Identifying Drug Targets, Saving Humans

Managing the Task of Discovering Breakthrough Treatments for Human Disease

Getting a new drug into the hands of patients is no easy feat. Pharmaceutical companies have to go through a myriad of tests in cell cultures, animals and humans before discovering and developing treatments for illnesses. Lexicon Pharmaceuticals, a biopharmaceutical company in The Woodlands, Texas, is focused on such a colossal task.

The researchers at the company identify drug targets through physiological analyses of mice, using a proprietary gene knockout technology whereby one or more genes are turned off. These genes represent potential targets upon which drugs would alter the gene products’ activity. The researchers use mice because their gene functions are very similar to those of humans.

“When a drug target is identified, we develop methods for screening hundreds of thousands of drug-like compounds for their ability to either inhibit or activate the drug target,” says William Paradee, a senior scientific group leader in the Discovery Technologies Group at Lexicon Pharmaceuticals.

These drug-like compounds are then “optimized” by chemists to improve their chances of becoming a real drug. Once a real drug is identified, it is moved into clinical trials—with an in-house clinical development team—to determine if it is safe and well tolerated in humans and if it has the desired clinical effect.

“Our group is using a new technology to generate and screen libraries consisting of millions of drug-like molecules that will complement the traditional screening of our drug targets,” Paradee says.

A traditional high-throughput screening approach will screen anywhere from hundreds of thousands up to 1 million molecules, each molecule screened in an individual reaction chamber. According to Paradee, this new technology can screen several million molecules all in a single reaction chamber, increasing efficiency and the number of molecules that can be screened at any one time.

Thus far, Lexicon has discovered the function of almost 5,000 genes; identified more than 100 promising drug targets; and created a unique, growing clinical pipeline in the fields of cardiology, gastroenterology, immunology, metabolism and ophthalmology.

Identifying drug targets

Researchers at Lexicon Pharmaceuticals identify drug targets by knocking out or turning off one or more genes in mice and then looking at how their bodies—physiologically and behaviorally— are affected by the disruption of the gene(s).

Dr. Paradee’s group uses a new drug screening technology that allows them to generate and screen millions of different drug-like compounds in a single reaction (micro-centrifuge tube; pictured). This differs from traditional high-throughput drug screening, in which up to a million individual compounds are assayed in separate reactions within thousands of multi-well plates.

“We look at hundreds of different characteristics of these knockout mice; we look at the blood profiling, body weight, body fat composition, bones and behavior, to name a few,” Paradee explains.

The scientists then evaluate whether the proteins encoded by the particular gene(s) can be used for pharmaceutical purposes.

“The vast majority of genes code for a specific protein. Some proteins are more amenable to inhibition by drugs than others,” Paradee says. “For example, enzyme activity can often be modulated by small-molecule drugs, while secreted and membrane-bound proteins can often be inhibited by antibodies or small-molecule drugs.”

Since inactivating a protein by switching off the gene that encodes that protein often has an effect that is similar to the way a drug inhibits a protein, this approach helps researchers model before a drug is made how it may react in the body.

Because of the comprehensive and broad nature of this method, which allows scientists to uncover functions of human physiology, Lexicon scientists do not have in mind any specific disease when they go to work on these genes. Rather, they start with a blank slate and go after different classes of proteins and enzymes.

“We don’t have a bias when we go into our analysis as to what we’re looking for,” explains Paradee of their process. “Now we have models in rodents that may give evidence that this would have an effect on Alzheimer’s disease or in learning and memorytype diseases, but we don’t start out looking for a cure for Alzheimer’s disease or mental retardation or anything like that.

“Those are things we discover after we look at the animals and their different characteristics; then we can say this drug target may have an effect or may be useful in these diseases.”

Paradee explains that proteins that are more involved with cytoskeletal structure don’t make good drug targets. That’s because structural proteins maintain the integrity of the cell, and targeting them could be harmful.

The overall process

The development of drug-like libraries is a multiphase process. During the first phase, a team of chemists conducts an initial screening of the drug-like molecules to see how they may fit together. Paradee’s group, which works on the second phase, will then interpret the initial data and will help re-synthesize the molecules in order to make a large collection of molecules for screening drug targets.

It’s a starting point for the development of the drug, Paradee explains. “It’s kind of like a puzzle; we take a bunch of puzzle pieces and put them in different orders.”

For example, if there are three molecules, A, B and C, one can put them in ACB order or CBA or several other combinations. Within these building blocks, researchers can find different functionalities. Each combination will generate its own unique characteristics.

A researcher loads DNA samples on an agarose gel for analysis.

“We’re making libraries with these ABC compounds, but we have hundreds of them, and [by using] different technologies and methodologies, we’re able to make millions of different combinations.”

Once Paradee’s team is done with a library entry, the information is passed on to the medicinal chemists, who take the molecules, scale them up and make enough for future experiments.

“The drugs we discover with our screens are just a starting point,” Paradee explains. “The chemists look at those chemical structures and say, ‘Well, maybe if we start moving some of these molecules around—like a methyl group over here, a benzene group over here—we can increase the potency.’ It’s quite an art, actually.”

By tweaking the chemical structures, the Medicinal Chemistry Group can affect the absorption and degradation of a drug as well. For example, if a patient is taking a drug as an oral pill, the researchers want to ensure that the compound is absorbed into the system and will reach the drug target.

After the drug is absorbed, it’s important that the enzymes in the liver— which has the job of degrading the drug—will not degrade it too rapidly.

“It’s a balancing act; you want to decrease the toxicity of your molecule while increasing the potency, the absorption, the amount of time it stays in the system. You don’t want to give a pill every 30 minutes,” explains Paradee.

The researchers also have to be wary of any adverse effects that the drug may have on a patient’s body, such as disrupting critical enzymes or proteins such as ion channels in the heart.

“You also want to make sure that your drug has specific activity for a particular target and isn’t having cross activity against similar family members,” Paradee says.

“There are families of proteins that have similar (but not identical) functions. In our initial screens, we try to ensure we are not affecting the similar proteins with our drugs, because there may be an additional effect by targeting more than one protein.”

These “off target” effects can lead to undesirable side effects.

Once a potential drug passes clinical studies and all the required applications and permits are completed and obtained, Lexicon will have to outsource the job of large-scale drug production to a partner or contract company with the necessary production capabilities.

Lab structure

Lexicon has approximately 350 employees divided into groups throughout the company. The Discovery Technologies Group has six employees, all biologists, divided into three groups—each one with a specific function and its own manager. Paradee manages one of these groups.

“I am responsible for testing new chemical reactions and overseeing the production of the new chemical libraries,” he explains of the new project he’s been working on for nine months.

But Paradee emphasizes that although his function is very specific, he and others never work in a vacuum and there’s a lot of cross talk between the groups in the company to increase productivity.

“We interface directly with groups all over the company. If you include everyone we rely upon, the number in our group would be around 20,” he explains.

The Discovery Technologies Group is housed in an 800-square-foot area in the company. But that does not include such common areas as chemical corridors and freezer rooms that the team routinely uses.

Unique challenges of a pharmaceutical lab

Prior to joining Lexicon in 2001, Paradee had worked only at university labs as a graduate student and a postdoctoral researcher. When he made the switch to a biopharmaceutical company, he realized how different the two worlds are.

“There’s more drive here; we’re under a deadline [and] trying to get things done in a certain period of time,” he says. “When I was a graduate student and a postdoc, I’d kind of set my own schedule, so I was really dependent upon myself [for] how quickly I want[ed] my experiments to run.”

“In an industrial lab, other groups are dependent upon us and we’re pretty dependent upon other groups. It’s more of a team environment. We’re trying to keep the team, the company, moving forward versus at an academic lab [where] it felt more individual.”

But this team-oriented effort doesn’t come without its own unique challenges. Because the groups that Paradee deals with are dotted across the company and work on different aspects of the project, communication is always a challenge.

“We’re all scientists, but we all have a different vocabulary,” Paradee explains. “Learning the lingo, learning how chemists talk, is a little different than how biologists talk, you know, when they’re trying to explain what they want done and how they would do it.”

This challenge is met through lots of interactions.

“We just talk it through,” he says. “I was on the phone last week with one of the lead chemists on our project and was explaining to him what I was doing, and he was just stopping me, saying, ‘We refer to that reaction as blah blah blah.’ So I start referring to the reaction that way so he understands me.”

Additionally, keeping everyone updated on daily expectations, data analysis and interpretation, and troubleshooting are Paradee’s other challenges. He overcomes these through proper planning and assistance from others in the company.

The team meets in the morning to work out what needs to be done that day, he explains of the daily expectations. As far as troubleshooting goes, Paradee relies on Lexicon’s highly trained chemists: “They answer all my questions.”

The details: instrumentation, maintenance, inventory and hiring

The Discovery Technologies Group routinely uses mass spectrometers to analyze chemical reactions and quantitative polymerase chain reactions for screens and assay development. The day-to-day maintenance of these machines is the responsibility of each lab individual. However, if major maintenance is needed on a piece of equipment, the team calls on the company’s maintenance department. If the job is too big to take care of in-house, it gets contracted out to specialists.

Each person is also responsible for taking inventory of general lab consumables— such as chemicals and test tubes—and for their own specific requirements. When an item is needed, the person will put a purchase request through to the group director for approval.

“If we’re looking at a big capital piece of equipment, that would be discussed and we would work with our purchasing department to work out the best deal,” Paradee explains. “We will [also] check different suppliers, [because] maybe different suppliers would have other options that we could consider.

“Consumable supplies that are used throughout the company are bulk ordered and stocked.”

Although he may recognize and suggest the need for new personnel, Paradee is not directly involved with hiring.

“In a biotech lab, hiring is coordinated through the human resources department,” he says. “Approval for a new hire must be granted by [that department].”

Worthy mission

Though the challenges Paradee and his team face daily are many, the overall mission of the company— discovering breakthrough treatments for human disease—makes the team push forward with renewed enthusiasm each day.

“I believe that with the power of the new technology we are developing and the robust pipeline of drug targets Lexicon has identified, we will discover those breakthrough treatments,” Paradee says.

Currently, the company has five drugs that are in preclinical and clinical trials. These include drugs for irritable bowel syndrome, carcinoid syndrome, diabetes, glaucoma and rheumatoid arthritis.