Monthly Archives: May 2014

The structural secrets of enzyme used to make popular anti-cholesterol drug

May_Part 2_LipidologyIn pharmaceutical production, identifying enzyme catalysts that help improve the speed and efficiency of the process can be a major boon. Figuring out exactly why a particular enzyme works so well is an altogether different quest.

Take the cholesterol-lowering drug simvastatin. First marketed commercially as Zocor, the statin drug has generated billions of dollars in annual sales. In 2011, UCLA scientists and colleagues discovered that a mutated enzyme could help produce the much sought-after pharmaceutical far more efficiently than the chemical process that had been used for years – and could do it better than the natural, non-mutated version of the enzyme. But no one quite knew why, until another team of UCLA researchers cracked the mystery.

Using a combination of experimental measurements and extensive computer simulations, the multidisciplinary team of researchers – from three chemistry, biochemistry and chemical engineering labs at UCLA – uncovered important structural features hidden in the modified enzyme that helped them unlock the secret of its efficacy. Their findings will be published in the June print edition of journal Nature Chemical Biology and are currently available online.

How the enzyme catalyst was first discovered

Simvastatin was already in widespread use when scientists found that a natural enzyme called LovD, originally harvested from a mold found in soil, could react and produce a drug similar to simvastatin. Yet as a catalyst, its rate of reaction was too low for commercial manufacturing.

So UCLA professor Yi Tang collaborated with scientists at Codexis Inc., a developer of industrial enzymes, and used a process called “directed evolution” to create a mutated version of the enzyme that was better, faster and more stable for industrial use.

“Directed evolution is a laboratory technique that mimics the natural evolution process but in a much more rapid fashion,” said Tang, who holds dual appointments in the department of chemistry and biochemistry and the department of chemical and biomolecular engineering.

Tang’s laboratory and the Codexis team created randomly mutated versions of LovD, each with a slightly different sequence of amino acids that altered its basic form and function. The team then selected those enzymes most capable of producing simvastatin and repeated the process to further enhance their reactivity.

After nine rounds of directed evolution, they had identified a mutated enzyme they called LovD9, which deviates from the original LovD enzyme through 29 distinct mutations and which produces simvastatin 1,000 times more efficiently than the natural enzyme.

“Simvastatin has several complicated structural features, so trying to synthesize it chemically takes time and money,” said Gonzalo Jiménez-Osés, a UCLA postdoctoral scholar and the paper’s first author. “If you can produce these compounds in a straightforward manner, it is a huge improvement for drug production.”

Prior to the development of the LovD9 enzyme, simvastatin had been produced through a multistep process involving expensive and hazardous chemical reagents and solvents; the introduction of LovD9 into the manufacturing process in 2012 changed all this, resulting in a far more efficient and environmentally friendly alternative to the chemical manufacturing procedure.

“Because it is an enzymatic process, no toxic chemicals or excess organic solvent are used,” said Tang, who received the EPA’s Presidential Green Chemistry Challenge Award in 2012 for his work with Codexis.

While making simvastatin using LovD9 provided clear advantages, the reasons why the mutated enzyme worked so much better than the natural one remained unclear. So Tang turned to colleagues Kendall Houk and Todd Yeates, both professors of chemistry and biochemistry at UCLA, to see if they could combine their expertise to come up with an explanation.

Cracking the structural mystery

“There are many different examples of directed evolution being used to produce catalysts that enhance the speed of commercial or synthetic processes, but the fact that you have a good catalyst doesn’t give you any information about how it works,” Houk said.

To determine the molecular structures of both LovD and LovD9, Yeates’ laboratory grew protein crystals from each enzyme and scattered X-rays off of them in a process called X-ray crystallography. These measurements gave Yeates an in-depth look at the molecular architecture of the enzymes, yet both appeared virtually identical, with no obvious structural variations to explain why LovD9 was more efficient.

While the two enzymes might appear similar when in solid crystal form, they behave quite differently when immersed in water, Houk said. The enzymes are composed of long chains of amino acids that can rotate and twist when allowed to move freely, yet this complex motion cannot be easily observed through laboratory experiments.

To quantify these minute molecular fluctuations, Houk and Jiménez-Osés used a computer program that simulates how the mutated and natural enzymes undergo internal motions when dissolved in water, and how this motion will influence the ability of the enzymes to cause the transformation that synthesizes simvastatin.

Determining why the mutated LovD9 enzyme works better than its natural counterpart involved simulating the movement of the complex enzyme in a fluid environment over a period just microseconds long. A microsecond may seem like a very short amount of time, but computations for the motion of such a large molecule required massive computing resources, Houk said.

The team was able to harness the tremendous amount of computing power necessary for these calculations by using the National Science Foundation-sponsored Anton supercomputer designed by the D. E. Shaw Research laboratory and located at the Pittsburgh Supercomputing Center.

“In the machines that we have for our routine calculations, each of the simulations in this timescale takes more than one month,” Jiménez-Osés said. “Using Anton, we can do the same amount of calculations in one day. It is a huge improvement.”

From the results of their computer simulations, Houk and Jiménez-Osés determined that part of what makes the mutated enzyme so effective is that it can function without the involvement of an additional protein that is required by the natural enzyme. Also, the mutated enzyme moves and twists in such a way that it remains in a configuration beneficial for simvastatin production far more often than its natural counterpart.

These calculations enabled the research team to understand how mutations located far from the active part of the enzyme can improve its performance.

“The directed evolution changes the nature of the amino acids that are in the protein,” Houk said. “The molecular dynamics simulations allowed us to trace how these changes in amino acids altered the structure of the protein and made it appropriate for use as a catalyst.”

In the case of LovD9, these small differences make the reaction to manufacture simvastatin vastly more efficient. Now that they know which structural features in the mutated enzyme help improve simvastatin production, the team hopes to directly engineer an enzyme with similar properties without resorting to the more random directed evolution process.

“What was special about this study is that we analyzed what happened during directed evolution in order to try to understand how these improvements are made within the protein,” Yeates said. “We hope in the future that it might be possible to make better enzymes in rational ways by understanding how it occurs in random ways.”

The mystery behind the mutated LovD9 enzyme may have remained unsolved without an extraordinary degree of collaboration between chemistry and biochemistry researchers across departments at UCLA.

“This project could not have been completed without four groups coming together to try and solve a problem that is really challenging by combining their different specialties and techniques,” Jiménez-Osés said. “By piecing together this puzzle from biochemical, engineering, structural, and molecular dynamics angles, it was possible to come to a fairly cohesive picture about how the directed evolution process worked in this case,” Yeates added.

http://www.medicalnewstoday.com/releases/276809.php

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Cancer spreads with help from ‘bad’ cholesterol

May_Part 1_LipidologyOnce cancer starts to spread to other parts of the body – a process called metastasis – it becomes much more difficult to treat. Now in a world first, an international study published in the journal Cell Reports and led by the University of Sydney in Australia identifies “bad” cholesterol as an important culprit in metastasis.

Cancer happens when normal cells start to behave abnormally, grow out of control and multiply to form lumps called tumors. If untreated, cancer cells can escape their primary tumors, travel to other parts of the body and grow into secondary cancers or metastases. Metastases are the major cause of death from cancer.

If we are to significantly improve cancer treatment, we need a better understanding of metastasis. One of the areas researchers are keenly investigating is what helps cancer cells escape primary tumors and set up new sites elsewhere in the body.

We already know that most of the cells in the body stick to each other because they have velcro-like molecules on their surfaces called integrins. In recent years, researchers have discovered that integrins help cancer cells to escape tumors and settle elsewhere in the body.

For instance, in 2012, Medical News Today learned of a study published in The Journal of Cell Biology that explained why migrating cancer cells often express integrins that provide better traction. The study revealed how a lipid-converting enzyme called DGK-Alpha helps cancer cells gain traction and mobilize.

So an important question in cancer research is how to block integrins so they stop cancer cells from moving and spreading. Some inhibitors of integrin have been developed, but they are not suitable for clinical use, say the researchers behind this new study.

‘Bad’ cholesterol helps integrins move, ‘good’ cholesterol keeps them inside cells

Researchers have discovered that integrins can move from the surface of cells to the inside, and thatcholesterol, one of the major lipids in the body, is needed to keep integrins on the surface of cancer cells. But the underlying mechanisms, until now, have been somewhat unclear.

Thomas Grewal, a senior author of this latest study and an associate professor in the Faculty of Pharmacy at Sydney, says they identified “that ‘bad’ (low density lipoprotein or LDL) cholesterol controls the trafficking of tiny vessels which also contain these integrins, and this has huge effects on the ability of cancer cells to move and spread throughout the body.”

He says they found high levels of “bad” cholesterol seem to help the integrins in cancer cells to move around, and in contrast, high levels of ‘good’ (high density lipoprotein or HDL) cholesterol seem to keep the integrins inside cells.

He and his colleagues conclude that fine-tuning of cholesterol levels could be a way to influence cancer cell migration and invasion.

Knowing “how to manipulate and lower ‘bad’ cholesterol could significantly help to reduce the ability of cancer cells to spread,” says Prof. Grewal, who with co-senior author Carlos Enrich, a professor in the Faculty of Medicine at the University of Barcelona in Spain, has been working on the link between cholesterol and cancer for 15 years.

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http://www.medicalnewstoday.com/articles/276606.php