A molecular hide and seek

Bruno Cenni, Executive Director of the immunology department at Novartis Biomedical Research, has some explaining to do whenever it comes to the covalent inhibitor he and his colleagues have worked on for nearly two decades. “Whenever we discuss this uncommon mechanism, many researchers are still surprised because the behavior is very counterintuitive compared to how drugs usually work,” Cenni said. “We had to rethink the methods we normally use to track what drugs do in the body,” added Cenni. Cenni is referring to a class of drugs that has previously been reserved for oncology treatments, but which Novartis has recently explored for a variety of immunological diseases. While the vast majority of drugs only temporarily interact with their protein targets, this class instead creates permanent bonds. “Typical drugs get flushed out of the body within a matter of hours, but in this case, the drug will stay in the body as long as the protein target does,” explained Cenni. “We only see the efficacy wear off when the protein itself reaches its natural lifetime and is replaced by the body, which for this protein is about one day.” Generally avoided because of concerns about side effects, these long-lasting, or covalent, drugs are now in advanced clinical trials for diseases such as urticaria, where they are already showing promising safety and efficacy profiles. But before human trials, the Biomedical Research team ran extensive preclinical studies to fully understand not only the safety and efficacy, but also where exactly the drug goes in the body.

Vanishing act “When you swallow a pill, the drug goes into your body and the classical model is that the amount of drug in the blood or tissue will determine how well the drug will work,” explained Cenni. “There is a very close correlation between drug levels and efficacy.” Normally, an oral drug will first be absorbed by the gastrointestinal system and, from there, it will enter the blood stream and be distributed at varying levels throughout the body. Researchers take samples from blood and different tissues to measure these drug levels and this information allows them to detect and fine-tune a drug’s efficacy. But Cenni went on to explain that this correlation between drug levels and efficacy is nearly absent in the case of a covalent drug – or at least it appears that way with the standard methods used for typical drugs. In fact, the drug is effective when there are no detectable drug levels in any of the places researchers normally look. At first, the behavior is the same – after swallowing a pill, there is a measurable increase in drug levels throughout the body. But then, the drug quickly vanishes, particularly from the location that is easiest to measure – blood. “We can still see the overall effects of the drug, but the drug itself is gone from all of the usual places we would measure it,” said Cenni. “This was somewhat surprising initially, but in fact this is consistent with how our teams designed this drug.” One of the inspirations for exploring this unconventional drug type was that all of its reversible predecessors were poorly absorbed in the body and were too rapidly eliminated to be effective. But once the team decided to design a covalent inhibitor, there was no need for the drug molecule to stay for long anywhere in the body except for the drug’s protein target. “It made all the difference when our chemists actually had this goal from the outset and it makes sense that there is no ‘free’ drug concentration anywhere in the body – all of the drug is very specifically located only where we find its protein target,” explained Cenni. “But that meant that we had to develop a new approach to understand the levels of this protein-drug complex where it occurs in the tissue,” added Cenni. Going where the drug goes The team knew that their target protein, called Bruton’s tyrosine kinase, is particularly enriched in a class of immune cells called B cells, which play an important role in how the immune system remembers and recognizes past infections. They also knew that a large source of B cells in the body is the spleen, and so they focused their preclinical efforts on that organ. “The key issue was to understand if the drug bound to the protein in spleen tissue and, if so, how long it stayed there,” said Cenni. “We also wanted to optimize our drug candidate by comparing which variations of our drug candidate did best.” To do this, the team’s chemists designed a molecular probe to specifically “fish out” the target protein from spleen tissue samples. This allowed them to isolate the target and measure how much of the protein had the drug attached – the goal being to have as much of the protein bound to the drug as possible.

“It was not so easy getting these assays running and it took the team a while, but in the end, this gave us a very good understanding of where the drug goes in the body and how it behaves,” added Cenni. Thanks in part to these studies, the team learned that their drug candidate was not only highly effective, but that the target protein itself was recycled after about one day. “This information helped us to determine the best dosing schedule for the drug, first in preclinical and later in clinical studies,” explained Cenni. Dogma-free thinking Cenni, who is a pharmacist by training, admits that his background outside of immunology helped him be open to exploring this somewhat misunderstood type of drug. “Similar to drug discovery’s avoidance of covalent inhibitors, for a long time the immunology field thought that it would be a safety risk to target B cells,” explained Cenni. “But since I came from a different field, I didn’t have those biases and it has since turned out that targeting B cells has been a very successful strategy, here at Novartis and by many others.” Eliminating B cells outright has provided significant benefits in diseases such as multiple sclerosis and rheumatoid arthritis, but the Novartis team hopes that their new drug candidate will offer a more flexible and nuanced treatment option. This is because this new drug’s effects will wash out after a day or two, whereas it takes the body many months to restore B cell populations after they have been depleted. “The jury is still out about the future of this type of covalent drug, but with every day and every patient that is doing well on the drug, we’re getting closer to achieving our goal of a new treatment to help patients,” concluded Cenni.

Research Thinking out of the box. A molecular hide and seek Text K.E.D. Coan. Photos Annick Ramp. Videos Stevie Michelsen. Most drugs only temporarily bind to their protein targets – interactions that last from minutes to hours. But what happens in the body if a drug forms a bond so strong that it lasts for the rest of a protein’s lifetime?

To make their case, scientists needed to develop new types of assays.

Sometimes, as Bruno Cenni said, it is necessary to keep a distance to the subject matter and be able to think out of the box.