Proteolysis targeting chimeras (PROTACs), a new class of drugs that can be orally administered, effectively targeted and degraded estrogen-receptor (ER) alpha as shown in preclinical breast cancer models. The study results were presented at the 2016 San Antonio Breast Cancer Symposium (Abstract S4-03).
Most breast cancer patients have ER-positive disease and, therefore, receive endocrine therapy. Endocrine therapies aim to mitigate the effects of estrogen, which is known to drive ER-positive tumor growth, by blocking the ER, degrading the ER, or blocking the production of estrogen. Eventually though, patients tend to stop because their disease becomes resistant to endocrine therapy.
Although still in preclinical studies, PROTACs that target ER alpha, also called ER PROTACs, have shown potential as a therapeutic option for ER-positive breast cancer.
“The oral ER PROTAC we developed really is the first oral degrader that affects the estrogen receptor,” Craig Crews, PhD, L.B. Cullman Professor of Molecular, Cellular and Developmental Biology, Chemistry and Pharmacology at Yale University, told Oncology Times.
PROTACs degrade a target protein, which in this study was ER alpha, but how degradation occurs is unique and inherent in their design. PROTACs are heterobifunctional molecules made up of a ligand for the target protein and an E3 ligase recognition domain. The ER PROTACs tag the receptor for destruction by binding to it and then recruiting E3 ligases, which ubiquitinate the receptor. The ubiquitinated ER alpha is then recognized and degraded by the proteasome.
This results in “reduced receptor levels and the subsequent recycling of the PROTAC to engage other estrogen receptors,” said John Flanagan, PhD, lead study researcher.
Although ER PROTACs have yet to be evaluated in endocrine therapy-resistance models, Crews and Flanagan are optimistic of their potential efficacy and told Oncology Times they believe the unique mechanism of action for PROTACs has the potential to overcome resistance mechanisms in ER-positive breast cancers.
Crews explained that 85 percent of resistance mechanisms still rely on ER expression but are insensitive to anti-estrogens, such as selective estrogen receptor modulators, or SERMs, that bind competitively to estrogen-binding domains.
“By simply making the protein go away—by making the problems go away,” Crews noted in regard to how PROTACs work, “we can address not only the initial ER-driven proliferation in these tumors but also address the majority of resistance mechanisms that occur with anti-estrogens.”
PROTACs were first developed by Crews in his lab at Yale, where he and his team also first developed the next-generation proteasome inhibitor, carfilzomib. Crews began exploring PROTACs in 2001, and he explained that his lab stopped working on inhibiting degradation, which is what carfilzomib does by inhibiting the proteasome, and “flipped to the opposite side” of inducing protein degradation, by activating the proteasome.
“What allowed us to really move this into the drug development arena was in 2009,” he said. “My lab came up with a small molecule ligand for one end of the first PROTAC.” This compound was “much more drug-like” and “had better pharmaceutical properties.”
The aim of the current study was to compare the ability of ER PROTACs to target and degrade ER alpha with that of fulvestrant, a selective estrogen receptor degrader (SERD) and commonly used therapy for ER-positive breast cancer, and other investigational SERDs currently in the clinic. The researchers used a variety of in vitro and in vivo experiments to characterize the activity of PROTACs in relation to these other compounds.
They found that the ER-positive, T47D cell line treated with either PROTACs or fulvestrant had “robust degradation” of ER alpha, whereas only slight degradation occurred for the current clinical SERDs, RAD1901, AZD9496, and GDC-0810.
Regarding activity in the nucleus, which is where ER alpha is most abundant, only PROTACs and fulvestrant reduced ER alpha levels in the nucleus, and PROTACs reduced levels slightly better than fulvestrant. AZD9496 and another SERD of a similar chemical class, both failed to reduce ER alpha levels in the nucleus, a surprising finding given these compounds have been labeled as downregulators or degraders of ER alpha.
“What we're finding is that so-called SERDs don't necessarily degrade, and so I think that people are beginning to re-define SERDs as they realize the limitations of their compound,” Crews said. He also noted that if these SERDs that failed to reduce ER alpha levels in the nucleus have clinical activity, “it must be through some other mechanism.”
The ability of PROTACs to inhibit cellular proliferation was tested by incubating T47D cells with PROTACs, fulvestrant, or different SERDs in an in vitro clonogenic assay.
“PROTACs and fulvestrant provided greater anti-proliferation effects when compared with the clinical SERDs,” Flanagan said. “We conclude that the additional degradation activity of the PROTAC and fulvestrant provided better growth inhibition than just the antagonist activity of the SERDs.”
One question Flanagan said is often asked is, how selective are ER PROTACs at degrading only ER alpha? The researchers answered this question by performing cellular expression proteomic studies to quantify reductions in proteins in the MCF7 proteome after overnight incubation with either PROTAC or inactive PROTAC. They quantified levels for approximately 7,600 proteins and demonstrated that ER alpha was reduced only with the active PROTAC. Moreover, proteins that are known to be regulated by ER alpha at the genetic level were similarly affected by the PROTAC and inactive PROTAC. This finding suggests that ER PROTACs do selectively degrade ER alpha.
“To demonstrate that the mechanism of PROTACs is through active recruitment of an E3 ligase, we used an inactive PROTAC, which still retains binding to ER alpha but cannot bind to the E3 ligase due to a compromised ligase recognition domain,” Flanagan said. “The inactive PROTAC did not reduce ER alpha levels.”
The researchers evaluated the effect of subcutaneous administration of ER PROTACs on tumor growth in MCF7 xenograft models and found that the ER PROTACs dosed at 30 mpk was well tolerated and provided 68 percent tumor growth inhibition even in the presence of exogenous estradiol required for growth of this xenograft model.
“Now, we recognize that a PROTAC that could be orally administered would be preferred over one that was subcutaneously delivered,” Flanagan said, “so we put a large medicinal chemistry effort into trying to understand and design PROTACs that would provide better oral bioavailability.”
They dosed MCF7 xenografts for 3 days with 30 mpk of one of the new oral ER PROTACs and saw a 61 percent reduction of ER alpha levels while there was a 50 percent reduction of ER with a single subcutaneous dose of fulvestrant, dosed at 200 mpk. A similar reduction of ER alpha levels was also seen in immature rat uteri with these molecules.
Advantages of PROTACs
“For almost 15 years, fulvestrant has been the only FDA-approved selective estrogen receptor downregulator for advanced breast cancers,” Flanagan said. “The last few years, a renaissance of discovering and developing novel oral SERDs has occurred due to the continued importance of ER alpha signaling in advanced disease and the proven clinical activity of fulvestrant. However, given the limited pharmaceutical properties of fulvestrant, an orally administered SERD is desirable and would be expected to benefit the patient community either as a single agent or in combination with other pathway inhibitors.”
Another advantage of PROTACs is they overcome two important limitations of traditional clinical inhibitors. First, unlike inhibitors, PROTACs do not require an active site. By not needing an active site to which to bind, PROTACs can potentially target “undruggable” diseases.
“We've demonstrated with other projects that we can find nooks and crannies—crevices on the surface of proteins—by using ligands that bind to those nonactive site grooves and crevices,” Crews noted. “We can use those as points for attachment, whereby a PROTAC can now bind to the nonactive site sites and recruit proteins for degradation.”
The second limitation of inhibitors that PROTACs overcome is not requiring a high dose and are able to do so because they work by a different paradigm.
“The paradigm that an inhibitor works under is occupancy-based, meaning that you have an active site or a ligand binding domain and that site has to be filled,” Crews explained. “Because the vast majority of these inhibitors are reversible, they can fall off, and when they fall off, of course, you don't get the clinical benefit, so you need to have excess of your inhibitor in case the initial inhibitor falls off.”
“This is one of the major challenges of drug discovery today,” Crews continued. “How do you achieve and maintain high excess levels of drugs chemically to make sure that at any given time you have 97 or 98 percent of all of the those sites filled?”
PROTACs instead follow an event-driven paradigm, he said, meaning you just need transient interaction between the PROTAC and the target protein to lead to permanent loss of that protein.
“It goes without saying that we are extremely excited having overcome this oral bioavailability hurdle with our ER PROTAC program and will be evaluating these ER PROTACs in a variety of clinically relevant endocrine resistant models,” Flanagan said. He explained that this approach using PROTACs to actively degrade target proteins is currently being employed for other programs. In the oncogenic space, they are developing PROTACs against the androgen receptor for the treatment of prostate cancer and also the bromodomain protein BRD4 for multiple myeloma and solid tumors.
As for what comes next for ER PROTACs, Crews noted, “We'd like to get into the clinic soon. We are working hard to get an IND filing done this year, and we hope to be in the clinic next year.”
Christina Bennett is a contributing writer.