by Christopher Gerry

Our DNA influences our height, eye color, affinity for sky diving and other extreme thrills, sleep habits, disease risk factors, and more. It’s no surprise, then, that scientists have found another job for our reliable genetic ledger: as a tool to aid the discovery of new medicines. The hope is that these DNA-based tools will enable researchers to find better starting points for drug discovery where traditional methods have failed.

Bullseye

To understand the value in these new tools, we first need to learn how scientists often begin the search for new medicines. The exploratory trial-and-error sequence often mimics a game of darts. Proteins, which are the large biological molecules that are tasked with many of our cells’ most important jobs, function as dartboards. And massive collections of small molecules—the class of chemical entities that often constitute the active ingredient(s) of a drug—act as darts. If one or more of the small molecules stick to the protein dartboard, it could change that protein’s activity in a way that imparts some sort of therapeutic benefit, such as slowing the progression of cancer or alleviating the symptoms of diabetes.

But proteins are not dartboards; proteins come in all different shapes and sizes, and they reject far more molecules than they accept. So, researchers throw millions of molecules—collectively known as small-molecule “libraries”—at their protein targets hoping that just one might stick. Even then, it’s difficult to determine when that one-in-a-million event occurs. If a molecule binds, or sticks to, a protein, it might change the protein’s behavior…but not always. Other times, the protein–molecule duo might perform their potential therapeutic function in a cell but not a test tube. Because it’s often easier to test small molecules in simple systems like test tubes, it’s possible that many potentially life-saving molecules have been left on the proverbial cutting-room floor.

Now, Stuart Schreiber, the Morris Loeb Professor of Chemistry and Chemical Biology at Harvard University, wants to improve the way scientists search for small molecules that can successfully bind proteins. He and his team have combined two techniques: DNA-encoded libraries (DELs) and diversity-oriented synthesis (DOS). Separately, both approaches have enabled researchers to study some previously murky corners of disease biology. In tandem, they could significantly increase the chances of finding effective small molecules, the elusive starting points for drug discovery.

“Hello, my name is…”

DEL technology combines synthetic organic chemistry with a technique called DNA barcoding. Although DNA is generally thought of as residing inside the cells of living things, scientists have been able to use its four-letter (A, T, C, and G) alphabet to spell out molecular “name tags.” In DELs, each small molecule is physically linked to a specific strand of DNA that allows researchers to tell it apart from the others.

This tagging system has several advantages. “[DELs are] much more efficient compared to traditional screening technologies,” said Dr. Wenyu Wang, a postdoctoral researcher at the Broad Institute. Traditional methods use molecules without DNA barcodes, so each molecule must be tested in isolation to monitor which chemical caused which effect. Expensive screening facilities are required to perform large numbers of these experiments simultaneously. In contrast, a trillion-molecule DEL can be tested in a single experiment in a cheap plastic tube smaller than your thumb—because each molecule is linked to its name tag, there’s no need to physically separate them from one another. “The whole technology is amazing” said Dr. Matthias Westphal, a postdoctoral researcher in the Schreiber lab. “There are so many reasons why it should not work, and yet it does.”

Another appealing feature of DEL technology—again rooted in efficiency—is the relative ease with which chemists can make very large DELs. Rather than synthesize each molecule separately and then attach its DNA-based identifier afterwards, researchers synthesize molecules and barcodes in parallel; each reaction, responsible for building a piece of the molecule, receives a small piece of DNA. When complete, the barcode reads like a reaction map, combining the codes designated to each step needed to create the molecule (Figure 1). This iterative, modular approach allows the team to make masses of DNA-barcoded molecules at once. And because these libraries can grow exponentially with each subsequent step, they can host billions or even trillions of molecules with minimal effort on the part of the researcher.

One of DEL technology’s greatest advantages, however, is also the source of its most profound weakness. The elegant parallel synthesis of a molecule and its barcode requires water. Most synthetic organic chemists do everything they can to exclude water from their reaction flasks, but DNA only behaves properly when wet. This constraint severely limits the types of chemical reactions that can be used to build DELs. As a result, many libraries contain molecules with similar structures; if a builder only knows how to lay bricks, their buildings will largely look the same. Even exceedingly large libraries are built using only a few reactions, so they often lack structural diversity. Because structure dictates function in molecular biology, it’s likely that many of these structurally redundant molecules will bind the same proteins, leaving other potentially promising targets untouched. These chemical shortcomings limit the number and variety of diseases that DELs can address.

Embracing (chemical) diversity

The Schreiber lab has a potential solution: Diversity-oriented synthesis (DOS). True to its name, DOS can help assemble libraries of molecules with a wide variety of complex, uncommon molecular architectures. Drug developers have exploited these atypical molecules to design new treatments for neurodegenerative disease, malaria, and cancer, among others. “Libraries that incorporate structural diversity lead to functional diversity,” explained Dr. Jeremy Mason, a postdoctoral researcher in the Schreiber lab. If you’re trying to drug the “undruggable,” you want as many chances as you can get.

When trying to apply DOS to DELs, water once again complicates things because many reactions that yield complex molecules have no chance of being suitable for DEL synthesis. To overcome the wet blanket, Westphal said the lab is “combining two approaches.” The first strategy involves pushing the boundaries of DNA-compatible chemistry to include reactions that generate diverse collections of complex molecules. Though the lab has shown that this type of chemical invention is possible, the whole process can take months with no guarantee of success. An alternative approach is to install structural complexity onto the molecule before attaching it to DNA. This up-front investment can’t take full advantage of the efficiencies of on-DNA chemistry, but it guarantees that the final DNA-barcoded molecules don’t simply replicate existing libraries.

The result of all this strategic weaving? The team has created their first library, a 107,616-member DEL with an endearingly practical name: DOS-DEL-1. Already, this achievement has inspired other researchers to design similar libraries, a DOS-DEL-2 or DOS-DEL-3 perhaps.

Playing the game

“Pipetting…so much pipetting.” Dr. Liam Hudson, a postdoctoral researcher and one of the first members of the DEL team, came from a background with minimal pipetting and no exposure to DELs. In fact, despite their recent successes improving the technology, “none of the people currently doing the work in the lab has prior experience with DELs,” according to Hudson. “I like to think this means we can operate without bias towards any given way of working and focus on what we think are the key questions.”

The lack of experience could be responsible for the team’s creative approach to constructing DELs. But even the most creative ideas often benefit from a solid foundation. So, to fill in any knowledge gaps, the Schreiber lab established a close collaboration with the Novartis Institutes for BioMedical Research. Schreiber is a Novartis Faculty Scholar, and team members Hudson, Mason, and Westphal work full-time in Novartis lab space. The entire cohort—including a Novartis DEL team in Basel, Switzerland—meet regularly to swap success stories, snags, and advice. “Working within the collaboration between the Schreiber lab and Novartis has been incredible,” said Mason. “We are really getting the best of both worlds.”

Moving forward, the Schreiber Lab will continue its hunt for new molecules with valuable tricks. Their first library (DOS-DEL-1) performed well with test proteins; now, the team will direct its small-molecule hordes at some of the Schreiber lab’s highest priority (and most challenging) biomolecular targets. Proteins involved in neurodegenerative diseases, such as Alzheimer’s disease and genetic prion disease, are first in line. Just one new chemical tool could lead to vital treatment for these intractable, debilitating diseases. To amplify the cause, the team hopes that other labs capitalize on the new technology to perform their own hunt for miracle molecules. “One of the advantages of DELs is that it’s easy to share libraries and perform binding experiments” noted Bruce Hua, a graduate student in the Schreiber lab. “I’m most excited by the prospect of creating a resource that many other researchers could find useful.”

In the drug discovery game of darts—with protein targets and small-molecule missiles—we forgot a crucial component: the people who play the game. Immense time, effort, and ambition help launch the hundreds of millions of molecular darts toward a potential target, with the hope that one could end up changing someone’s life for the better.

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For more information:

• Check out this article from Chemical & Engineering News that describes how the pharmaceutical industry is increasingly looking to DELs for solutions to some of their most challenging problems.
• Explore the Schreiber lab website to read about some of the other projects that the group is working on.
• The academic research paper describing the design, synthesis, characterization, and validation of DOS-DEL-1 can be found here.
• Read this article from genomeweb to learn about molecules synthesized using diversity-oriented synthesis that can inhibit CRISPR-based gene editing.