Rewriting the genetic code in the name of medicine:

Like language instructors introducing new words to their students, Harvard Medical School researchers have “taught” cellular ribosomes – a cell’s protein factory – to create new compounds using foreign substances.

The advance promises to open new frontiers of drug discovery, as scientists seeking to harness this approach free themselves from nature’s stricture that anything a cell makes is created from the same list of 20 amino acids.

This will allow incorporation of different chemicals that would give the biochemically created compounds properties that would previously have been impossible. It will also allow the creation of numerous new compounds that will increase researchers’ chances of finding treatments or cures.

“Drug discovery today is somewhat like a lottery, whereby you take as many tickets as the technology allows and you hope one of them is a winner,” said Instructor of Pathology Anthony Forster, who conducted the research along with Associate Professor of Pathology Stephen Blacklow.

Blacklow and Forster, both of Brigham and Women’s Hospital, said that in addition to practical applications, their work will also help illuminate the protein-making mechanisms at work inside the cell. The two worked closely with collaborators from Virginia Cornish’s lab at Columbia University and also credited the guidance of Herbert Weissbach, a professor at Florida Atlantic University who has done pioneering work in this field. Their results were published in the May 27 issue of the Proceedings of the National Academy of Sciences.

Over the years, scientists have repeatedly sought to use a cell’s protein-making process to create new drugs and other compounds. They have had some dramatic successes, such as inducing bacteria to produce human insulin by splicing human insulin-producing genes into the bacteria’s DNA. They have been limited, however, to creating only natural substances like insulin by nature’s insistence that anything a cell makes is drawn from the 20 naturally occurring amino acids.

Blacklow and Forster’s findings have now changed – in a test tube anyway – a fundamental law of biology termed the “central dogma.” The central dogma says that information flows in a rigid way within a cell, originating in the DNA, moving to the RNA, which then couples with a ribosome to create proteins out of the naturally occurring amino acids according to the universal genetic code.

Blacklow and Forster figured out a way around the system’s natural constraints by essentially hijacking the DNA’s messages in transit. They did this by switching the chemical adaptors that respond to the DNA’s instructions. Instead of delivering the natural amino acids that the DNA calls for, these new adaptors introduce their custom-made unnatural amino acids.

This allows the researchers to essentially order the ribosomes to begin creating protein-like molecules that contain their unnatural amino acids.

These new creations are called peptidomimetics and incorporate different chemicals that give the molecules properties with potentially far-ranging characteristics. Besides creating entirely new compounds according to the modified genetic code created by the researchers, the process might also be used to add pharmacologically desirable characteristics – such as, for example, increased durability, or making a medicine available orally rather than by injection. They could also increase a drug’s effectiveness by improving its adherence to a target bacterium, virus, or cancer cell.

Inspired by nature

Blacklow and Forster’s work is not the first in this field. In fact the two point out that the idea underlying their work was first proposed by DNA’s co-discoverer Francis Crick in 1955. Previous attempts have managed to coax ribosomes to create proteins with only one or two unnatural amino acids in specific locations on the molecule. Taking a different approach that prevents cellular reactions that have blocked previous attempts, Blacklow and Forster’s process is the first that allows creation of peptidomimetics with several unnatural amino acids in a way that could potentially be expanded for use in drug discovery.

The two said they were inspired by the process that nature uses to fight disease. When a bacterium or virus invades a body, the body’s immune system sends teams of antibodies to the site. Using a process that mimics evolution, the body amplifies the most effective antibodies and then tweaks them until it creates an even better one that defeats the infection.

Nature has a huge advantage over medical researchers, though. Instead of the million or so compounds in drug companies’ libraries that can be tested against a new disease, nature can create a staggering diversity of antibodies – more than a billion different types – to throw against an infection.

“Antibody diversity is vast compared to the compound library [at drug companies],” Blacklow said.

Though scientists have managed to create artificial antibodies, they’re of limited use because they are expensive, they cannot move inside a cell, and they have to be given intravenously.

“We were thinking, ‘What if we’re to take the lead of the immune system but make something more like a drug than an antibody,’” Forster said.

The two have so far only managed to make very small peptidomimetics, but Forster said that may not be a problem because small size is an advantage for a drug, as it helps it move through the cell wall.

An important advantage of using peptidomimetics versus compounds created in other ways is their ability to be amplified, or replicated repeatedly using the genetic code. Peptidomimetics that bind to a target bacterium, virus, or cancer cell could be selected, amplified, and potentially even evolved repeatedly, enabling the screening of libraries of peptidomimetics as vast as nature’s antibody library.

Technological trickery

The two started with the basic premise that the difficulty scientists have had creating these artificial peptidomimetics doesn’t lie with the original instructions contained in the DNA, but rather with the cell’s protein-making system that uses those instructions.

In nature, a protein is created from blueprints contained on a cell’s DNA. Those blueprints are taken by a kind of RNA called a messenger RNA, or mRNA, to a ribosome, which is a cell’s protein-making factory. The ribosome reads the blueprints using a different kind of RNA, called a transfer RNA, or tRNA. Different tRNAs collect different amino acids, which are strung together by the ribosome to create a protein.

Working with E. coli bacteria, Forster and Blacklow decided to try tricking the system into accepting unnatural amino acids by creating their own tRNAs loaded with unnatural amino acids for delivery into the protein.

One of the hurdles they had to overcome is the cell’s own proofreading system, which ensures the right proteins get made. Trying to remove these proofreading elements would be difficult, so the two decided to isolate just the cellular elements they wanted and then add them together.

“In order to remove so many things – it’s almost impossible – so we put together the system from scratch,” Forster said.

The two did so with the advice of Weissbach on the elements the system should contain, and with the chemistry know-how of Cornish, whose group created the unnatural amino acids to connect to the tRNAs.

The two said the experiment proved that their process could be useful in creating these new substances and that the next step would be to begin building compound libraries.

“What we’d like to see, ultimately, is drugs coming out of this,” Forster said. “Ultimately, we hope to make a difference.”