The turbulent chemistry behind smarter, safer therapeutics

A stone tossed into a lake will create ripples, which travel outward, reflecting the stone’s impact on everything in its path. To effectively combat disease, it is vital to view each therapeutic drug like a stone tossed into a lake: reflecting its influence throughout the body in constructive and destructive ways. Optimizing therapeutics is not just about understanding the drug, but also everything it interacts with, which ultimately dictates its effect.

Researchers at St. Jude are working to better understand these drug-induced ripples, from those observed within proteins to those that activate entire regulatory systems.

Alternative binding sites represent drug-discovery conduits

Scott Blanchard, PhD, Department of Structural Biology, uses single-molecule imaging to observe how drug binding creates ripples within proteins; from this experience, he suggests a change in perspective. “Typically, the focus when measuring drug activity is how tightly it binds its target. But we should be thinking of activity in the context of what we are trying to achieve, and how we want to achieve it,” Blanchard said. 

While drug discovery efforts typically target the orthosteric (primary) site where molecules bind, it is often not the only place where molecules can attach to a protein. A molecule that binds to an alternative, or allosteric, site on a protein can have knock-on effects, sending ripples through the protein, even altering how it binds other molecules at its orthosteric site.

Through his work on the cystic fibrosis transmembrane conductance regulator (CFTR), Blanchard has seen the impact of allosteric modulation. CFTR controls ion flow (the movement of electrically charged particles) across the cell membrane. Mutations to the CFTR protein can lead to salt imbalances in the epithelial cells of the lungs, causing the sticky, airway-clogging mucus associated with cystic fibrosis.

For CFTR to function properly, adenosine triphosphate (ATP) binds to it at a site on the inside of a cell, sending a signal to open a channel gate on the outside of the cell. While rational drug design might focus on the ATP binding site, a 2023 collaboration between Blanchard and investigators at Rockefeller University, published in Nature, tells a different story.

“The binding site for a lot of CFTR effectors is actually in the middle of the protein; the part within the plasma membrane that you would never think would do something functional if targeted,” explained Blanchard. 

In the study, the team identified an allosteric pathway running from the ATP binding site to the channel gate on the other end. “ATP hydrolysis causes the gate to open by communicating over this long conduit of protein,” Blanchard said. “These drugs can bind to that conduit and either potentiate it or attenuate it; it’s remarkable.”

Understanding how drug binding ripples through its target reveals new opportunities for drug development, such as allosteric binding sites. Still, researchers must consider the broader complex cascade of effects within cells, including what else the drug activates, and how it changes as it breaks down.

One family of proteins, nuclear receptors, uses small molecules such as vitamins and hormones to activate genes, forming a vital communication network. But what does this mean for therapeutics?

Bolstering therapeutic efficacy with bodyguards

Pregnane X receptor (PXR) is a nuclear receptor in cells that detects external and internal chemicals and activates a toxin removal system to eliminate them. Cytochrome P450 3A4 (CYP3A4) is an example of such a janitorial protein that removes undesired substances from our cells. Unfortunately, these substances can sometimes include therapeutic drugs. Taosheng Chen, PhD, Department of Chemical Biology and Therapeutics, seeks ways to prevent PXR from sniffing out therapies prematurely.

“CYP3A4 metabolizes more than half of all drugs. Paclitaxel (Taxol), the anti-cancer drug, for example, is a PXR activator and also a CYP3A4 substrate,” Chen said. “It’s almost like a suicidal pathway: The drug activates PXR, which upregulates CYP3A4, which degrades the drug.”

Increasing the drug dose or modifying it can help, but drug breakdown produces metabolites, which cause their own ripples. “Some metabolites are not neutral and have unexpected toxicity,” Chen explained. “So, instead of just case-by-case chemical modification, we propose using a PXR inhibitor as a co-drug.”

Drug-binding activates PXR, but Chen wanted to better understand how to inhibit it because a PXR inhibitor could be leveraged to act as a bodyguard for many therapeutics. In two studies published in 2023 in Proceedings of the National Academy of Sciences and 2024 in Nature Communications, Chen and colleagues mapped PXR’s active site. They identified “hot spots” where chemicals can bind without activating it. 

This allowed them to design a panel of potent and specific PXR inhibitors rationally. However, in noting that small structural changes can turn helpful inhibitors into potentially harmful activators, the team began looking into alternatives.

“Since we know that we can change a PXR inhibitor into an activator with a structural change or even a mutation, it’s very dangerous,” Chen said. “So, we thought that if this is going to be a problem, instead of inhibiting, maybe we should degrade PXR.” This led Chen to proteolysis-targeting chimeras (PROTACs), which can simultaneously bind PXR and recruit protein-degrading machinery.

In a study published in the Journal of Medicinal Chemistry, combining Taxol with a PROTAC designed to degrade PXR significantly improved outcomes. PROTACs present their own therapeutic challenges, though, especially their bulky size. Chen continues to pursue multiple strategies, including PXR PROTACs, PXR inhibitors, and CYP3A4 inhibitors, to optimize drug efficacy.

But what happens when drug-removal mechanisms evolve with drug discovery? This is the challenge with antimicrobials, especially antibiotics. Years of overuse and underdevelopment have produced bacterial strains broadly resistant to antibiotics. Richard Lee, PhD, Department of Chemical Biology and Therapeutics, believes knowledge is key.

Antibiotic exodus addressed with chemical manipulation

“Drug discovery efforts usually favor a brute-force strategy: by screening millions of molecules and hoping to get lucky. I favor a knowledge-based approach; exploring the limitations and bridging them, whether it’s efflux, metabolism, or target mutation,” Lee said. “By understanding what drives resistance, we can adjust our chemistry and discovery pipelines.”

Lee’s team focuses on nontuberculous mycobacteria and Gram-negative bacteria, which rapidly develop resistance and pose major risks to immunocompromised patients. “Many bacterial infections are acute, so short treatment isn’t a big deal,” Lee said. “But our patients are immunosuppressed for months, and infections don’t clear because of the lack T cells. Some bacteria, such as Mycobacterium abscessus, are also harder to treat, leading to long-term off-target toxicities.”

Treating M. abscessus is difficult due to its robust resistance mechanisms. Many promising drugs fall victim to bacterial efflux, the process cells use to physically remove drugs by pumping them out. Increasing the dose only raises toxicity, leaving clinicians and patients with few safe and effective options.

Drug optimization often takes the form of streamlining drugs to fit their target active sites better. To avoid efflux proteins, Lee and his team explored options to physically bulk up antibiotics. In a 2024 Proceedings of the National Academy of Sciences study, the researchers optimized the antibiotic spectinomycin by adding an extra chemical side-chain to its molecular structure. This bulky addition meant the drug no longer fit the efflux protein, TetV, allowing the antibiotics to bypass efflux completely. They maintained their efficacy, however, making the modified antibiotics 64 times more potent against M. abscessus than traditional spectinomycin.

“This study helped us understand how new strategically designed analogs overcome TetV-mediated efflux while still inhibiting the mycobacterial ribosome target,” Lee said. “This was vital to reactivating the activity of spectinomycin against Mycobacterium abscessus.”

The body’s interconnected complexity means drug development is just the first step in understanding and balancing the ripples therapeutics cause when administered. However, by tracking these ripples, whether through allosteric modulators, efficacy-boosting co-drugs or knowledge-guided analogs, St. Jude researchers are charting a course towards safer and more effective treatments.