson’s team mapped out two large clusters of atoms―known as domains―in the protein and is running additional studies to uncover more. Strung together, all the domains will give a precise picture of an extremely large protein.

Satchell notes the boon that CSGID is for Northwestern: scientists don’t have to wait years for protein imaging and don’t need grant money for structural biology investigation because the center already does this.

“Wayne’s group has really expanded biochemistry on this campus,” Satchell explains. “One of the big advantages he brings is that biochemists are interacting a whole lot more with structural biologists, and these one-on-one interactions are invaluable.”

Bacteria Behaving Badly

It’s not just how proteins look, but what they do and how they do it that are key pieces of information.

CSGID has partnered with the non-profit Novartis Vaccines & Diagnostics in Siena, Italy, for five years to map the surface proteins on Staphylococcus aureus and groups A and B Streptococcus bacteria.

Understanding the location and action of proteins on the surface of pathogens is the key to creating strong vaccines and also part of the new design approach known as structural vaccinology. Other researchers have put the first vaccine candidates developed through structural biology into the development phase and clinical trials. Guido Grandi, PhD, senior project leader for research for Novartis Vaccine Development, looks to do the same.

“The efficiency of Wayne’s group is outstanding, and now Wayne and I have a long-term vision and project,” says Grandi, who also sits on CSGID’s target selection committee.

Some infections caused by these bacteria have become antibiotic-resistant, which has led scientists to focus on creating a vaccine instead of another antibiotic.

So far, Grandi has submitted 100 targets to CSGID, and about 15 percent have been successfully mapped. From that information, he  is determining  which domains in the larger protein are important to produce an immune response.

“We will weed out domains that are irrelevant to work on and focus efforts on those that are possibly worth pursuing for a  universal vaccine. I am sure this is going to be a big contribution to vaccine development in the near future,” Grandi explains.

Precursor to Drug Discovery

CSGID does not do the actual chemistry for drug discovery. In fact, it is too soon for new drugs to be discovered or reach clinical trials following the center’s findings.

However, studies point researchers in the right directions to find the best drug candidates faster than conventional methods,helping scientists quickly sift through millions of potential compounds.

Wayne Anderson at the Advanced Proton Source at Argonne

Anderson came to Chicago mainly because of the capabilities he foresaw with the Advanced Photon Source, and emphasizes that he appreciates Northwestern’s support for the facility.

“Making crystallography available to any biomedical scientist who is interested in doing it has been very rewarding,” he says.

His findings expand opportunities for investigators throughout the world to work on new drugs and vaccines because CSGID’s results are available in the public domain. Scientists can speed up research instead of waiting for peer-reviewed publication or proprietary information. As word got out about the service, Anderson says requests for imaging escalated from outside Northwestern starting in 2010.

High demand for the CSGID led him to step down this year as director of the Midwest Center for Structural Genomics, an international organization dedicated to mapping proteins through crystallography and other methods.

The days of serendipitous drug discovery are long gone, and past experiments through traditional methods have been disheartening. But new and better information provided through Anderson’s teams are prompting scientists to look again for new antibiotics.

“Detailed structural information on protein targets will increase the efficiency of new drug discovery.  I am very optimistic that we are refocusing on the right protein targets that will bring us new antibiotics. This is imperative,” Anderson emphasizes.

CSGID is funded with Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contracts No. HHSN272200700058C and HHSN272201200026C.