 Good morning. I am Jeff Green. I am the chair of the Venme Department for Cancer Research at the University of Chicago and I have worked in the steroid receptor field for more than 40 years and in particular on developing and characterizing receptor antagonists and Sean Fanning will come next and he is a senior fellow in my lab. Good afternoon. It's really my pleasure today to tell you about our work with Bayes Doxafine, which is estrogen receptor alpha antagonist and we're interested in it for use in overcoming hormone-resistant breast cancers, really for treating these cancers. Even still today, breast cancer remains a significant health challenge for women. One in eight women will be diagnosed with breast cancer in their lifetime. In the US alone over 40,000 women will die every year as a result of their disease. We care about estrogen receptor, in particular estrogen receptor alpha and breast cancer because over 70% of these diseases are classified by their expression of the protein. When we think about how small molecules modulate estrogen receptor in breast cancer cells, we typically classify them in three different ways. First is the classic hormone binding, which leads to increased pathology by the breast cancer. Here, estradiol, shown as a green square, binds to estrogen receptor, which makes it shed heat shock proteins in the cytosol, form head-to-head homodimers, and it elicits a structural rearrangement that leads to the recruitment of co-activator proteins that go to thousands of different target genes and essentially reprogram the cells transcriptome leading to the pathogenic phenotype. Now, drugs like selective estrogen receptor modulators or CERMS, the most probably well known is for hydroxy-tomoxifen, or tomoxifen, and for hydroxy-tomoxifen is active a tabillite of it, it competitively binds to estrogen receptor alpha and also lets us a structural change, but it's a different structural change than when hormone binds, leading to recruitment of co-repressor proteins instead of co-activators, where it again reprograms the transcriptional profile of the cell, but in this way it leads to decreased pathology instead of increased pathology. So it prevents the metastasis in the adjuvant setting. Now, selective estrogen receptor degraders like full vestrette, or also it's also called ICI, they can also antagonize receptor like for hydroxy-tomoxifen, but they disorder its structure as well, and this leads to recruitment of E3 ligases, both ubiquitin and sumo, which then leads to its proteasional degradation. So these cerds are more thought of as being pure antagonists in some ways because they antagonize receptor in every tissue, whereas selective estrogen receptor modulators have tissue specific antagonism. We use extra crystal structures to understand how these molecules elicit these structural changes and lead to different receptor activities inside the breast cancer cell. On the left, we see estrogen shown as green sticks on the interior of the protein. This is the ligand binding domain of the estrogen receptor in its homodimer state. I've highlighted in red what we call helix 12. This is the C-terminal helix of the receptor, and it's really the main molecular switch that governs this co-activator mediated activity in the breast cancer cell. On the left in the active state, helix 12 is capped over the hormone binding pocket. This opens up what's called the AF2 cleft or activating function 2 cleft, where co-regulator bind by LXX, LL motifs. On the right, we see four hydroxy-tomoxifen bound, which is again one of the most widely used selective estrogen receptor modulators in disease. And you can see here that helix 12 instead of binding over the ligand binding pocket is essentially prohibited from doing so by the arm of tomoxifen and now finds a new home AF2 cleft where it blocks co-regulators for binding. Now, tomoxifen is again frequently used in the adjuvant setting following chemotherapy and radiation. It's also frequently given as a course of anti-estrogens for several years. Unfortunately, many patients will present new metastatic hormone-resistant lesions or cancers, metastases, following these prolonged treatment regimens. Kind of paradoxically, many of these patients retain the expression of estrogen receptor even though they're insensitive to treatment by these anti-estrogens. So around 2013 or 2014 several studies in particular by our collaborators shot Storat-Shonorlopidae at Memorial Cell Catering in Miles Brown at Dana-Farber did deep genomic sequencing on these hormone-resistant metastatic lesions and found that there was a prevalence of somatic mutations to ESR1, which is the gene for estrogen receptor alpha in many of these patients. In particular, these mutations seem to lie at the ligand binding domain in particular at positions 537, where it's typically tyrosine to serine mutation, and position 538, which is an aspartate to glycine. And interestingly, both these mutations lie right at the end terminus of Helix-12. So you can see on the right, I kind of highlighted in yellow the positions of where these mutations are. These mutations, but in particular Y537S, create a receptor that's active in the absence of hormone and resistant to 4-hydroxy-tomoxifen. Well, the only molecule that seems targeted estrogen receptor alpha antagonist that seems to be able to fully ablate the transcriptional activity is the selective estrogen receptor degradable vestrant. In 2016, we published a paper in E-Life that looked at the molecular basis for this dysfunctional receptor activity that's brought on by these activating somatic mutations. And what we found is that using X-ray crystal structures and biophysics and computational methods was that the 537S and 538G somatic mutations produce a receptor that can adopt the agonistic confirmation in the absence of hormone. But it still can be activated by the binding of estrogen. So essentially, these mutations introduce this new equilibria seen at the bottom, whereas you can see at the top with the wild type receptor that this equilibria is not present. We also found that by preforming the agonist confirmation in the receptor, it reduces the ligand binding affinity of both hormone and tomoxifen alike. In this, we saw, we kind of postulated that selective estrogen receptor degraders could be more potent because they act on Helix-12. They're well known to act on Helix-12 by disordering the structure, which is what leads to proteasal degradation. While the vestrant looked certainly promising in our early studies, its clinical utility is somewhat limited by its poor solubility and lack of oral availability. With that, we wanted to look at maybe some other potential clinical candidates and see how they would act in this ESR1 mutation setting. We chose to work with based-oxypherin. It seemed like the logical choice to us because it's a molecule with certain properties. It's orally available and it's already been examined for another use in the clinic and it's approved in combination with the premarin for hormone replacement therapies and I believe it's also approved in Europe to help with osteoporosis. I guess most importantly of all, worked by Donald MacDonald at Duke, showed that based-oxypherin is a potent anti-tumor properties in vivo in preclinical models that had the Y537 as mutation. First, we wanted to look at its properties and the wild-type setting and see how that eventually translated into the Y537 and its D538G. This was done by Renath Jusselsen at Dana-Farber, who's a co-author on this paper and she showed that basically based-oxypherin is a highly potent inhibitor of wild-type estrogen receptor alpha transcriptional activity and it also induces receptor degradation similar to fulvescent or ICI and compared to ICI and for hydroxy tamoxifen, you can see in panel G, based-oxypherin is just as potent, if not a little bit more potent at inhibiting the breast cancer cell proliferation. Being a structural biologist, I was interested in uncovering how does based-oxypherin interact with estrogen receptor alpha ligand binding domain to elicit this SIRD profile and as I kind of alluded to earlier, one of the main ways we know that SIRD's induced proteasalum degradation is by disordering helix-12. This exposes hydrophobic residues to the surface, which then leads to proteasalum degradation. So we were able to solve an extra crystal structure to 2.5 angstroms of based-oxypherin and complex with the wild-type ear alpha ligand binding domain and here I've overlaid it with reloxifen, which is a selective estrogen receptor modulator. It doesn't degrade protein, it stabilizes it and it's the most chemically similar kind of clinically relevant molecule. If we look at panel B, you can see that based-oxypherin in cyan pushes up against the loop preceding helix-12 and this in turn appears to disorder the helix. It looks less helical early on by positions 537. Now going to how it acts in the context of the activating somatic mutations, we see that based-oxypherin is highly potent in cells that actopically express the Y537-S mutant and it also potently inhibits its transcriptional activity. It does induce degradation or it's more disordered compared to reloxifen and faridroxy-tomoxifen, which are both the cerns. And interestingly, for the 538G mutation, we saw that it appears to disorder the receptor a little bit more than fulvestrant or at least as good as it. What I don't show is that we did comprehensive computational modeling as well as in vitro dynamics experiments using HD exchange mass spectrometry as well as ligand binding affinity. And through all these experiments, what we saw is that based-oxypherin's CERD activity, its ability to disrupt helix-12, is really what makes it a good potent inhibitor in this setting, whereas tomoxifen in particular seems like its activity is substantially reduced. So overall, again, it's the CERD activity of based-oxypherin that makes it a potent antagonist of these Y537-S and D538G breast cancer cells. So there's a lot of people to acknowledge in this, in particular Surat Chanderlopides group at Memorial Sloan Kettering, Miles Rounds group at Dana-Farber and Harvard Medical School, and the second author on this paper, Radnaath, did a ton of work and was really helpful on this. There's also significant efforts from John Katzenlobogen and Imata Chorkid's group at University of Illinois as well as Pat Griffin and Kendall Nettles at Scripps Research Institute. Thank you very much.