MDA Grantee Christian Lorson's research on SMA has helped to lead to a high-tech screen for drugs to treat the disorder.

Fast-Track
Pharmacy

New Technology Could Propel Rapid Drug Discovery for DMD and SMA

In the mid-1980s, when MDA-supported researchers began searching for the defective gene underlying Duchenne muscular dystrophy (DMD), the scientific community at large was pessimistic about the outcome.

In contrast to previous gene hunts, this one was launched with no clue about the gene's identity. Lacking the option to select a handful of candidate genes, the researchers chose to conduct a broad sweep for the DMD gene using markers — genetic signposts that were inherited along with DMD in different families. To many scientists, the effort was a typical "fishing expedition" — casting out a line with little chance of catching anything.

But by 1986, a team of researchers had reeled in the DMD gene, an accomplishment that paved the way for research into gene therapy and stem cell therapy for the disease.

Today, MDA-supported researchers are continuing to fine-tune those therapies, but they're also launching a second wave of fishing expeditions — this time, to uncover pharmaceutical therapies for DMD and other neuromuscular disorders, including spinal muscular atrophy (SMA) (see "Fishing for Drugs").

Rather than use traditional pharmacological approaches to evaluate just one or a few drugs at a time, they're using new technology that rapidly sifts through thousands of drugs and drug targets with potential clinical value.

This approach — called high-throughput screening in the research world — is again drawing skepticism, but even the skeptics have to be impressed with its possible rewards. These high-tech searches have the potential to significantly add to the limited drug treatments currently in use or in clinical trials for DMD (see "DMD Drugs in the Pipeline") — all with the same remarkable speed of the DMD gene hunt.

Making Up for Lost Dystrophin

The DMD gene encodes dystrophin, a protein normally found beneath the surface (membrane) of muscle cells. As illustrated at right, dystrophin appears to provide an essential link between proteins within the membrane and proteins that make up the cell's skeleton (or cytoskeleton).

Many researchers believe that dystrophin and its associated chain of membrane proteins, together called the dystrophin glycoprotein complex (DGC), form a scaffold that protects the muscle cell from the mechanical forces of contraction. In any case, genetic defects that cause a loss of dystrophin — or essential pieces of it — destabilize the DGC and lead to the characteristic muscle wasting of DMD.

The obvious aim of therapy for DMD is to compensate for dystrophin deficiency, and researchers have envisioned several pharmaceutical treatments that might accomplish that feat. One strategy is to search for drugs that could stimulate intact dystrophinlike proteins to take dystrophin's place in the DGC.

A related idea is to custom-design a drug that could directly take over some of dystrophin's function or perhaps rearrange other proteins in the DGC so they're not as dependent on dystrophin.

And a final approach is to single out entirely new targets for drug intervention, by identifying unknown genes and proteins that contribute to the progression of DMD.

Delivering a Boost to Dystrophinlike Proteins

At least two proteins have a function similar to that of dystrophin, and efforts are under way to identify drugs that could boost those proteins in dystrophin-deficient muscle.

Utrophin is a small protein that looks a lot like dystrophin; it actually stands in for dystrophin in fetal muscle, but gets largely replaced by dystrophin and ends up in small patches in mature muscle. Alpha-7-beta-1 integrin (shortened here to "integrin") is a protein found in a unique complex that's functionally related to the DGC.

MDA-supported researchers have shown that when dystrophin-deficient mice are genetically engineered to overproduce utrophin or integrin, they're protected against DMD. Kay Davies, at the University of Oxford in England, did the work on utrophin, while Stephen Kaufman, at the University of Illinois in Urbana, did the work on integrin.

Since there's no obvious drug that can boost utrophin or integrin, Davies and Kaufman are independently preparing to screen thousands of chemicals that might be up to the task. Because these high-throughput screens require massive resources, including a comprehensive stock of chemicals, both Davies and Kaufman are working with biotechnology companies.

For his screen, Kaufman is collaborating with Aurora Biosciences, based in San Diego. Brian Pollok, vice president of Discovery Biology at Aurora, says the company has a chemical library that "tries to cover as much chemical space as possible." Some chemicals in the library are traditional druglike molecules and others are less conventional, derived through a process known as combinatorial chemistry, he says.

Developing an efficient way to screen for an integrin-boosting chemical will take several months, says Pollok, but once a screen is set up, it's possible to test as many as half a million chemicals in just one week.

Marius Sudol uses three-dimensional computer models of dystrophin and beta-dystroglycan to help him understand how the two proteins interact.

With help from the Long Island, N.Y.-based biotechnology company OSI Pharmaceuticals, Davies has already done a pilot screen to identify chemicals that stimulate the "A-promoter" — an on-off switch in the utrophin gene. She's screened hundreds of thousands of chemicals, and found some promising candidates that are now undergoing further testing. Recently, she discovered that the utrophin gene has several other promoters, so she's in the process of revamping the old screen.

"We're hopeful that if we can design a screen to cover all of the promoters, then we'll have a multiple-target screen that is more likely to work," she says.

Though drug screens for DMD are still at an early stage, Aurora Biosciences and MDA-supported researchers have already seen glimmers of success with a drug screen for SMA (see "Fishing for Drugs"). Since drug screens for DMD will probably follow a similar design, that's good news not just for SMA treatment, but for DMD treatment as well.

Custom Designing Drugs to Repair the DGC

While Davies and Kaufman pursue drugs to boost utrophin and integrin, MDA grantee Marius Sudol is taking a closer look at dystrophin and other proteins of the DGC. Sudol, a biochemist at Mount Sinai School of Medicine in New York, believes that examining the physical interactions between dystrophin and its partners in the DGC could enable the design of drugs that fit into the DGC and compensate for missing dystrophin.

For years, Sudol has focused on a tiny region of dystrophin that acts as a critical attachment point between dystrophin and another DGC protein called beta-dystroglycan (see illustration). Studying that tiny region, called the WW domain, "gives researchers a new tool to probe the [DGC] and might lead to the discovery of small molecules that can regulate [DGC] assembly," says Sudol.

The WW domain is present in many proteins besides dystrophin, including caveolin-3, the protein that's defective in one type of limb-girdle muscular dystrophy (LGMD). For all proteins that have it, the WW domain appears to be an important site for interaction with other proteins.

To gain a better understanding of the WW domain's function, Sudol is collaborating with AxCell Biosciences Corp., a biotechnology company in Newtown, Pa., that specializes in charting protein-protein interactions. Using a patented technique, AxCell recently completed a high-throughput screen and identified more than 69,000 protein interactions of the WW domain. At least a subset of those could hold clues to drugs that might compensate for the loss of dystrophin and its WW domain, Sudol says.

In his own laboratory, Sudol's research on the WW domain has recently led to progress toward drugs for Alzheimer's disease. In recent experiments, he designed small pieces of protein (peptides) to inhibit a WW domain-containing protein involved in Alzheimer's, and found that the peptides had beneficial effects in a cell culture model of the disease.

A designer drug that mimics or enhances the function of the WW domain might make an appropriate treatment for some cases of DMD, Sudol says.

Sudol predicts that large-scale screens for other protein domains that regulate formation of the DGC will yield further insights into drug treatment for DMD and other types of muscular dystrophy, like LGMD.

"I believe that protein-protein interaction screens will have a tremendous impact on muscular dystrophy research," he says.