Scientists help build better antivirals

April 02, 2010

The last big flu pandemic in 1918 infected one-third of all humans on the planet and killed about 100 million people. As with earthquakes, it's a question of when, not if this will happen again. A new paper published in Nature Chemical Biology by SFU chemist Andy Bennet and PhD student Jeff Chan describes a clever method to assist scientists in the rapid design of anti-viral drugs. Their strategy targets a vulnerable point in the life-cycle of the flu virus, and it can be used even after the virus mutates to become drug resistant.

The H1N1 flu gets its name from two proteins on its surface that stick out like spikes. The "N" stands for neuraminidase, an enzyme that breaks down a type of sugar molecule. Neuraminidases are found in most life forms, sometimes even in human saliva. Influenza uses it to plow through mucus in the respiratory tract to infect human cells, and later to help release groups of fresh virus particles after they emerge from an infected cell, thus spreading the infection. Anti-influenza drugs such as Relenza and Tamiflu work by blocking the action of viral neuraminidase to slow the infection and make it easier for the body's immune system to dispose of the virus in big clumps. But the problem is that neuraminidase occurs in many different variants. At least 137 are known and nine are found on flu viruses. Also, viruses tend to mutate and change their neuraminidase ever so slightly to become drug resistant.

Enzymes with names ending in "-ase" cut molecules in two. Neuraminidase cleaves a molecule called sialic acid—from the Greek word for saliva sialon—found on cells throughout the nose, mouth and lungs where the flu virus thrives. The enzyme works by lowering the energy required to break up sialic acid in a chemical reaction. The highest energy point in a reaction is called a transition state (transition from reactants to products) and it only lasts for one tenth of a trillionth of a second (10-13). "That's equivalent to the duration of one bond vibration within the molecule," says Jeff Chan, the SFU graduate student whose doctoral thesis is based on this research.

Chan, who grew up in Richmond, BC, will be graduating in the Spring of 2011. He likens a chemical reaction to the act of getting up from a chair. "When you are sitting, you are very stable, and when you are standing, you are also stable in a different way. But what about that moment when you are in between? There is a point where your instability is at a maximum. That is when the enzyme breaks the molecular bond," he says. It's this brief transition state that Chan and Bennet study. If they can characterize the atomic bond distances, the electrical charge distribution and the geometry of sialic acid as it passes through the transition state, they will have a blueprint for the design of new molecules that block the action of the enzyme; in other words, a drug that can stop the virus called a transition state analog inhibitor.

Their new methodology uses a technique called Nuclear Magnetic Resonance (NMR) spectroscopy, which is based on the same technology as MRI scanners for medical imaging, but is much more powerful and highly tuned to specific atoms. Bennet devised a way to simultaneously compare a series of NMR spectra taken during enzyme cleavage of normal sialic acid to that of modified sialic acid molecules containing heavier isotopes of carbon (C13) and oxygen (O18). The SFU chemists carefully synthesize the "heavy" sialic acid with C13 and O18 atoms strategically located next to the cleavage site. The isotopes’ bigger nuclei resonate slightly differently during the NMR run, yielding valuable information about the nature of the molecular bonds at the precise instant they are breaking. "The perfect small molecule therapeutic has to look like the transition state of an enzyme reaction," says Bennet referring to potentially new anti-viral drugs.

Bennet and Chan have perfected their method so it only takes about two weeks to analyze a drug-resistant neuraminidase to predict the design of a drug that works. "If you give a person a cocktail of two or three anti-viral drugs, then the virus would have to mutate two or three times before it would be fully resistant," says Bennet.

The research was made possible by the acquisition of a heteronuclear cryoprobe for an NMR spectrometer in 2007 thanks to funding from Western Economic Diversification, the BC Knowledge Development Fund, the Canada Foundation for Innovation and SFU. It was a first for Canada. "The cryoprobe improves the signal to noise ratio so we can measure things in much greater detail. It’s like having a way more expensive NMR," says Bennet, who had the idea for this research in 2004, but was not able to realize it until the tools improved. The probe takes advantage of a phenomenon that increases sensitivity and lowers noise at very cold temperatures near absolute zero. The isotope-caused differences in molecular vibrations in Bennet’s experiments are so small that they cannot be measured accurately in a standard NMR device. "To turn these ideas into reality you need a combination of expertise, good tools, good graduate students, good collaborators and above all perseverance," says Bennet. His research suffered a major setback when a steam pipe broke in the Chemistry building on July 1, 2008 flooding the NMR room.

Jeff Chan points out that their technique employing NMR spectroscopy and isotopes to characterize the kinetics of reactions can be used to study non-enzymatic systems and all sorts of fundamental chemistry. In the meantime, let's hope it leads to a few new anti-viral drugs so we will all be better prepared for the next flu pandemic.

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