December 21, 2018
Could hijacking one of the body’s own systems be the key to finding new targets for hard-to-treat diseases? Dane Mohl, Ph.D., scientist at Amgen (Thousand Oaks, CA) believes it may be. With the advent of genomics, drug development has focused on using genetics to identify therapeutic targets, which has led to targets that aren’t necessarily easily drugged through traditional methods. “Letting genetics guide our choices for treating disease means we have to use whatever modalities are available,” Mohl says. “We have to be more creative and that means we need a larger tool box to get to those new targets.”
Protein degradation is essential to maintaining the right balance of the many different proteins required to keep cellular processes going in the human body. Mohl explains that all metabolically active cells are constantly producing proteins. Once these proteins have performed the function they’re designed to do, they are no longer needed. That’s when the proteasome machinery steps in and degrades those proteins, sometimes as soon as they’re made. The ubiquitin proteasome pathway is a key part of this process. Protein degradation via this pathway involves first tagging the substrate protein with one or more ubiquitin molecules to form a ubiquitin ligase. The ligase then signals the proteasome to destroy the protein.
Mohl’s interest in protein degradation began while he was working on chromosome partitioning in bacteria as a graduate student at the University of California, Los Angeles (UCLA). Ray Deshaies, Ph.D. who was a visiting professor at the time and is now head of research at Amgen, gave a lecture on the role of ubiquitination in cell cycle programmed degradation of proteins. “That was a really inspiring talk. It brought into focus the dual role of gene expression and programmed degradation and the timing of the cell cycle,” Mohl says.
After getting his doctorate degree, Mohl began post-doc work in Deshaies’s lab where he was able to pair his interest in DNA segregation and chromosome partitioning with the lab’s interest in programmed protein degradation. Around that same time, researchers in Deshaies’s lab were collaborating with members of Craig Crews’s laboratory to try and design molecules that could stimulate the degradation of proteins. That’s when the immense power of the ubiquitin proteasome machinery became evident. According to Mohl, “At the point of regulation, a protein could go from a half-life of hours to minutes.” Early efforts to tap into the ubiquitination system showed this power could be harnessed. And that, says Mohl, is really the goal of targeted protein degradation.
Mohl says the idea of targeted proteolysis really began in the early 2000s with the approval of the anti-cancer drugs Faslodex and Revlimid, which both utilize the ubiquitin proteasome system. Faslodex, or fulvestrant, was approved in 2002 for the treatment of hormone receptor-positive breast cancer. It works by binding the estrogen receptor of cancer cells and preventing estrogen, which is required for the spread of the cancer, from binding to the receptor. But as Mohl explains, “It not only blocks estrogen from binding to the receptor, but it also changes the configuration of the protein in a way that may reveal hydrophobic surfaces that then signal the cell to degrade that protein, causing estrogen receptor levels to go down.”
Revlimid or lenolidamide, is the drug that Mohl finds especially interesting. First approved for the treatment of multiple myeloma in 2005, lenalidomide is a thalidomide analogue that inhibits proliferation and induces apoptosis of multiple myeloma cells by binding to cereblon, part of a ubiquitin ligase. Once bound, it recognizes certain proteins including Ikaros, a protein vital to the survival of multiple myeloma cells, as substrates that are targeted for ubiquitination and, ultimately, degradation. Mohl says once the drug’s mechanism of action was understood, researchers realized they could design molecules that would do the same thing and tap into the ubiquitin proteasome machinery using hybrid molecules to cause the degradation of proteins that we know are required for the growth and proliferation of cancer cells. This was the initial idea that Mohl had seen years ago in Deshaies lab played out and it gave rise to the PROTAC strategy.
Proteolysis-targeting chimeras, or PROTACs, are molecules that have a ligand that binds to the target protein on one end and a ligand that binds to the ubiquitin ligase on the other end with a linker connecting the two ligands. When the target protein gets near the ubiquitin ligase, ubiquitin units are transferred to lysines of the target protein. That protein is then recognized by the proteasome degradation machinery. Mohl says at the time the first PROTACs were created, the tools available were a bit crude in comparison to today’s technology and the molecules produced were based partly on peptides, so they weren’t as potent as the molecules available today. “In fact,” he says, “if you had asked anybody 10 years ago how feasible this was, the response would have been very negative. But now, there’s been a huge revolution in building PROTACs, with some that can catalyze the degradation of proteins in minutes or hours, which really shows how effective they are at hijacking the ubiquitin proteasome system.”
Although scientists can now generate molecules with amazing activity, Mohl points out they are untested at this point. “Until we actually see PROTACs make it to the clinic and used to successfully treat patients,” he says, “we won't really know whether this is something that can be applied broadly or something that will have a narrower use.” One company, Arvinas, is getting close, says Mohl. They plan to begin phase 1 studies in early 2019 with ARV-110, their androgen receptor PROTAC for the treatment of metastatic castration-resistant prostate cancer.
Now that scientists can generate these active molecules, the next step is finding the molecule that binds the protein responsible for causing disease. Mohl says, “One advantage we have with the PROTAC strategy is that we’re really agnostic as to where that molecule binds the protein. It doesn't have to be to a specific active site or block any particular function of that protein. We just have to find a molecule that sticks to that protein in a way that we can present it to a ubiquitin ligase.”
Finding the right ubiquitin ligase is a challenge in itself. According to Mohl, there may be as many as 600 ubiquitin ligases in the human genome. With that many to choose from, it’s important to know disease types in which those ubiquitin ligases could be used. That question is what inspired the work that Mohl is presenting at the upcoming 2019 SLAS International Conference and Exhibition. As he explains, “We know that these new PROTAC molecules are really, really powerful but we don't know how we could apply them to different cancer types. Would there be cancers where we just couldn’t use them?”
The next hurdle Mohl sees is designing molecules that not only mimic the activity of Revlimid, but are also drug-like enough to be good therapeutics for patients. Generally speaking, small molecule therapeutics fall within Lapinski's rule of five, one of those rules being that the molecule has a molecular weight of less than 500. But PROTACs, by their very nature, have molecular weights closer to 1,000. As Mohl points out, it requires a great deal of engineering to get these molecules into a form that’s orally available. “That is a tremendous hurdle with any molecule that is very large,” he says.
Still, he thinks that PROTACs have so much potential there’s no doubt in his mind that medicinal chemists and academia collaborating with pharmaceutical and biotechnology companies can solve some of those problems through creative strategies. “In fact,” he says, “in the last several years we’ve seen an increase in the number large molecule therapeutics, including bispecific antibodies and antibody therapeutics, that are revolutionizing the treatment of serious diseases.” For Mohl, it’s very gratifying that this area is giving medicinal chemists new territory to expand their work and make use of their expertise. That’s something he has seen firsthand at Amgen. “The medicinal chemists here are excited about this technology,” he says. “And we're definitely tapping into their efforts and their hard work to move into this field as rapidly as possible.”
Mohl suggests that the potential of PROTACs could be limitless. He thinks cancer will be the first field to see PROTACs as therapeutic agents because there are clear opportunities to eliminate proteins rather than just blocking them. But he says it’s too early to put limits on where they will be useful. He can see the PROTAC strategy being applicable to many different disease types, including neurological disorders like Alzheimer’s disease, and cardio-metabolic disorders. Eventually he expects to see PROTAC therapeutics span the entire spectrum.
That’s one of the things that Mohl finds so exciting about his work. “I don't think anybody here at Amgen sees boundaries at this point,” he says. “We just see the problem and the potential of the system to tackle that problem. It's an incredible opportunity to do research for a living, where every day you are allowed to chase something that you find to be intellectually stimulating that will hopefully help patients.”