Visualization of transcription regulatory complexes in animals and Development of drugs for rare cancers

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Video in TIB AV-Portal: Visualization of transcription regulatory complexes in animals and Development of drugs for rare cancers

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Visualization of transcription regulatory complexes in animals and Development of drugs for rare cancers
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Visualization of transcription regulatory complexes in animals and Development of drugs for rare cancers Investigation of molecular mechanisms under physiological conditions requires new methods for the detection of macromolecular complexes in tissues and organs. Our laboratory has developed methods to visualize protein interactions in cells and tissues thatare based on complementation between fragments of fluorescent proteins (bimolecular fluorescence complementation analysis). We present an adaptation of these methods to study protein complex binding at specific genetic loci on Drosophila polytene chromosomes. These methods facilitate genome-wide visualization of protein complexes on chromatin in single cells. One area where the investigation of molecular mechanisms in animals is important is the study of responses to drugs and synthetic compounds. Animals can recognize and respond to xenobiotic compounds, including drugs, and can protect cells from the effects of such compounds. These mechanisms mediate the resistance to drug treatment that often develops in response to cancer chemotherapy. We investigated the mechanisms of transcription regulation by the xenobiotic response regulators dKeap1 and CncC (homologues of mammalian Keap1 and Nrf2) in Drosophila larvae. Visualization of chromatin binding by dKeap1 and CncC separately and in complexes on polytene chromosomes revealed that they bind and regulate genes that are transcribed in response to endocrine hormones (ecdysteroids). The mechanisms whereby dKeap1 and CncC regulate these genes differ from the mechanisms whereby they regulate transcriptionin response to xenobiotic compounds. The limited understanding of molecular mechanisms that determine drug pharmacodynamics and pharmacokinetics is a major cause of the low success rate of drug development. We present the initial results of our efforts to repurpose compounds for the treatment of rare endocrine cancers. We focus on compounds that have undergone advanced pre-clinical characterization to reduce the likelihood of unfavorable outcomes during late stage development and in clinical trials. The initial results suggest that consideration of bioavailability and bioactivity at an early stage of drug development can improve the likelihood that the prospective drugs benefit patients.
Keywords Molecular machines cellular pathways and mechanism intra- and extra-cellular coordination and communication genomes and cell fate disease, cancer and aging
[Music] my talk will have minimal mathematics in it which may be a relief for a few of you that mention a few things which might be more mathematically applied investigated by not by us but by others one thing I'm delighted to participate in such a broad topic from spanning from molecules to human health and taking that at heart I will present actually two different stories I'll warn you these two stories have nothing to do with each other so if you miss the transition I'll try to make a warning when I make it but but there will be two different talks I want to thank Boone for pointing out the the distinction between these talk that we're gonna be talking about interactions and we're very much interested in regulatory protein interactions and the reason I should say that the reason that these interaction networks end up putting putting order the regulatory molecules in the middle is that they interact with everybody it's not uncommon to find proteins that a hundred have a hundred interaction partners and the reason these interactions are so critical for these regulatory proteins is that the regulatory proteins lack identity they lack specificity individually they acquire their specificity through their interactions and is it it is that very process of acquiring the specificity that we're interested in studying the reason we use direct imaging is also because for us the localization where something happens the responses to stimuli when it happens in the prelate of two other processes in the cell those are the main part of the biology if you will of regulatory proteins it is not sufficient to know who interacts with whom we need to know what controls the interaction and and where it happens in the cell and we have a number of examples in the lab of studying these kinds of problems and finding out through the investigation of the interactions that we were completely ignorant of a whole area of biology in the process so the two talks I'll be or the two stories I'll be telling our one one done by who I'd end postdoctoral fellow not at the University of Minnesota whose expertise in Drosophila provided the opportunity of translating these imaging approaches which we've done in in in living cell systems for many years into live animals and the other one which is going to be our attempt at making some impact on human health is in the area of developing drugs for rare cancer by Veronica burns and Eva Marie Chiang now my introductions is I have two very different topics I apologize it's somewhat presumptuous but bear with me I'm going to start with asking a question which to me has been puzzling I think most of us in this room who have been at our area least investigation for some time are proud of the advances that we've made over the last five or ten years we think of things that happened ten years ago they were really hard and now we can do things that are amazingly much more than we could 10 years ago now if we compare with what we're doing in many basic sciences to what's happening in drug development this is a plot of a number of drugs approved please apologize or other ancient slide but the pit the picture hasn't improved despite enormous effort has measured here by a number which is beyond my sort comprehension in terms of financial impact the success rate of drug development is woefully low and the puzzle here is why is this we are making such enormous advances in science why is this not translating into patient benefit and this is a too big question for me to really answer it let alone to solve today but I want to give some flavor of the reasons for this and perhaps where things might go now this is sort of the state and drug development program if you will and this is not the skill but nevertheless the point here is that we're starting with identified lead compounds through some large-scale screen and then through these individual steps we screen out the ones that do not fit some criterion and the reason this is not the skill is that this is in fact a much less efficient or much lower yield process than we know about the ones that we actually have numbers for the ones that involve u.s. federal registration it's an order of magnitude at every step and it's at least as bad at the lower levels which means that we're simply losing almost every hit along the way and we have no solution for improving this well the solution at the lower levels is in fact already solved we can easily screen we're efficient at going through large numbers of compounds through these steps the problems happen here at the higher levels where our ability to make any sensible prediction any sensible analysis of what kinds of processes may are involved is really limited so one of my first stories is going to focus on the area of pharmacodynamics that is to say how animals respond to foreign compounds and of course the targeted effect is the one that we are striving for in terms of health and benefits there are many non target effects and
what I'm focusing on today is these induced response pathways which is something that is initially rather surprising that all of us all of our cells have the ability to recognize foreign things it's almost like an immune system at a molecular level at the molecular level what's amazing about these systems is that they're able to recognize the chemical characteristics of foreign compounds and respond in a purposeful manner typically by inactivating exporting somehow getting rid of a foreign entity in order to protect a cell we because of course things that come from the outside is generally bad for us it's hazardous however drugs of course we have to get into the cell in order to have an effect so we have to somehow understand and overcome these pathways which are again one of the steps in this drug development enterprise which are really failing it's at least later steps in this this pathway if you will that are the reason why drug development they become really expensive here so we cannot afford being so ignorant about what's causing the failures now from a biological point of view there's a lot and I don't expect you to read this list it's busy again admission here that we're looking at a very small part of this picture there are many biological mechanisms that protect us from the outside being evil our skin our the step separation of self from the environment what's amazing about these things is that many of these things can discriminate newly synthesized compounds compositor comes from chemical factories things that have never been present during evolution how did how did they or how did evolution develop the capacity to distinguish these kinds of compounds that we're very familiar with many kinds of biological stimulus response pathways we can recognize much of normal biological stimuli because they have been present over eons billions of years in some case and have had all that time to find to in the response but how about the next drug which has never been present during evolution how can our cells recognize it and respond in any purposeful manner not to speak of the pesticide or what other compounds that were exposed to and this is the problem that we've been really interested in addressing and because it's such an you evolution development in my mind are two sides of the same coin so we've studied this in a developmental context in Drosophila the model system and again I'll emphasize that this is a tiny little part of this big picture is this system which has been characterized for xenobiotic responses xenobiotics being compounds that are artificial which is based on a protein called keep one which acts as a sensor and it is dedicated to control of this transcription factor nrf2 and under basic conditions to degrade it and I'm going to admit right away that I'm putting up something of a strawman here with the purpose of knocking it down there is much data to support this model and I don't say want to say that this is wrong merely incomplete one thing that's amazing about this sensor keep one protein is that it's able to recognize this extreme variety of structural chemical diversity which allows us to function as a mechanism for xenobiotic responses again the canonical model is that this modification and/or structural change in keep one releases the NRF two transcription factor which then now activates this wide variety of response pathways which act at the level of protecting the cells now there are some flies in the ointment if you will in that the keep one protein is also seen in the nucleus and exactly what it has with what it's doing there at present or formerly was was was thought to be an export factor for nrf2 protein as you'll see it does much more interest and and one of the things that is was intriguing for us and the reason that we got into this game is that the coupling between the stimulus and the response was seemingly mediated by very tenuous link this nrf2 transcription factor which would limit the ability of selective activation response to particular classes of compounds and particular response subsets and this is something that's observed if we treat a cell or an animal with a particular compound we don't get the identical response pathway so therefore there seemed to us to be something missing as I mentioned we're studying this problem in drosophila drosophila has homologous proteins to these mammalian proteins that have been studied extensively in drosophila these proteins do what we might think they do they support they they they they they they protect the fly from pesticides etc these are the pathways that the the pesticide makers concerned themselves with the the reason we use the fly is that it has many nice advantages in studying what to us is rather a mysterious process and it also allows us to ask in hindsight somewhat the question of where did these pathways come from how was this this ability to respond to something that is unknowable or at least initially not evolutionarily present developed again we like using
imaging so the first question we asked is where these major actors are both keep one and CNCC these are salivary gland cells this is a nuclear stain with a DNA binding dye our nuclear which initially was somewhat surprising remember we usually think of the keep one protein as being cytoplasmic sense or at least this was the model just to make sure that our antibodies were specifically we also make transgenic my flies in this case and and and the trench jean genet express proteins are clearly nuclear one of the tools we like to use to look at interactions and this will become relevant toward a second later in the talk is that we like to look at how an interaction between two partners alters the specificity of their biology and in this case we use a tool that we developed some decade ago which allows us to selectively the protein-protein complex whereas the individual subunits are invisible by fusing them to fragments or fluorescent protein now the fragments of the protein just like half an enzyme doesn't have any activity when brought together provide us a reporter a fluorescent signal which we can now visualize and see where the complex is and here perhaps no surprise the complexes are present in the nucleus now again we're using this Drosophila system
because Drosophila imaging is really powerful now those of you in the audience we use imaging know that the instrumentation the power of optical visualization has grown by leaps and bounds and this is really due to these enormous instruments that are very useful for for many purposes in our lab we tend to take a rather more pedestrian approach of just adapting our system of visualization to the particular problem at hand so in this case we take advantage of something that is characteristic of fruit flies particularly the salivary glands of
fruit flies the classical observation over are called polythene chromosomes what these are are basically strands of the genome the entire genome stretched out and copied in thousands or copying and aligned in parallel and what this does for us is that it positions individual genes in thousands of copies in a physical location now this is not to say that we couldn't do a similar experiment in a mammalian cell and seeing a spot correspond to an allele but what this quality in chromosome system does is that due to classical work on the part of many people individual genes have been mapped relative to physical reference maps on the chromis chromis team so that we can by simply looking at one of such spread get a genome-wide map of where the proteins are bound and in this case we were really quite surprised that the pattern binding here by these proteins which have this function known function in protection from foreign compounds corresponds to a very characteristic developmental program which is something called a ectis own response pathway this is the pathway that the fly uses to conduct what's called metamorphosis the remarkable process of the frog fly larvae to completely reorganize its own body structure now to confirm that in fact these patterns are binding meant something biologically we look at knockdowns eventually loss of function mutations in these proteins and we find that this class of classical response genes is reduced in expression relative the wild-type flies suggesting that indeed they require both the keep one and CNCC proteins for their expression this is not because we have compromised development at least at the level of the celery gland because the late genes are expressed and therefore the the fly in general is intact now i had to do a little little education here in terms of fly biology so so fruit flies developed from embryo and in response to these pulses of an endocrine hormone a steroid hormone called egg the steroid egg die sonne undergo specific transitions in development and it was studying these so so i'm gonna have to sort of cut out a big part of my talk here in the middle and say that we found that when we were depleting these regulators in the fly we were finding that development of was arrested so it seemed like something was going wrong beyond the inability of the salivary glands to produce what are rather pedestrian glue proteins things that make that make me make the larva stick to the surface where it's going to pupate so we suspected that perhaps the production with the steroid was being compromised so we looked a depletion experiment of these proteins in a particular tissue where the actor steroid is produced and we found that indeed the depletion of these proteins in this case the CNCC protein results in a delay and reduction of synthesis of this fly endocrine hormone this is the time scale of fly development these are they ultimately for MS pupae as you can see the pupil formation is significantly delayed in this context this is because in this pro rata gland an endocrine gland in the fly a pathway that leads from cholesterol to just like we make steroids for our use in reproductive and other regulatory processes there's a pathway encoded by a number of different proteins which convert cholesterol to this endocrine hormone and we find that the genes that are encoding these these these these these the p450s are again regulated by these proteins watch which we think of primarily as as as protective as as as providing protection from and from the external impacts so it appears that in this context these regulatory proteins are functioning to control the fly development and again a control we are showing that the gland itself is intact so it's not that taking away these genes has caused development the halt but rather in this case it's this bio transformation that is compromised in the absence of this this regulator so to actually look at this in a biological context we can look at the time at which a larva after hatching forms the pupae and look at what happens when we deprive the larvae of one of these regulatory proteins and show that there is a shift a delay in time of hatching we can rescue this fully by feeding the larvae the endocrine hormones so we can add that the steroid into the food and the larvae hatching is restored to a near wild-type timing so this indicates that it is the form production of this actor steroid which is regulated by these regulatory proteins that is essential for a for for for maintenance of the development and rather to confirm that it wasn't that we had somehow compromised somehow made non-functional larvae these are the larvae that have been depleted and as you see they keep growing in fact they become larger than the wild-type larvae because again they keep eating and not pupae ding and it is this absence of the actor's third which is essential for privation so summarizing this part what we find is that these proteins which we know as regulators of a of a of xenobiotic responses function in two different tissues or in several tissues both the central endocrine gland that is synthesizing the ectis own dysfunction is in response to a neuroendocrine hormone ptth and via the distribution erectus own in the circulation targets peripheral tissues where these proteins again function in concert to regulate the the peripheral response this endocrine hormone so these data suggest that this regulatory network has existed
or at least also exists as a function to control developmental progression and we think that it occurs because the development has to respond to the environment it needs to be able to modulate the timing of the phi development depending on external conditions now I mentioned that we like to look at the protein-protein interaction using this imaging but I haven't told you anything about what this complex and in fact we were surprised to find that the distribution of these complexes is distinct from the distributions of the individual proteins so here is the distribution of the complexes again visualized on one cell the nice thing of this tool is that we can look at individual cells and variations among individual cells compared to the binding of the individual proteins and although these are different methods it should be obvious to you that the patterns are quite different even without the annotation of these bands so we were curious what these genes were which selectively bind the complex this is again one of these features of these regulatory protein interactions where it's the protein-protein interaction that alters the targeting I'm going to
skip this in the interest of time simply to say that we can quantify these differences and show that it is not just a qualitative ability to detect butBut but a shift in specificity which is occurring this makes a more qualitative image again without trying to look at the individuals the left half of this graph here shows the patterns of binding of the individual proteins by various different imaging strategies the right half shows the patterns of binding of the complexes and again the patterns are quite different telling us that the protein-protein interaction re targets the specificity of binding of these proteins now we look at particular Dean's as a sort of a biological response to this pathway some of these are have developmental context Jun hormone juvenile hormone hydrolysis are regulating the stability of other developmental endocrine or moans the d keep one protein itself is a target for transcription regulation by this complex and again so the question for us was
well what is the biological process that is that is reflected by this complex formation by these proteins and we looked at the effects of drugs on these flies and found that there are several bands which are absent here in control flies but when we feed these flies particular drugs are now present telling us that in fact the binding of these proteins is reprogrammed in response to the drugs in a way that is similar that is to the reprogramming of the binding by the protein-protein interaction so this turns out to be specific to particular drugs other compounds which are also regularly in the keep one protein do not regulate binding at this particular load side they regulate binding at other low side so this appears to be a case where the binding of the drug we think in the context of chromatin no this is one thing we have yet to demonstrate that the drug actually acts on the DNA bound complex alters the binding specificity of the complex we look at the functional
consequences of this and again this upper group of genes are the ones which are directly bound by the complex and we measure the levels of transcripts of these genes and find that indeed phenobarbital one of the drugs that induces is binding increases transcription of these genes and other drugs which do not induce binding at this gene do so to a much lesser extent this is the control group of transcripts that are regulated by that do not bind the complex so these are the genes which we chose us sensitives or the binding of the complex selectively and again we can also measure the protein products of these to say that they are significant to look at the functional necessity of this binding insofar as does it alter are these proteins in fact required for the transcriptional function we'll look at the basal expression here in the absence of phenobarbital and we compare the genes where the proteins are bond is a complex and other protein other genes where the CNCC proteins appears to bind independently of the keep on protein where we don't detect keep on binding and in the context of in a Barbie tour both groups of genes are activated the CNCC protein depletion of their of causes a decrease in this phenobarbital activation of both groups now in contrast when we do this same experiment with a keep one protein we find that again keep one is require that the feel more Barbie tour activates transcription but that keep one protein is required for activation of this group the ones that are bound by the complex but not for activation of this group of genes where CNCC appears to function independently so there seems to be a selective activation of different groups of proteins depending on this particular protein protein partnership we can do the gain-of-function experiment the same way in that by expressing keep one independently we can activate transcription and CNCC with some of these genes functions as a synergistic regulator such as here so the combination has greater activity than the individual proteins whereas keep one acts as an antagonistic inhibitor reducing transcription of these genes where CNCC functions independently of keep one so again the protein-protein interaction at this specific locus see to have a distinct effect depending on the binding so to put this half of my talk in context this is the classical model which can very easily explain how a compound a drug or some other external molecule functioning through a pathway activates a set of genes which in some ways help detoxify this compound now the challenge becomes as I mentioned more difficult to explain when we start talking about many different compounds which have partially overlapping but distinct responses so how does this system mediate this kind of response and further as I mentioned we find this group of developmental signals which regulate a very slightly overlapping set which also depends on these same mediators so how can this sort of bottleneck if you will be tolerated we say that it probably is not and in this case that it is the complex acting on chromatin which mediates through some we imagine allosteric effect in other words through binding different co-regulators the specificity of this complex is altered such that it regulates different sets of targets depending on the small molecule mediator and the mystery remains to us that how the system has adapted to respond essentially unknown inducers by ways of modulating specificity in a way that now allows it to target genes that are specific or or effective for each of these targets so we like to think that this system occur operates on many different levels and I just want to thank the people participating this part of the work and our collaborators who helped us with some reagents now for the second which will be a shorter half of my talk I want to return to this problem of I have barely scratched the surface of the problem in the sense of I told you that I would address this pharmacodynamics problem but really all we've addressed this well we have some hazy idea about where this might have come from evolutionarily which does very little to address the practical problem of well if we're going to develop a drug how do we approach the problem such that we're not going to get tangled up and tripped along this way and this becomes especially critical in drug development programs that don't have the tens or hundreds of millions of dollars that are typically devoted to developing and drug these days and that is the case for in fact the majority of diseases which are rather rare and attract very little interest on the part of her methodical community and the problem of course is that it is not just going back and forth and optimizing each of these steps one at a time but rather that a failure at the late stage here often leads the abandonment of the entire pro prob program and you read about the drug companies that shut down an entire division say cardiac diseases and you know that there are going to be no follow-up on these DVDs projects so what can be done well this problem sort of has a similarity to me with the children's game of snakes and ladders I'm not sure if you play this it's it's one which is can be quite frustrating especially in the context where in this system at least there are very few letters you keep going back and and and go starting over so how can we if you will put back some ladders into this system and allow us to anticipate or perhaps even plan ahead for the challenges of dealing with these steps in in in the drug development which are which are rather poorly understood so I'm going to tell a story of a project at my laboratory and I should tell you that I'm have very little background in drug development but there are many rare diseases adrenocortical carcinoma being one which there is essentially no drug development going on which means that this is still treated in the same way that it was treated 50 years ago with compounds that were approved in the 1950s and there's really no company has that is doing any development and and the outcome is predictably unfortunate there are many challenges to this and many such drug development problems diseases are often complex and despite the genomic genetic advances that are made the feedback the turnaround of using that information for drug development is is very slow one key aspect of this cancer drug development problem is that the tumors in fact retain a very unique metabolism they have characteristics that are akin to their progenitor cells which in this case is a one that we take advantage of as an Achilles heel in other words we try to target the unique metabolic properties of these tumors in order to potentially treat them and we do this by using compounds that have been identified that have a very selective tissue or cell type-specific toxicity this is a very classical way of doing cancer drug development but I think it has lost perhaps some of its cachet in in our case we take advantage of the fact that this kind of information is if not entirely publicly available there are certainly many drug companies who discard drug leads which have very extensive characterization and here is sort of the key to our development plan is that we need to start effectively very close to the finish field we need to start with compounds where we know that if we sort of get over the last few jumps we will actually have something to release in the the clinic we cannot afford to have the risk of losing away along the way so I'm going to since time is short going to sort of give you the answer and then give you some of the steps that we got here and some of the problems that we face so we've identified mainly through literature investigation a compound discarded in the 1980s which has a selectively eternally activity and this compound through some pre preclinical studies that we've conducted that are describing brief has been now in three phase patient trials and so far we know that the compound is well tolerated has no potency which is one challenge that we're facing in in in in in the in these preclinical trials so briefly going over that kind of characterization many cancer drug development projects undergo so we use a xenograft model which is to say we take cancer cells we put them in a mouse and we give the mouse a compound in this case this ATR 101 compound and this decrease in the growth of the xenograph isn't astounding but it is at least a clue that we have something that might have some benefit we weighted xenograft at the end and very importantly and and here's the critical aspect of trying to decide on which kind of compound would be appropriate the patients that are treated for disease this disease are quite sick given that it is a late label the diagnosis is often at a late stage and therefore we have to choose a compound which has no adverse effects this is not true of course for the major majority of chemo therapies which are essentially toxins which you use at a sub toxic sub acute toxicity level but this compound was chosen selectively because it is so well tolerated here simply represented by the body weight of a mouse that is not decreasing the compound can also be relatively effective in this case we're looking at the same kind of xenograft assay but we're starting administration early now this is not really a realistic cancer model cancer is usually treated at the time when the tumor is quite large but in this situation we can affect the press to tumor development almost entirely this might be perhaps representative of a situation of a of a adjuvant therapy after after a surgery or such this compound acts by inducing apoptosis it induces the cells to kill themselves and this apathetic stimulus has been the area of interest and you might wonder well we have these compounds in clinic what are we doing trying to understand what they do well the critical problem for us is that the compounds potency is not really adequate or at least is going to pose some difficulties for use in the clinical so given that we have no clue how this compound has this-this-this potential therapeutic benefit we have to go back and try to understand how it works in order to improve on it very briefly and without giving you much background we found that this compound causes cholesterol accumulation so this is a cholesterol stain where this compound the active compound causes the accumulation of cholesterol in the membranes whereas a controlled compound like this dimethyl amine is not causing the cholesterol cumulation this occurs in many cancer cell or many cancer cell lines of the adrenal and the adrenal is rather a unique site in that the cholesterol metabolism of the adrenal that makes all of our corticosteroids the ones that regular blood pressure it has has its own very dedicated cholesterol metabolism which is why we think that this cholesterol accumulation turns out to have such tissue-specific effects it is very rapid so this compound within a few minutes causes a queue of cholesterol which is in fact faster than the time at which the cells are starting to die losing ATP and increasing the caspase signaling suggesting that this is in fact the potential mechanism of toxicity what makes it most compelling to us is that using this polysaccharide cholesterol chelating agent methyl beta cyclodextrin we can remove the cholesterol effectively treat the cells with this compound but not cause the cholesterol accumulation which saves the cells effectively by adding this cholesterol chelating agent we can restore ATP to cells telling us that it is in fact the cholesterol accumulation which is essential for this ATP depletion in the absence of this chelating agent and conversely prevent caspase activation which results in the cell death and and and indicating that that in fact the way that compound works is by causing a excess cholesterol accumulation how does it do it I was pleased to see that in the afternoon we were here one of the presentations of ABC transporters which are in fact the target that we've identified for this particular compound the compound prevents cholesterol efflux so adrenal cortical cells export excess cholesterol and in the presence of this drug the cholesterol accumulates with a timeframe that is similar to the ATP depletion and this is in contrast control compound which does not influence the cholesterol export and this group of ABC transport is a complex one I won't try your patience bye-bye or my lack of knowledge of them by describing them but there are literally dozens of them there are multiple substrates here that are affected in addition to the cholesterol influx were also finding that the export of cortisol of the products of steroid synthesis is inhibited in the presence of this compound and this inhibition seems to also be essential for for for for for that for that for the inhibition of viability so that we can mimic the effects of this compound so the compound effect here on ATP levels is quite substantial by combining multiple inhibitors of ABC transporter with different specificities we can somewhat mimic though we don't quite reach the efficiency of this compound indicating that this particular set of ABC transporter substrates in other words these particular inhibitors target different ABC transporters must be inhibited in order for this compound to have its its its its cholesterol accumulating effect and an therapeutic benefit what has this gained us so far well so far we only find that by adding to this compound known inhibitors of particular ABC transporters we can increase the potency so we can do what was mentioned earlier previous talk effectively combination therapies combination therapies have certainly an appeal the problem is that they are problematic in clinical trials then and also well you have to get many companies to work together to underwrite such progress the other thing it gives us is that it tells us that there are specific limiting activities in other words if we can increase find a compound which increased targeting specificity for some of these ABC transporters we may be able to do a get better job with patients so at present this is the model we basically have identified that cholesterol accumulation is a process that's driven by many inputs and outputs in the presence of this drug candidate we caused a accumulation of cholesterol by blocking several of the pathways of export causing toxicity many people have participated in this project veronica burns and even her chen leaning groups of students in the project a drug development program requires participation of people of right their different interests from expertise in in in in in drug development which which is not me and again the clinical trials are run by a startup company that we found it and we've had its systems from from many clinicians which is something which again compliments my expertise and has been essential for the project so i thank you all for your attention and happy to ask questions well what was known before is that there are for us yeah for for us what's valuable is that there are very large data sets of compounds that have been tested in animals in this case thousands of compounds these compounds typically come from projects such as heart disease drug development and often these projects come up with discarded compounds that have been administered to guinea pigs dogs monkeys animals which are relatively good models for humans I think what mathematics could do for us that isn't happening right now is that although we know the structures of these thousands of compounds that have been tested and we know something about the physiological outcome it is context for example we knew that the compounds had by availability they were got into the animal they had bioactivity in the form of their cholesterol lowing in fact and in fact in this case the operative word we knew something about the toxicity we knew something about how these compounds caused the adrenal - we knew that they cause the Selective effect on the adrenals now relating the one to the other is something that is currently completely empirical in other words we don't we have no clue about what it is about the difference between these groups of compounds that make some other than adren toxic and others not I think that is a problem which there should be much more information about not obviously just for adrenal toxicity but questions in general in terms of drug distribution what causes a compound to accumulate in one tissue versus another again a problem for which there's really minimal information it's empirical we give a compound to an experimental animal and we find it in a target organ we say good this is a compound that could have benefit a priori we have no way of predicting those things so that's perhaps that the challenge and the difference that we made in this project of making sure that we understand roughly how the compound works continuous question is there nothing known about the physiology of adrenal cortical carcinoma a pathway beginning of several genes heredity family etc etc etcetera because if I understood correctly what you did is the very very old way trust by other taking everything yes and the way now people see I think 10 years the companies this if they have a pathway they have some enzymes or phosphorylation or something like that and then they choose active site they will take advantage they drive with many compounds just specifically to see Blake this so I've sort of glossed over many diseases like I don't accord car carcinoma are certainly not highly studied but that doesn't mean we don't know anything in fact we have quite a bit we have tumour sequences from hundreds of patients which as some of you know we can identify candidate genes those candidate genes them tend to be the usual suspects they are in pathways that influence genome integrity p53 they're in signaling pathways beta catenin etc the success at targeting those pathways has been quite spotty in other words we've known about pathways like rats for thirty years we still don't have a drug we don't know how to target Mick well those will certainly come but the other problem with many diseases such as a different cortical carcinoma is that they are highly heterogeneous in other words the molecular causes the clinical presentation of the disease varies quite broadly so although we may you know not-too-distant future be able to treat 1% 5% maybe 10% of the patients no no no no seemingly reasonable timeframe will we be able to treat a large majority of the patients by targeted mechanisms the other thing is here I mean we can get past these but it is up here where all the failures happen so by taking the approach that we're taking we are somewhat say these stumblings up here are so catastrophic that we have to focus on doing these instead of spending all of our time here I mean it is nice to have a specific target but unless you have a way of dealing with the problems that will come up here you will never get to the finish line that is my argument yes so target was founded for tender Pacific viability becomes over your problem they start to stick to everything and then their pharmacodynamic is a mess it is very very hard to do that and get an ID card it's very expensive so there's not enough resources that come from there are big challenges no there are big challenges I wouldn't call it impossible it may not be and I think I want to contrast the two halves of my talk there's a very different approach that we take when phasing a problem of developing a therapy then we take when we try to address a problem having to do with mechanism in even an animal and I think that that difference in approach it doesn't solve the problem but it puts our emphasis on thinking about these problems early and trying to anticipate trying to I wouldn't say that our solution this case is generalizable I mean I think every disease has its own peculiarities understanding that disease context I think contact with clinicians has been key for us in other words talking with people who treat the patients to understand these are very sick people in order to develop a drug we cannot start with a compound that's toxic we have to sort of rethink of what a therapy might be so so challenge is about but I would say that yes except for this part it doesn't have to be outrageously expensive but that requires sort of accepting compromises and taking shortcuts along the way questions about me giving really more clarification so I didn't quite understand what the keep one CNCC are they direct binders to all these in a bio box is that where they feeding it upstream somewhere yes yes so keep one is a remarkable molecule and there are crystal structures of keep one it operates by many mechanisms one of the major one being that it contains cysteine residues that are hyper reactive those cysteine residues react with electrophiles compounds that are more reactive and most bio active compounds so that's one big class of keep and reactive or keep on activation mechanisms this model here of a changing conformation of state of keep one has been somewhat corroborated by biophysical data the the piece that we've been trying to work on is well how do we get from this reaction to a target and that has been the thing that has had sort of a missing link which we think we've established I still think that the question of how keep one especially the physiological environment in a Cell let alone an animal is able to discriminate between molecules that are hazards than molecules that are mainly active intermediates of some intermediary metabolism it doesn't do a great job in some respects humor rate for example is a great activator to keep one but it just operates at a sub hey so so it's tuned to react with compounds in a very specific way it also binds polycyclic hydrocarbons and it does that with a very sort of relaxed specificity that again discriminates between steroids and things that otherwise might seem like it would activate high high levels of glucocorticoids will activate keep on but not the levels that are present most house the second question the clarification was so the the two proteins that are going to these puffs these sites and a leaky chromosomes maybe I didn't understand this properly we did the localization initially of each one they both look like the dicen ones and yet when you have them presumably they're forming a complex there ah that's presumably what is different about the individual binding of the proteins and when we see them together a big difference is we only see the redistribution when we overexpress the proteins or when we treat the animal with drugs so there's something which admittedly is a bit nebulous here between the proteins individually at their endogenous levels and the proteins over expressed as a complex now that something isn't the trick that we play this complementation as it's not the fact that we've trapped the proteins on the chromatin because we can also over express them in ways which don't allow them to form this stable biomolecular complex so for us the over expression is sort of a it's a it's it's it's a unfortunate but perhaps in format tool we think it reflects because it produces a similar redistribution or at least some aspects of our industry distribution can be reproduced by drugs we think it has some relevance relationship to that drug induced targeting but in general I think that the effects or expression should be considered problems so to continue Mark's question to really know if the complex has a function in not just another an expression artifact you probably need to do an interaction no no it is easy so in fact all of our experiments we do this a lot and we often find that overexpressed proteins have characteristics that are non biological that we can't handle waves such as hear and say maybe this over expression actually does something that is relevant most of the time when we find over expressed protein doing something weird it's bad and indeed our prime strategy including this project is to show that a mutation in the prot protein prevents the signal we don't get fluorescence and hence that the fluorescence that's at a certain level represents a biologically relevant complex formation so what you need to see that now that you know the genes which are attached so in our case the mutation isn't quite as subtle a step in our case the mutation in this case is one that simply disrupts the complex so it's a yes yes what you're asking is for us to show that retargeting is something that depends on the specific interaction where's all that we've shown is that disrupting the complex prevents us from seeing a signal and and that we don't know partly because we don't really know what the signal is what the confirmation of whatever the mechanism is that converts this protein from one that is targeting one set of genes to a different set of genes thank you [Applause] [Music]