Dr. Marianne D. Sadar Video (Text Version)
Title of Talk: Developing Inhibitors Directed Against the "Achilles' Heel" of Androgen Receptor Action
Speaker
Our next speaker is Dr. Marianne Sadar; she is a Senior Scientist at Canada’s Michael Smith Genome Sciences Centre in Vancouver. And the Provincial Program Leader for Prostate Cancer Research at the British Columbia Cancer Agency. She received her Ph.D. in a joint program at the Universities of Bradford and Göteborg in the UK and Sweden, respectively. She has been a continuous grantee of the DoD Program since 1999 when she was first granted a New Investigator Award. Dr. Sadar’s research has focused on discovering therapeutics for advanced prostate cancer that target the N-terminus domain of the androgen receptor and developing decoys, peptides, and small molecules from marine sponge extracts to yield new drugs with strong potential for clinical development.
Dr. Sadar’s presentation today is on developing inhibitors against the Achilles heel of androgen receptor action; Dr. Sadar.
Marianne D. Sadar, Ph.D., Senior Scientist, Michael Smith Genome Sciences Centre, BC Cancer Agency
Thanks very much to the organizers for inviting me to this meeting that has just been wonderfully organized; it’s been a real delight to be here and kudos to the organizers and Natasha Kyprianou for such a fantastic job.
So looking at inhibitors to androgen receptor, one needs to know that the activity of the androgen receptors and the N-terminus domain. So I first need to make a disclosure; all of the compounds that I’m going to talk about today have been licensed into a company that I’m the Chief Scientific Officer of, and I do have stock equity in that company. Also as mentioned, the U.S. Department of Defense New Investigator Award was the first grant I ever received in my career and I really do give credit to this award mechanism as launching my career, and so thank you. And thank you for the continuous funding as well. So much of the work that you see today was considered extreme high-risk in 1999 and it’s been funded all along the way not in renewal grants but in individual grants and so hopefully the high-risk has been turning into high-gain.
So looking at prostate cancer and the therapies, if we look at how the stages of the disease progress from invasive disease to castration-resistant disease, there are wonderful therapies for local disease. These include brachytherapy, external beam radiation, and radical prostatectomy. For many patients this will be curative therapy but 20 to 40% of patients will recur and for these patients there’s hormonal therapy and these include compounds or methods to reduce ligand, reduce androgen or testosterone, or by using anti-androgens. These initially work but eventually unfortunately they will all fail. And so when we’re developing therapies as I’m going to be speaking about, we’re talking about a hormonal therapy that would be included in here. For those patients that fail, we then go onto chemotherapy.
So castration-recurrent prostate cancer it is still considered to be an androgen receptor-dependent disease. Some of the mechanisms thought to be involved in this are gamma function mutations and this is where the androgen receptor can have mutations that make them activators in response to anti-androgens. There are amplification overexpression of androgen receptor, residual androgens—either the tumor can make them themselves or adrenal over-expression of the co-activator such as CBP or steroid receptor co-activators. Like an independent activation of the androgen receptor, so this is in the absence of testosterone. There’s these alternative pathways that can activate the receptor. And then perhaps one of the most exciting developments has been the discovery of these constitutively active splice variants of androgen receptor that don’t have a ligand binding domain.
And so this is one of the biggest caveats, the downfall of the current therapies because they can't actually inhibit these types of variants and so an inhibitor to the immuno-N-terminus domain such as I’m going to describe can actually address all of these mechanisms.
So let’s look at the androgen receptor; it is a transcription factor. There are domains of it; these particular domains have a structure. They’ve been crystallized. The N-terminal domain where we’re developing inhibitors is intrinsically disordered. How this works on the N-terminus domain is it requires interactions with other proteins for an induced fold. So I’ve drawn CBP in here because it’s essential for the activity of this transcription factor and many transcription factors.
The N-terminus domain is the business end of the androgen receptor. You absolutely have to have this part of the molecule for a transcriptional activity. It contains activating function. One so it really is the important part of the molecule. If you take away the ligand binding domain, this receptor is still active, and this of course is the theory behind the constitutively active splice variants that lack ligand binding domain. If you take away the N-terminus domain, the receptor is dead; it still binds ligand but it isn't transcriptionally active. So as mentioned, all the current therapies are targeting the ligand binding domain either by reducing ligand or by creating anti-androgens that work through this region.
So back in 1999, we proposed that you could make inhibitors either by creating decoys and a decoy would be an identical copy of this region that would mop up these essential co-activator proteins or you could create small molecule inhibitors that would also work to block those interactions. So we did develop a decoy; it was regions 1 to 558 of the N-terminus domain. We also have identified a number of small molecules, the sintokamides that were isolated from a marine sponge originally collected in Indonesia. This is a chlorinated peptide that’s been very difficult to synthesize. It’s—we got to name the compounds ourselves because they had never been discovered before.
And the compound I’m going to speak mostly about is EPI-001 that was also isolated from a marine sponge from Papa New Guinea, and it too is a chlorinated compound; it has two [kyro-centers], so in the middle of my talk I’m going to be talking about stereoisomers and that’s because this compound—two [kyro-centers] so it has four stereoisomers.
So just showing you a bit of data on each of the approaches to inhibit the N-terminus, we published this back in 2007 and this was using the decoys. And this—this was an important piece of work because it was the first time anyone had shown that if you target the N-terminus you can get a therapeutic response. And so what we did is we created—we did this two ways and I’m just showing you one way—we used a viral delivery of the decoy to establish tumors or we created LNCap cells that are human prostate cancer cells. We created LNCap cells to express the decoy. And that’s what I’m showing you here. So these cells were implanted subcutaneously, and we castrated the animals when the tumors were a particular size, and you can see that in each of these cases, the tumors with the vector you get a PSA response and eventually it comes back. I’ve lost my battery—eventually it comes back and just like we see in the clinic, when you have tumors that have the decoy you can see down here that these tumors don’t really come back; they just sort of hover.
If we look at the actual tumor size in these matching samples, we can see that with the—just the vector, the tumors are growing, and with the decoy those tumors stop growing. We actually get a little bit of regression but then they just sort of hover.
The sintokamides—this hasn’t been published; the paper just describing the sintokamides was published in 2008, but if we use the sintokamides and also test this on LNCap tumors this was done by inter-tumoral injection, when the tumors were 100 millimeters cubed, we castrated the animals. We did an inter-tumoral injection at time-zero and with our control tumors, shown in the blue line, you can see that they’re growing still in the absence of testosterone. When we do inter-tumoral injection of the sintokamides, so these lines represent the individual animals with individual growth curves, we get good regression in some animals, and in the other animals, it just holds the tumors in check. And when we harvest the tumors you can see how they look. The DMSO tumor, the control tumor is on the top, very bloody; the bar represents 10 millimeters and the sintokamides—these tumors are actually not growing and—and you can actually see a physical difference of the tumor as well. They tend to go to this whitish color.
So now looking at EPI-001 how does this work? This is the compound I’m going to be talking about. What happens is it interacts with the N-terminus, and it causes a change in confirmation such that it inhibits essential proteins interacting. And so you have an inactive receptor. This work was done at the University of Aberdeen and what this—this slide is going to I hope convince you is that although the N-terminus is intrinsically disordered, it has some structure. So there are some regions, of alpha-helical region and you have to have some of this very minimal structure in order for EPI-001 to interact with the N-terminus.
So, in this particular case, we’re using just the AF1 region which is amino acids 142 to 485 its recombinant protein; when you incubate this protein with EPI-001, you get a shift in the confirmation which can be measured by a change in the fluorescent emission. So that’s what’s being measured here; we’re looking at the fluorescents between 300 to 400 of—of—thank you very much—of the tryptophans and tyrosines present in this molecule. So if we just look at—if we just look at the recombinant protein curve for itself, it’s up here. If we add in the EPI-001, we get this big shift meaning it’s interacting. It’s causing a change in the fold.
If we denature the protein with urea, we lose that, so meaning you have to have some structure. This was done in another way; now what we’ve done is we’ve mutated a region in an alpha-helical region. This is particularly the mutation is between the 236 and up in the 255 and so this destroys the structure. And so the same thing; if—if we have just the AF1 protein with this mutation, EPI-001 no longer causes that shift. So there has to be a little bit of structure.
How else can we prove that EPI-001 truly interacts with the androgen receptor? Well, the next question was let’s show it—try to show it at least in living cells. So we created probes and this is a probe that has a little alkene tag on it. We incubated LNCap cells with these probes; these probes are active. They inhibit androgen receptor if you use a reporter assay. So you incubate the cells with it. It presumably binds to the androgen receptor; then what you do is you—you lyse the cells, you do click chemistry to put on a biotin tag to this little region in here and you have a spacer in the biotin tag. And then what you do is you run a strip [inaudible] column to pull out the biotin-labeled probe and any protein that it’s interacting with. And we were hoping it’s androgen receptor of course, but it will pull out any proteins that this molecule is interacting with.
So it tells you as well how clean this drug is. Is it hitting a lot of other proteins? And so the first thing you notice when you run this gel and we are probing for biotin so we’re probing for the actual EPI-tag, you’ll see that it’s very clean. There’s—there were some non-specific fans that just came up with the biotin that you can see across here as well as down here, so in the lanes we have before the—the IP and then after the IP, A1 and A2 refer to these different stereoisomers of the molecule. And so what we have right here is androgen receptor—or at least we think it is; it’s at 110 kilodaltons. It runs exactly where androgen receptor runs, and it’s the predominant band. There’s these two other bands here; we don’t know what those are. We’re trying to identify them now. But otherwise it’s extremely clean, meaning that there would be less toxicity in patients, very specific.
We then took this gel—or took this western blot and we stripped and we re-probed for androgen receptor. Androgen receptor was right underneath those bands. So we’re pretty confident it is androgen receptor. We’ve done this experiment about 20 times, poor post-doc, and in this particular case, we’ve used all different analogs, all different probes, and I just want to show you this one here again, identical experiment but we only have—we have a few more extra controls in here. We have the inactive analog and the inactive analog is identical except that chlorine atom has been changed and so it doesn’t bind at all so it was a very nice control to see.
So indeed we think that we are getting real interaction in living cells with the androgen receptor.
So just to show you a little bit of data that it is active on the full-length receptor so this is LNCap cells using the endogenous receptor and the PSA promoter. When you add in androgens, you get androgen receptor binding to response elements so you get this big increase in activity. Bicalutamide inhibits and EPI-001 also inhibits.
It’s specific for androgen receptor; we don’t see this inhibition if we look at other steroid receptors. This is glucocorticoid receptor. When we add in dexamethasone we get an increase in GRE luciferase activity and EPI-001 doesn’t inhibit that, so it seems very specific and that’s important because these receptors interact with many of the same proteins such as CBP.
So now going into splice variants. As I mentioned this—this is perhaps one of the best niches for these types of inhibitors because they don’t have a ligand binding domain so the current therapies shouldn’t be effective on them. So this is Charles Sawyers’ work; it’s just showing the effects of Medivation drug, the MDV-3100, on splice variants, so he’s looking at D145 cells that have been stably expressed to either have the full-length human androgen receptor, full-length murine androgen receptor, and in the presence of androgens the MDV-3100 drug works extremely well. When we have the variants that lack the ligand binding domain, this is the variant seven or the murine variant four, no ligand binding domain, and Medivation drug is not inhibiting. You can see this is still at 100% activity with androgens.
If we take Stephen Plymate’s variant which is also lacking the ligand binding domain, so this is a naturally occurring variant, and we transfect it into COS-1 cells using a probasin reporter also an androgen receptor driven reporter, the black bars represent with androgens—white without. The first thing you notice is that androgens don’t have any effect on the variant. Why should it; there’s no ligand binding domain. And here we’re showing EPI-001 works very well at inhibiting these variants or this particular variant.
This is work courtesy of Stephen Plymate; this is just looking at the LNCap cells first, looking at the endogenous full-length receptor using Medivation drug at 1 micromolar and 10 micromolar and so in the absence of androgens we have on the left; in the presence of androgens, we see that the Medivation drug works extremely well on the full-length receptor that has ligand binding domain, so 10 micromolar just knocks out the activity completely.
What happens when you have a mixture of the full-length receptor with the splice variant? Here this is LNCap cells that’s just been transfected to express this variant. The first thing you notice is that androgens are having no effect. These are transiently transfected. I should mention that and Medivation drug as well has no effect on these mixed populations of full-length receptor with the variant. So we repeated the experiment; we didn't have the Medivation drug so we used bicalutamide. This lefthand side is just the full-length receptor on its own LNCap cells, androgens induced, bicalutamide inhibits, EPI-001 also inhibits. When we have the variant expressed at a one-to-one ratio, we did a western blot to show this. First you see this huge increase in activity. Androgen has no effect just like what Dr. Plymate shows and bicalutamide also has no effect. And look at this, EPI-001 was very effective at blocking this, so we were very excited about this data. It seems like it’s EPI-001 really may be the first inhibitor, good inhibitor of the N-terminus and splice variants.
Oops; so now looking at castration-recurrent prostate cancer—that was the whole point. So these are LNCap xenografts that have been planted subcutaneously when the tumors are 100 millimeters cubed. We castrate the animals; 7 days later we normalize to 100% and start our first injection of EPI-001. There are two experiments here; the first experiment was done more than I think 2 years from the second experiment. So the first experiment was inter-tumoral injection. We injected the tumors directly, and the control arm is the black one, so DMSO inter-tumoral and the matching EPI-001 treatment is shown in green. We did this every 5 days, and 5 days after the last injection we harvested, and some of these tumors were completely going away. So that’s why we stopped the experiment there because we wanted to analyze the tumors.
With the intravenous treatment you can see that these growth curves are superimposed. With the intravenous we treated every other day by tail vein injection; we had to stop after 14 days because the tail vein became quite chewed up. When we harvest the tumors from the intravenous treatment now you can see what they look like, so this is after 14 days. The black bar represents 10 millimeters. We stained these tumors for TUNEL which is a marker for apoptosis and any brown cell is undergoing apoptosis and you can see that these cells are really undergoing program cell death. They’re regressing so you would expect this; while it’s in the control it’s very hard to find any staining. The measurement for proliferation using ki67 very few cells proliferating; very difficult to find any in the EPI-001 treated and in our control these tumors are growing—lots of cells are lighting up.
If we look at potential toxicity looking at behavior of the animal, whether the animals are losing weight we didn't see any of that. So we looked at body weight before the treatment and after, and we didn't see any change in body weight with the EPI-001 treatment. So this was a very good sign and also supportive that it truly is specific and not hitting other targets.
If our molecule is truly specific for androgen receptor, it shouldn’t have any effect on tumors that are not dependent on androgen receptor for growth and survival and so in order to prove this, we used the PC3 xenografts. They have some androgen receptor; it’s not functional. We did castrate the animals however and we started treatment. This is intravenous treatment, tail-vein injections, and you can see from the growth curves that we don’t get that regression. Remember the regression in the LNCap were like this; so the growth curves aren't significantly altered. And for those of you that are interested, that’s what PC3 tumors look like.
So ultimately, we would like to have a drug that you could give orally and so we sent our compound; we sent EPI-001 to a CRO in order to look at some of the pharmaco-kinetics and the bioavailability of this compound. So they did single dose of 50 milligrams per kilogram body weight by tail-vein injection or a single oral dose of 100 milligrams per kilogram body weight. They then measured the levels in the blood of these compounds, and this compound and over this time and you can see that with the oral dose, so these are three animals per—per time point—with the oral dose we’re able to achieve blood levels that were at the concentration we needed for our in vitro experiment. So that was a really wonderful sign. All of the in vitro experiments you saw were at 5 micrograms down here or 10 micrograms per milliliter. So we can achieve the effective concentration.
Importantly, with 86% bio—well bio-oral dosing which is really wonderful news and the half life was three and a half hours so that too is a reasonable—you can work with that. If it’s orally available, it should mean if you give this compound orally to the animals we should see efficacy on the tumors and indeed we saw that. This was a very small study looking at the growth from the start of the treatment in our controls. They almost tripled in size; with EPI-001 they were reduced and again you see this—this physical change in what the tumor looks like and bicalutamide just kind of held the tumor in check.
So in conclusion, we have inhibitors to the N-terminus and they are feasible for drug development and potentially for the treatment of castration-recurrent prostate cancer. With EPI-001, it is the first drug shown to interact with the N-terminus domain. It inhibits variants lacking the ligand binding domain. Its mechanism of action binds to the N-terminus and it inhibits essential protein—protein interactions. I didn't show you some of that data because I showed it yesterday. It’s specific for androgen receptor and it causes cyto-reduction of prostate cancer tumors which is the most important point with no apparent toxicity. And currently we have it in clinical development.
So I just want to thank our chemist; he’s made more than 100 analogs of EPI-001 and has worked with me since 2003 when we had a Screening Grant from the U.S. Army to find these drugs. [Inaudible Wong], I haven't shown any of his data; he’s tested our drug in some of his models and [Ian McKeown], Jane Dyson is doing NMR for us and, in particular, I want to thank the University of Washington—very supportive group that have been working with our group at the splice variants as well as some of the clinical trials that we’re developing, and importantly the people doing the work in my lab, so thank you.