Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Novel method could improve the performance of proteins used therapeutically

10.03.2011
Whitehead Institute scientists have created a method that uses the enzyme sortase A to site-specifically modify proteins.

Using this technique, researchers were able to increase potency, slow the metabolism, and improve thermal stability of several proteins, including interferon alpha 2 (IFN-alpha 2) and granulocyte colony-stimulating factor 3 (GCSF-3). IFN-alpha 2 is used to treat a variety of diseases, including leukemia, melanoma, and chronic hepatitis C, while GCSF-3 (known as filgrastim and marketed as Neupogen®) is administered to patients with neutropenia.

RELEVANCE: Sortase could be used to improve other proteins used as therapeutics, for example those with IFN-alpha 2's characteristic four-helix bundle structure. Such proteins include erythropoietin (EPO), interleukin (IL) 2 (known as aldesleukin and marketed as Proleukin®), IL-4, IL-7, IL9-, and IL-15. This same strategy could likely be applied to other therapeutically important proteins, but with a different folding pattern, as well.

Novel method could improve the performance of proteins used therapeutically

Whitehead Institute scientists have created a method that site-specifically modifies proteins to exert control over their properties when administered therapeutically. The technique should be useful to increase potency, slow metabolism, and improve thermal stability of therapeutically useful proteins, such as interferon alpha 2 (IFN-alpha 2), which is used to treat variety of diseases, including leukemia, melanoma, and chronic hepatitis C.

The method, reported this month in Proceedings of the National Academy of Sciences (PNAS), uses the enzyme sortase A and can be applied to tailor proteins that possess a structure found in IFN-alpha 2, referred to as a four-helix bundle. Such proteins include erythropoietin (EPO), granulocyte colony-stimulating factor 3 (GCSF-3, known as filgrastim and marketed as Neupogen®), interleukin (IL) 2 (known as aldesleukin and marketed as Proleukin®), IL-4, IL-7, IL9-, and IL-15.

"In the course of this work, the first author of the PNAS paper, Maximilian Popp, together with other members of the lab, has put together a nice palette of sortase-based techniques that now allow us to modify a large variety of different proteins, and equip them with properties and behaviors that cannot be easily specified by more standard molecular biological techniques," says Whitehead Member Hidde Ploegh. "I see the value of these approaches first and foremost in their general applicability and ease of use."

IFN-alpha 2 is a cytokine, a hormone-like substance that usually acts on cells other than those that produce the protein. Upon binding the cytokine, the recipient cell responds, for example by starting to divide and proliferate, or by exercising certain functions of benefit to the organism. Like other cytokines used for therapeutic purposes, IFN-alpha 2 can be a finicky drug. It is thermally unstable and must be continuously refrigerated to maintain its potency, a requirement that limits IFN-alpha 2's use in areas with intermittent or no electricity. Also, IFN-alpha 2's relatively short half-life (and resulting rapid clearance from the body) often necessitates frequent injections when the drug is used to treat certain conditions.

To keep therapeutic IFN-alpha 2 active in the body longer, the current strategy is to tack long polyethylene glycol (PEG) chains onto the protein to turn them into effective drugs. This so-called PEGylation not only masks IFN-alpha 2 from the patient's immune system but also increases the time the body needs to break it down. However, because current approaches to PEGylation are not specific, the PEG chains can block or alter the protein's normal binding site—an unintended consequence of this modification that can diminish IFN-alpha 2's potency by as much as 90%.

Seeking greater precision in attaching PEG chains, Popp, who is a graduate student in the Ploegh lab, used the enzyme sortase A to cleave IFN-alpha 2 at a specific site on the protein, engineered so that it would be recognized by the sortase. Then, a small molecule bearing the PEG chain was attached at the site cleaved by sortase. When Popp tested for biological activity, the resulting IFN-alpha 2 was highly potent, indicating that the PEG chains were not interfering with the drug's binding ability.

Popp also used sortase A to suture PEG chains to the cytokine GCSF-3. When he tested the PEGylated version in mice, it remained in the bloodstream significantly longer and evoked a more robust and prolonged response than a non-PEGylated version. By using sortase A's inherent precision to attach PEG chains, Popp could replace the less precise chemistry-based technique with a highly effective method that should have broader applications.

Next, Popp addressed IFN-alpha 2's thermal stability. Previously, the Ploegh lab stabilized linear polypeptides like IFN-alpha 2 by molecularly gluing their ends together to form circles. A few such cyclic proteins are found in nature. Once circularized, cyclic proteins are often more stable than their linear precursors. This forced looping can interfere with the function of some cell-signaling proteins, but because IFN-alpha 2's binding site is not near its ends, the function of IFN-alpha 2 is unaffected when its ends are joined to form a circle.

To create a cyclic version of IFN-alpha 2, Popp used sortase A to join the two ends of IFN-alpha 2. When he heated the cyclic form of IFN-alpha 2, it was more resistant to breakdown than its linear counterpart and remained biologically potent even after boiling. Popp then tested the circular, PEGylated version and the linear version in mice. The modified version was metabolized more slowly than the linear version and maintained its thermal stability, demonstrating that this simple technique can significantly enhance desirable properties of a therapeutically relevant protein without sacrificing its potency.

"We really take advantage of the site specificity of the sortase enzyme. Placing a molecular suture like that can't really be done by other means. So I think this method is of value to the protein engineering field in general," says Popp. "The reaction itself is easy, but it took some time to actually figure out how to do these transformations. Once we figured that out, the technique was robust and reproducible."

This research was supported by the National Institutes of Health (NIH).

Written by Nicole Giese

Hidde Ploegh's primary affiliation is with Whitehead Institute for Biomedical Research, where his laboratory is located and all his research is conducted. He is also a professor of biology at Massachusetts Institute of Technology.

Full Citation:

"Sortase-catalyzed transformations that improve the properties of cytokines"
PNAS, online February 9, 2011.
Maximilian W. Popp (1,2), Stephanie K. Dougan (1), Tzu-Ying Chuang (1), Eric Spooner (1), and Hidde L. Ploegh (1,2)
1. Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142

2. Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02142

Nicole Giese | EurekAlert!
Further information:
http://www.wi.mit.edu

More articles from Health and Medicine:

nachricht A 'half-hearted' solution to one-sided heart failure
24.11.2017 | Boston Children's Hospital

nachricht New study points the way to therapy for rare cancer that targets the young
22.11.2017 | Rockefeller University

All articles from Health and Medicine >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: New proton record: Researchers measure magnetic moment with greatest possible precision

High-precision measurement of the g-factor eleven times more precise than before / Results indicate a strong similarity between protons and antiprotons

The magnetic moment of an individual proton is inconceivably small, but can still be quantified. The basis for undertaking this measurement was laid over ten...

Im Focus: Frictional Heat Powers Hydrothermal Activity on Enceladus

Computer simulation shows how the icy moon heats water in a porous rock core

Heat from the friction of rocks caused by tidal forces could be the “engine” for the hydrothermal activity on Saturn's moon Enceladus. This presupposes that...

Im Focus: Nanoparticles help with malaria diagnosis – new rapid test in development

The WHO reports an estimated 429,000 malaria deaths each year. The disease mostly affects tropical and subtropical regions and in particular the African continent. The Fraunhofer Institute for Silicate Research ISC teamed up with the Fraunhofer Institute for Molecular Biology and Applied Ecology IME and the Institute of Tropical Medicine at the University of Tübingen for a new test method to detect malaria parasites in blood. The idea of the research project “NanoFRET” is to develop a highly sensitive and reliable rapid diagnostic test so that patient treatment can begin as early as possible.

Malaria is caused by parasites transmitted by mosquito bite. The most dangerous form of malaria is malaria tropica. Left untreated, it is fatal in most cases....

Im Focus: A “cosmic snake” reveals the structure of remote galaxies

The formation of stars in distant galaxies is still largely unexplored. For the first time, astron-omers at the University of Geneva have now been able to closely observe a star system six billion light-years away. In doing so, they are confirming earlier simulations made by the University of Zurich. One special effect is made possible by the multiple reflections of images that run through the cosmos like a snake.

Today, astronomers have a pretty accurate idea of how stars were formed in the recent cosmic past. But do these laws also apply to older galaxies? For around a...

Im Focus: Visual intelligence is not the same as IQ

Just because someone is smart and well-motivated doesn't mean he or she can learn the visual skills needed to excel at tasks like matching fingerprints, interpreting medical X-rays, keeping track of aircraft on radar displays or forensic face matching.

That is the implication of a new study which shows for the first time that there is a broad range of differences in people's visual ability and that these...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

Ecology Across Borders: International conference brings together 1,500 ecologists

15.11.2017 | Event News

Road into laboratory: Users discuss biaxial fatigue-testing for car and truck wheel

15.11.2017 | Event News

#Berlin5GWeek: The right network for Industry 4.0

30.10.2017 | Event News

 
Latest News

IceCube experiment finds Earth can block high-energy particles from nuclear reactions

24.11.2017 | Physics and Astronomy

A 'half-hearted' solution to one-sided heart failure

24.11.2017 | Health and Medicine

Heidelberg Researchers Study Unique Underwater Stalactites

24.11.2017 | Earth Sciences

VideoLinks
B2B-VideoLinks
More VideoLinks >>>