Good day, everyone. My name is Kristen and I'm going to be the moderator for today's webinar on Nanobrett for exploring like Edible Space in the Dark Kinome. Thanks for joining. And before we get started, we have a little bit of housekeeping items to take care of. So on your screen, there are multiple windows, all of which are movable and resizable. So feel free to move them around to get the most out of your webinar experience today. And in this webinar, we're going to have many ways to be interactive. So you can submit a question at any time during the webinar, and there's a little box for that on your screen. And we'll answer those during the live Q&A session at the end. In the resource library is a list of helpful materials, which also includes a copy of today's presentations. So feel free to download any of those resources and share them with your colleagues that would be interested. And you can also get a reaction. So that's another way to provide feedback and let the presenter know what you're thinking. And last, we're going to have a series of questions that we're going to start right now. It's just only two, and we'd really appreciate if you would take a moment to answer these questions. And you know, your feedback is really valuable to us. So let's get started with those questions. So the first question is, do you have any experience with the Nanobret target engagement technology? There is only three, three choices here. So A no experience yet, it's new to me. B, I've heard about it, but I haven't used it yet. And C, I'm a current user. So I'll give you a few minutes to to provide some responses. Again, it's do have any experience with the Nanobref target engagement technology. A is no experience, it's new to me. B, I've heard about it but haven't used it yet. And CI am a current user, so we have some responses coming in right now. So I'll just give it another minute or two and then we'll just flip it over and see what we have. You can continue to provide responses, but so far we have a good mix. This is great, good mix of people that this no experience yet all the way to a new user. So thank you all for joining and providing responses. And my second question before we get started with the webinar is what methods you currently use for measuring compound binding to a target protein. Now there's six options here and you can check all that apply to you or your research teams. The first one is SPR or surface plasma in residence. B is DSF or differential scan inflammatory, C is TR FRET or FRET, D Sepza or similar cellular thermal shift assays, E Nanobret target engagement or F affinity based proteomics. So again, the question is, what methods do you currently use for measuring compound binding to a target protein? And you can check all that apply. And there are six, six different responses from SPR all the way to affinity based proteomics. So I'm going to give you all a few minutes. I know that's a lot of potential choices, especially if you're using multiple methods, but your input is really is valuable to us. So we'd love to hear from you. So I'll give you one more minute and then we'll flip it over to see what responses are are coming in as as we're working through things. And let's see what we have. So, so far, we have a lot of a lot of target engagement users, sets of users to your Fred SPR Great. All right, this is super helpful. Thank you. And now I'm gonna get started, and I have the pleasure of welcoming and introducing our speakers, Matt Robers and Allison Axman. Matt is a distinguished scientist and associate research director at Promega. He received his initial postgraduate training at the University of Wisconsin, Madison, and he received his PhD at Gulf University in Frankfurt studying target engagement at biomolecular complexes with Doctor Stefan Canal, Matt focused on technology development and cellular pathway analysis at Life Technologies. Since joining Promega, Matt has built a team focused on the development of new technologies to assess intracellular target engagement, residence time, and drug polypharmacology. Dr. Allison Axman is an assistant professor in the Division of Chemical Biology and Medicinal Chemistry and a principal investigator of medicinal chemistry in the Structural Genomics Consortium at the University of North Carolina, Chapel Hill. She's a highly experienced synthetic medicinal chemist and a core scientist within the SGCUNC. The SGC is an interdisciplinary team of researchers that designs and performs iterative medicinal chemistry in order to turn promising chemical starting points into selective chemical probes and or degraders. These compounds are then shared openly with the scientific community. Allison is dedicated to carrying out the basic research that will lead to the new therapies for diverse diseases. Her research has focused on the synthesis of small molecules that selectively modulate or degrade proteins implicated in disease propagating pathways to drive medicinal chemistry optimization. Elson's lab has also been involved in development of small molecule tracers that enable cell based assays. And with that, I will turn it over to Matt. OK, hello everyone. Yeah, my name is Matt Robers. And today I, I really hope that we can give you a story really with the focus on demonstrating that we can measure target engagement across the humankind home with this technology called Nanobret target engagement. But more importantly, that when we use this method to to query engagement across the kind of, we can actually discover new interactions and new vulnerabilities for understudied proteins. So when my my group was founded almost 10 years ago, we were really focused on this conundrum that cell free or biochemical pharmacology often doesn't translate or predict what happens when you move inside cells. And this is just one example of that using AFD, an FDA approved drug called crizotinib that is a multi kinase inhibitor that targets met in ALK. And we, we always like to show this example, but there are many of these in the field in the literature that really show that the, the potency in a cell free assay with the multi kinase inhibitor chrysotinib really just doesn't correlate with perfectly and doesn't predict the rank order target engagement selectivity of chrysotinib. If you measure a downstream biomarker of kinase activity using a cellular assay, in this case a cellular phospholyza that that measures kinase activity. And so, and I think this is not shocking. I think that the field is, is now recognized that what happens inside the cell is often difficult to simulate with pure protein. So we've grown, you know, accustomed to thinking about permeability causing potency offsets, but it can be more nuanced. I think we wrecked the field recognizes for kinases that high concentrations of ATP that are often unpredictable in cells often just don't match what's used in a biochemical system. Also protein protein interactions can influence pharmacology at a target protein, the targets activation state, the targets localization and so on and so on. And I really feel that the the composite sort of influence of all these factors really just makes it difficult to create a cell free system that would simulate all that complexity. And so I was really kind of, you know, excited to see that there's a lot of people here on the call that are, you know, not familiar necessarily with nanobred target engagement, because that gives us an opportunity to introduce that method in some detail. But I was also excited to see that there's a lot of groups out there that seem familiar with H TRF and, and TR fret like assays. I think that the, the Brett system, this bioluminescence resonance energy transfer assay that quantitatively measures target engagement is very similar to cell free methods that use HTRF. Fundamentally, these probe displacement assays. The difference in this case is that it's like the TR Fred assay can be moved inside of a cell and you can still get that same level of quantitation, but but with all of that cellular Physiology at play. And so briefly, the way that this method works is that we, we create a, a luminescence reporter at the target of interest by genetically tagging the target with a small and very bright luciferase called nanoluck. And what this, this is an enzyme that produces intense very bright luminescence inside intact cells. You can see, you can even, it's so bright, you can even image where the luminescence is coming from. If it's an RTK, you can see plasma membrane localization of that light. If it's a cyclone dependent kinase, you can see nuclear localization of that light. But that gives us this local optical signal at the target that then we can combine with the sort of magic of the assay, which are these fluorescent drug tracers that are designed to permeate cells and and occupy a binding pocket on the target protein. These are typically derived from for kinases, they're typically derived from ATV competitive kinase inhibitors that have SAR and allows us to create this fluorescent adduct and still retain binding potency at the target. We optimize these reagents for cell permeability. When they bind to the target, they form a Brett complex and the luminescence from the luciferase that's that's blue transfers into the fluorescent dye that becomes red. So now the cells grow a glow with red light when these tracers bind. That's really just how the assay is, is designed And, and these complexes are, are based on a dynamic equilibrium. So they're reversible complexes. So if you introduce a test compound to the assay, you want to measure its binding characteristics. Now what you really have is, is that that HTRF like assay that measures probe displacement. So when the compound binds, it competes with the tracer leading to a loss of red cells, go from red cells back to blue. So just to give you an image or a visual of what actually happens in these wells that have these live cells expressing the luciferase. This was one of my very first bread experiments with a kinase. This was with CDK 2 and a kinase tracer that could permeate cells and you can really see that energy transfer happening and so bright that this allows us to have very low level of expression of the target protein. So we can go to low expression levels, be endogenous or even sub endogenous. And this assay still works quantitatively. So we don't use, you know, images or or cell phones to run our experiments. We, we use HTS compatible plate readers and we also don't treat these tracers in kind of a we, we, we, we give a lot of consideration to the tracers affinity when we use them to report quantitatively on target engagement. So we always dial these Brett tracers down to a, a concentration of tracer that is at or below the KD apparent for the target protein. And when we do this, the assay is quantitative. And so it, it, it, it, you know, I methods that are out there for target engagement, like cellular thermal shift are incredibly powerful, but, but sometimes you don't get that accurate quantitation of target affinity. This method is quantitative for Ki apparent it's more than just an IC50, but again, that's really based on using these tracers appropriately and operating in that window of quantitation that I just described. So what you can then do when you've got tracers for many different kinases throughout the kinome is you can kind of redo these experiments like the one I showed you on the first slide. Take the multi kinase inhibitor chrysotinib, measure target engagement across all those exact same kinases. And now when you plot engagement potency versus phosphorylation potency all in a cell, you get a much better agreement and the rank order makes more sense. And so it was these data that really got us excited about building a kinome wide platform to explore target engagement, polypharmacology and also vulnerability inside living cells. And so this is the summary of what I'm going to present on today. Just briefly, I'm going to initially just talk about how we've combined all these assays into one assay to measure target engagement across about half of the kinome in cells. And really with that allows us to demonstrate the impact of cell Physiology on kinase TE at a global level. I'm going to then kind of do a deep dive into an interesting story. We can kind of learn about the conformational landscape of a unique type of kinase inhibitor called the type 2 kinase inhibitor that is kind of conformationally selected within the kinome. It binds to this kinase when it's in the the DFG out conformational state. And really with this kind of chemical probe, we can really observe novel interactions that we don't see using cell free assays and discover potentially new vulnerabilities. Then it within that type 2 kindness inhibitor story, I'm going to do a deep dive into how we can kind of observe not only novel vulnerabilities, but also escape mechanisms within a very important signaling complex called the Ras Ras signal zone, which is a very important a protein complex involved in cancer. And then lastly, kind of present that Ras Ras story as a new platform to measure target engagement of biomolecular complexes, which really represents the next generation of Nanobret TE assays that we're excited about rolling out. And this is kind of new iteration on this platform. So before I get into the the the vulnerability story, I think it's really important to recognize that this method Nanobret TE when you operate inside cells with these full length kinases who are sampling all these different confirmations and and protein interactions. This does allow us to have a platform that can do more than just measure target engagement of molecules that are similar to the tracer. We can actually use our tracers, which are typically derived from type 1 ATP competitive binding ligands. We can actually use those to query all of these different types of interactions at kinases that that are not just the Type 1 ATP competitive binders that that bind oftentimes to the kinase when it's in the the active state with that aspartic acid pointing in toward the pocket. We can also measure more confirmationally selective inhibitors that bind via a Type 2 mechanism with the DFG out conformational state being sampled. But it gets more interesting. We can even use these tracers to measure binding events that are adjacent to the ATP pocket where our tracers reside. And even maybe the most provocative observation which I think the field is really starting to embrace now is we can use these tracers that occupy the ATP pocket to often times sample allosteric engagement that happens many angstroms away from where our tracers sit really kind of supporting that that these kinases are breathing, they are sampling these different confirmations and our assay is really able to to detect these these types of of interaction events. And on the right hand side, I'm showing a few examples for different kinases where we've been able to query all four different modes of of engagement, including the type 4 Allosteric. And I think my favorite example is this example with able kinase where we can see engagement to GNF 2, which is a Marista lazy Marista pocket Finder. So so how do we then use this method to ask this question? How does cell Physiology impact kinase target engagement at a global level? What we've done as we've taken almost our entire kinase library of assays, which is over half of the kinome, it's, it's approaching 300 assays at this point for wild type kinases. This experiment I'm going to describe was done with more of like 240 kinases. But what we can do is we can take all of those individual kinase plasmids and array them out into plates and now query target engagement in one experiment against the almost, you know, half of the human kinome. So we've got this library of kinase nanoelect fusions. You can obtain this library through our our catalogue or you can come to Promega and run these this experiment as a service. Both are fully supported, but the idea is what you do is you do reverse transfection of that kinase library into your cell type of interest. We typically use Heck 293 out of convenience because they're very easy to manipulate cells. But you know, any potential, any cell type could be used that's transfectable. If you can, you can do this reverse transfection seamlessly. You then combine those cells that have been transfected with the luciferase and the kindness of interest. You then introduce these Brett tracers at a quantitative concentration again at or below KD apparent for the target. And now when you put your test drug into the assay, now you can look for competitive displacement and actually get fractional occupancy of your molecule of interest against almost half of the humankind home. And then you can build these interactive maps like you're used to seeing in a cell free assay with good, you know, reliable methods like, you know, discover X kind of scan. But we can now build these inside intact living cells and compare the pharmacology in cell with the same target pharmacology in a cell free assay and see if we can actually learn something new about, you know, intrinsic vulnerability within the kinome. And this was an experiment that was run against 240 kinases using the nano bred assay. And we ran this experiment with a multi kinase inhibitor to satinib, which is a very promiscuous tyrosine kinase inhibitor targets PCR able and many other kinases. We can actually measure target engagement in a potency mode against about half of the kinome. And we can compare the potencies in the nano bread assay against the potencies in a biochemical assay. Again, a very robust and quantitative assay like the the the kinome scan assay. But we can now kind of see the extent to which the cell impacts that engagement pharmacology. And every dot in blue represents a kinase that's right shifted by one to two orders of magnitude in a cell compared to a cell free system. And we, we have really grown accustomed to seeing these types of results with type 1 kinase inhibitors like disatinib. And it's very easy to explain this now retrospectively based on ATP interference. Of course, ATP levels are very high in the cell. In the cell free assays, they're very low or even absent like they are in the kinome scan assay. So it's not that shocking that you see this kind of compressed spectrum of activity of disatinib in a cell compared to a cell free assay. But it's not really that simple for other types of kinase inhibitors. If you look at more confirmation, we selected kinase inhibitors like serafinib, which is a type 2 kinase inhibitor. It kind of enforces this confirmational change in the kinase. You see the movement of the aspartic acid from the, the, the position where the that residue is pointed in toward the ATP pocket in a way where the the kinase is kind of poised for phosphotransfer into a confirmation where the aspartic acid is pointed out away from the ATP pocket. We call these DFG out inhibitors. Now what you actually see is something very different than dasatinib. Now you see a number of kinases where their pharmacology or their potency is enhanced inside of a cell compared to the cell free assay. So if you just take one example, RIP K2, you can see it's 40 nanomolar in cell with nano bread, it is 1.3 micromolar in the biochemical assay. That's not a subtle shift. That is a major shift. Implying at first you know, based on kind of initial assessment that RIP K2 is more intrinsically vulnerable to this type of inhibition inside of a cell compared to a cell free assay. Opens up a lot of questions like is are there conformational changes at play? Are there protein, protein interactions at play? What could be causing this enhanced engagement inside of a cell compared to the cell free system? It's not unique to serafinib. That result kind of motivated us to look at a number of different type 2 kinase inhibitors that are fairly well characterized in cell free systems. And for for retinib, AST 487 as well as serafinib, we pick up novel interactions that are invisible in the biochemical systems. And so this is really intriguing to us and this really got us even more excited to kind of dig deeper into to what could be happening here. So we, we started to run these experiments like this one that we ran with Matthew Solner at the University of Michigan who specializes in creating creating hybrid molecules that you allow you to kind of survey the DFG out landscape within the kinome. And so we did in this experiment with Matthew is we took the satinib, which is a well known multi kinase inhibitor, highly promiscuous in cell and we actually turned it into a type 2 inhibitor by adding this one of these DFG out features that enforces that conformational change in the aspartic acid and kind of pushes the kinase into often times into inactive confirmation. And this would allow us to have a matched pair of chemical probes that allow us to ask, are there kinases that are potentially more intrinsically vulnerable to a DFG out mechanism of engagement compared to a type one or desatinib based mechanism of engagement? And so in other words, if we were to do a correlation plot of the type 2 dash DFG out IC50 versus the type 1 desatinib IC50, would you see molecules of the upper or left quadrant or the lower right implying that that kinase is more prone to a a type one or type 2 mechanism of binding? So what we what we observe was fascinating. And so this is one example. This is taking rich RIP K1, which is you know in that RIP K family and taking the satin have been seeing that you barely see target engagement with the satin at RIP K1 up to about 10 micromolar in cell. But if you add that DFG out feature, you get a a a striking increase in potency at rib K1. And if you look across the kinome, you see tons of examples of this and you see molecules like or targets like those in the lower right hand quadrant here. All of these red dots represent kinases that are one or more orders of magnitude more vulnerable to the DAS DFG out molecule compared to its parental molecule Dasatinib, which is our already a highly promiscuous kinase. And so you what what I think was striking is you pick up a number number of interactions of this vast DFG out type 2 hybrid that are actually target kinases that have known described potent inhibitors in the literature. So two of those examples are shown here, although there are many of them SRMS kinase or SDK 36, which is kind of an bulk family member cousin, there's no describe potent inhibitors to these kinases. And by turning a type 1 inhibitor into a type 2, we can discover these not novel vulnerable vulnerabilities inside cells. I mean, so we were really excited by these types of results, but it really kind of opened up a number of fundamental questions about intracellular pharmacology and the Physiology of a cell that could be impacting this. So, so is it, is it simply that our kinase is sampling more conformational diversity inside cells maybe because we're using full length kinases compared to cell free assays that use kinase fragments, our protein, protein interactions potentially drivers in target engagement potency. But I think most importantly from a, you know, tool development standpoint, it's this last bullet. How can we actually measure target engagement at protein complexes? Is there a way that we can turn our assay into one that can conditionally query or interrogate target engagement at a complex of a kinase instead of just a kinase that's monomeric or in a way that you wouldn't know what kinase is interacting or what protein is interacting with your kinase? And so to kind of do that mechanistic deep dive, we've really been interested for a number of years now in the Ras Ras, sorry, the Ras Ras signalisome, which is a well studied kinase complex that is vulnerable to type 2 target engagement with molecules such as Sarrafinib. And there's a number of FDA approved drugs and molecules that are being queried in, in clinical trials that target this signaling complex. But I think the reason why we were so interested in applying it to this complex complex is because of all of these protein, protein interactions and conformational changes that we anticipate and expect our drivers in pharmacology of these types of compounds. And so just in brief, the way that the signaling pathway and the signaling complex Flex functions is when Ras is activated or bound to GTP Guani nucleosides, you get this activation, the stimulation of this signaling complex where you get recruitment of the RAF kinases to Ras GTP. And again that Ras GTP could be could be activated through somatic mutations like codon 12 or it could be stimulated through upstream RTK activity like EGFR. But what this causes is a protein, protein interaction between the rafts typically HOMO or heterodimerization can occur. They interact with, with Ras GTP and this then sort of facilitates a signaling complex where you get phosphorylation of MEC, phosphorylation of IRK and ultimately transactivation of gene expression. We can inhibit this complex in a number of ways. There's a, we have a, a big story about targeting Ras. I'm not going to have time to get into that today, but this complex is druggable through the RAF kinases which are the proximal of downstream effectors of Ras. And the way that you inhibit this complex complex is by engaging both proteomers of RAF, either homodimers or heterodimers in complex with rash GTP. Now what's really interesting is not that much is known about a RAF. Almost all of our knowledge and historic sort of understanding of the pharmacology of this complex is driven through B RAF and C RAF, typically through heterodimers of those two RAF paralogues. And so one thing that we see that I think is really striking it with a nanobret target engagement assay. And this is kind of our initial inkling that we should probably spend more time trying to understand what's happening is that when you when you just express a nanoluck fusion with B RAF and do a target engagement assay just in, in HECT 293 cells, you, you get, you know, kind of a modest potency, nothing too high, highly potent in terms of target engagement. But when you add mutant RASP to the system, when you just run the B RAF assay in a a AK Ras GTP biased lineage or you take you over express K Ras like and use AG12C allele that's biased or the GTP state and is is competent to form a complex with RAF. What you see is a huge enhancement in target engagement at B RAF almost two logs of an increase in potency of these type 2 RAF inhibitors when you add oncogenic RASP into the system, really supporting the target engagement truly is cooperative when RAF GTP is in complex with with the RAF. And So what what what kind of got us really, though, I guess interested in digging more into this is when you look at the potencies that have been described against RAF, you compare those 2A phenotypic readout or a a downstream biomarker in a cell that measures RAF activity. You really don't get great agreement. So we we can measure the pathway using a phospho ERC assay. This is a gold standard assay to measure inhibition of of mutant Ras raft signaling. And so we can run this kind of an experiment with a, with a LUMIT assay that looks at phospho ERC status. We can run that in AG12C lineage like the pocket 2 cells. When we compare that potency, this is where it gets really confusing to what's been reported at A RAF, B RAF and C RAF using one of these type 2 inhibitors. What you see is a huge disconnect. It's almost a three log disconnect between pathway inhibition and biochemical inhibition with pure protein. And these are the, these are the types of challenges that we gravitate toward and try to use our technology to understand. And so there was another paper that came out along these lines that really kind of galvanized our interest in trying to understand target engagement at these individual RAF paralogues. It was this really provocative finding that in the one of those G12 CK Ras lineages, what appears to be a potency limiting factor is, is a RAF. It was really striking. So this group, this group at Novartis did this this really excellent study where they looked at targeting, they looked at genetic did genetic knockout of all these different RAF paralogues. And what they noticed uniquely that was that knockout of ARAF or genetic ablation of ARAF which really uniquely sensitized these K RAFG 12C lineages to the anti proliferative effect of these type 2 RAF inhibitors. Implying that maybe ARAF is actually an important signaling kinase and maybe an open end up the possibility that these type 2 kinase inhibitors actually spare ARAF in many of these different cancer cell types. And So what we were really interested in doing is trying to develop an assay that could dissect the mode of action of these inhibitors. So give us an understanding of target engagement and all of these different individual raft paralogues in this signaling complex. But to do this, we couldn't just use our our standard Nanobret TE assay that uses nano luck as a fusion partner. So the way that we really had to think about this as we weren't really interested in just looking at Target engagement at total RAF, whether it's B RAF, C RAF or a RAF. What we really needed to do is, is kind of eliminate that, that readout from the cell and really get at RAF dimers. So really understand are these ligands binding to and and and sort of symmetrically engaging both proteomers in these these different mixed RAF heterodimers. But more importantly, we had to do that in a complex, in a, a context where RAF is in complex with RAF GTP. And we needed to also get a a programmer specific readout for target engagement to understand whether this a RAF sparing mechanism is actually happening. And so to do this, we really needed a new way of doing target engagement. We couldn't really just use nano luck that measures total population of TE. We really needed a TE assay that we get specifically a protein complexes. And to do this, we utilized a new or a more recently developed technology based on nano luck called nano bit that's based on structural complementation. And the idea is if you can use the nano bit system. So the nano bit system is really 2 subunits of nano luck that are non luminescent, but when they're brought into close proximity through a protein protein interaction, you kind of restore nanoluck luminescence and get light output similar to native nanoluck. And so the idea was if we can put the small subunit of nano bit onto 1 protein in the system and we can put the large subunit of nano bit onto the other protein. When these two proteins interact, you make light at the complex. And now you can bring in one of these kinase tracers to query target engagement just like you normally would in the nanoluck assay. But in this case you're looking at APPI instead of a total population. And so here is really the finding that I think we were, we thought we found very intriguing and we think is going to be very impactful for RAF inhibitor research is really supporting this, this notion that these Type 2 RAF inhibitors do indeed spare A RAF compared to B RAF and C RAF. So what I'm showing you here is just B RAF programmer engagement or C RAF programmer engagement at the signaling complex and and ask working whether target engagement is sufficient to sort of inhibit or or match that of the pathway readout which is phosphoric. And what we see is there's almost a 2 log shift between B RAF and C RAF engagement compared to phosphoric supporting the B RAF and C RAF are perhaps not the limiting factor or the the potency limiting factor of these molecules. But it wasn't until we looked at A RAF, but we really started to see the potencies matching really supporting the A RAF could be a missing piece of this puzzle. And when we expanded that to a broader cohort of these Type 2 RAF inhibitors that are undergoing clinical evaluation, you see they all kind of have this potency limiting factor that seems to match that of A RAF unlike B RAF and C RAF. So we did then as we took a broader a sample of these type 2 RAF inhibitors, many of which are undergoing current clinical evaluation and you see they all engage a RAF to varying degrees, but they all show much stronger engagement to B RAF and C RAF supporting that maybe to truly have molecules that can inhibit the Ras RAF signalling complex in a variety of Ras driven cancers, you may need you may need molecules that are more potent at a RAF. And so we're very excited about this as a technology that can potentially support drug discovery efforts at the RAF kinases in complex with Ras. And we have since gone on to to turn this assay into one that can be used for for to query a lot of different types of protein, protein interactions. I'm not going to have time to get into these. These are just four different examples. I just told you the RAFT story. We also have a story where we've queried target engagement at Ras timers and exposed kind of novel vulnerabilities within the Ras hotspot mutants. We've applied this assay to measuring CDK 12 cycling complexes in a way that has not worked well for us just using nano luck, the nano bit assay really salvage that that system. And then most recently this is really hot off the press data that were made were able to look at EGFR homodimer target engagement where we're able to do this across a broad collection of the clinically observed mutants of EGFR. And we're even going into a direction now where we're going to look at Target engagement at mixed EGFR heterodimers with other her family members. And we hope we can learn something with this new assay system that we just developed there. I also want to touch on this concept that we are very excited and we want to encourage DIY kinase tracer development. Now, people are probably familiar with the basic assay system. We enable building blocks for building your own tracers. We recognize that Promega can't develop tracers for all the kinases. We need help from the field to do this. We are seeking collaborators. We are seeking people that want to kind of do this on their own. We recognize there's a need for this and we would, we would encourage that. We would also encourage you to check out a database that is has been created by the SGC that shares information about many different kinase tracers that Promega has not offered commercially. And if anyone is interested in recreating those or collaborating with the groups that submitted these tracers to the Tracer DB, you'll be able to do that through this free sort of crowdsourcing initiative. And I, I urge you to check out Tracer db.org if you want to check, find other examples of Brett tracers for your target of interest. So to summarize, we hope this, we hope, I hope you've recognized and appreciated that novel kinase interactions can be observed inside cells that you might not see with other methods. Many kinases favor particular conformational states inside cells that you might not see in a cell free system and that supports novel vulnerabilities. We see that the Type 2 story really kind of allowed us to to understand target engagement at the RASP RAP signaling complex and how many different inhibitors can spare a very important kinase called a RAF. We can now use this new concept that I showed you as POC with the Ras RAF complex as something that could be applied to other biomolecular complexes. We hope this becomes kind of the next generation target engagement technology. We are very interested in expanding beyond PP is we have an effort now to do target engagement at protein metabolite and protein nucleic acid complexes. I have a lot of people here to thank, too many to list individually, but I, I, I thank all these individuals. I thank you for your time and I look forward to addressing some questions in the Q&A. So thank you. All right, I'll take it from here. Thanks everyone for joining from around the world. I'm going to build a little bit on what Matt has been telling you and talked about some specific examples where we've used cellular target engagement assays to drive our science, specifically looking at targeting dark kinases. So I'd like to start by introducing my lab and kind of how we run things. First of all, the people in the top right hand corner, their names will go by as I'm going through my slides. I will fail to acknowledge them, but trust that they've done all this exciting science and I'm thankful for all of them. The main focus in our lab is to try to address the the fact that there's no good available drugs to treat the causes of neurodegeneration. So we're trying to use our expertise in developing chemical probes and using cellular targeting engagement assays to address areas of interest and areas of need like Alzheimer's disease and ALS. And we're using stem cells and other models to be able to model these diseases and and test our small molecules. And so today specifically, I'm going to tell you about how we fuse the this idea of chemical probes and cellular target engagement assays to get at some ALS important proteins that are implicated in ALS. Specifically you'll hear a little bit about PIC 5 and TTB K1 and two. So since establishing the SGC at UNC in 2020 15, we've really taken on this approach where we have a three prong approach. We're trying to identify chemical starting points for all kinases, develop a mechanism to profile them and then engage with a collaborative network. So firstly we've we've developed thousands of kinase inhibitors and so we've broadly profiled them to to make a database of binding affinity for certain molecules versus a large number of kinases. So we have this really huge database of chemical starting points for various kinases including those that we consider understudied or dark. We have a long standing collaboration with Promega with Matt and his team specifically that where we've worked to develop cellular target engagement essays, Nanobra assays for kinases where there haven't been one to expand the portfolio for of kinases that you can profile using this technology. And because we work on dark kinases, kinases where there's less than 1010 papers in the the public domain, for example, we're very quick to send an e-mail to a collaborator that has published one of those 10 papers and say, hey, we've made this really good chemical tool we've tested in your cell based system. So because we are, we're constantly doing that for, for kinases. We have a huge collaborative network and people who are interested in working with us. So chemical probe is used ubiquitously in the, in the literature. This is the SGC definition of a kinase chemical probe. We have to have biochemical potency of with an IC50 or KD less than 100 nanomolar. We do broad screening and in the Discover X panel or more recently using the Promega K1A2 assay that Matt has talked about the the panel of of many kinase assays, many Nanobra assays. And we're looking for a specific criteria, let's say less than 7 kinases if it's it's screened broadly, but really less than 5 kinases that a compound engages with. We're looking for selectivity within the kinase family versus non homologous kinases. We're looking for a 30 fold window. We do cross screen against other protein families such as GPCR to see if we have reactivity or binding at those those protein targets that also engage ATP for example. We are using that Anabra assay exclusively to to gauge our our engagement with our intracellular targets and we're looking for an IC50 of less than one micromolar and we develop a structurally related negative control That means that it looks like the kinase chemical probe, but it's inactive on its target. So for our kinase inhibitor, that would be maybe methylating the the hinge binding region so that it no longer binds, but the chemo type is the same. And because we're the SGC, everyone can use our molecules. We don't have any restrictions. Please publish, please contact us and get these molecules. And while this is pretty stringent criteria, we've been pretty successful and we've delivered 9 chemical probes that meet this criteria since 2020. And we have others in the pipeline for darker kinases that you'll see hopefully this year. And many of you on the call probably know kinases for cancer. But I'm here to tell you that kinases are important in the brain too. There's kind of more studied repurposing efforts that are going on for kinases like Antor and Lurk 2 and and P38 alpha. But there's also this idea that there's a dark kinome these this 80% of the kinome that hasn't been extensively studied that we know by in small molecules. And maybe these these kinases, if we unlock their potential could present a new direction for neurodegeneration. So I'm going to focus on dark kinases. And the reason that we got interested in illuminating the function of them is because we are funded in 2017 by the NIH to illuminate the druggable genome. What does that mean? While there is 3 arms to this project of one on ion channels, one on GPCR's and one on kinases. Not surprisingly we were funded to work on 162 NIH dominated dark kinases. We could have a whole webinar on what the NIH definition of a dark kinases, but we'll just say there's a list of 162 that we had to go after. And for our part of the project what we needed to do was to identify high quality chemical tools for these kind dark kinases, not probes, but high quality less than 20 kinases inhibited by these molecules, a chemical starting points really and then also to enable these nanobra assays for kinases where they weren't enabled. And so we look to the literature as we always do to see what's out there, what kinases, what, what molecules have been described where there might be kinase off targets that were not really pursued. And we found this compound from Amgen that in the MRC panel that is about 200 or so kinases. There were many of these IDG, these understudied kinases that were inhibited by this chemical starting point. You can see the bar graph there. All of those orange bars are kinases on our list where there was at least a chemical starting point with modest activity on these kinases. So we thought, hey, there's a great chemical starting point, maybe we should diversify this and it'll give us a lead for many of these dark understudied kinases. And so we did that including TTB, K1 and two, which as I prefaced are, are two of our kinases interest. So that was just another interest because these kinases are important in ALS. And so how did we do this? We had to remake the parent, which is up in the top right there. We did a lot of different permutations on this chemical scaffold. We we made the ring that shown with the N123. We made that bigger or smaller, either 6 to 8 member rings. We changed the terminus of the alkyne. We opened up that ring system altogether and then we eliminated the alkyne. We did find a sweet spot in that 7 membered ring on that top left of the box. And so we did make a few more analogs with that 7 membered ring intact. And then once we made our 39 compounds, we sent them for enzymes profiling. We did broad screening at Discover X we and then we of course used the Nanobra assay on kinases of interest to see are these molecules engaging with their targets in cells. And so once we have this compound in hand, we could expand on that MRC panel that was about 200 and and profile it against more than 400 human kinases. This is the largest cell free screening panel that it's a commercially available. And when we introduce this molecule in this assay panel, we saw that it bound about 2021 kinases with less than 10% POC. What that means is that really tight binding at one micromolar to about 20 kinases. This is not what we would define as a chemical probe. It wasn't even good enough for what we would define as a meeting the IDG metric. And so we are hopeful in, in looking further into our data, we could find more and we wanted to see which kinases we could find more. And so here is the results from this larger screening panel where now you're seeing the the IDG kinases once again are orange. Some of the overlap with the MRC panel that I showed you that was public and some new kinases as well came into the mix such as pick 5 MYL K4 and pick 5K2 CS. Some of these kinases that maybe we're not present in the MRC panel and now seem like really good chemical starting points at least for this compound. So and then also we're still working on the packs. You can see those are, are are are represented across Pack 456 as Type 2 packs. And we do have an ongoing project based on the scaffold on the packs. What about the rest of the library? So I told you that chemical starting point was was not a chemical probe. Well, maybe we could find some treasure in the 39 compounds that we made. So here's our chemical starting point. What about for pick 5? Sure enough, what we used, we had broad profiling data to support this compound was pretty selective across the larger kinome when against 403 human kinases. We ran it in the nanobret assay that matched so nicely introduced to you all and we showed it was a very potent binder in the to pick five in cells. And so it met the probe criteria and we published a nice paper and I'll tell you a little bit more about pick five in, in a few minutes. Looking at another member of this library where we've eliminated the alkyne altogether. We had a really nice chemical probe for another lipid kinase. Turns out this the scaffold really likes lipid kinases and also maintained activity on MYLK 4. So we reintroduced it to the field as a dual probe of these two kinases. And then I mentioned already that we had TTV K1 and two of interest. We knew that that that it was a potent hit across the across the number of compounds, but there was no Nanobra assay available. So we had to make it work. And so the first thing we wanted to do was enable that Nanobra assay for TTB Q1 and two, we solved a Co crystal structure of one of our ring open analogs and showed that the indole nitrogen was solvent exposed in a good exit vector for for covalent attachment of a linker and a dye. So that's what we did. You can see that green molecule is the one that's in the green circle. We had put a linker on to Bodipi, we made our nanobret tracer and sure enough we showed that we could enable our Nano Brett assay for TTPK. One and two competed off with the parent compound that is in the green circle and it it worked pretty well at two micromolar to run our compounds, although they were pretty, pretty weak in this assay. We did enable an assay that we can still use today for more potent compounds. And we we had this really nice compound. It's a chemical starting point. It's not a chemical probe. It does inhibit too many kinases, but we still used it in stem cell based studies to study ciliogenesis. And So what we showed is our compound when used at one micromolar phenocopy TTB K2 knockout in that stem cells did not IPS CS did not grow cilia at all. So it was really nice as a chemical tool to show that ATTBK one or two inhibitor when treated for prolonged period of time could phenocopy TTBK 2 knockout. And as I mentioned briefly, we have continued to work on this project and now we have inhibitors that are sub nano or sub micromolar where in the sub 200 nanomolar and then TTBK one and two nanobra assays still using that tracer. So it's still useful to us. We're starting to get some glimpses on whether or not it's selective across the larger chinome. Spoiler alert, we're running it in the K-192 assay in the coming weeks. So we'll know if this broader selectivity is, is this small panel is indicative of the larger panel of kinase assays. And that curve just shows it versus TTPK one and two. That's in that paper down in J Med Chem is it's kind of the gold standard TTPK 1 inhibitor in the in the field right now. And so we have continued a little bit with this tracer. Just as to to Matt's point, this is a, a repurposing of the tracer to see what else it could be useful for these. If we put these things into the tracer DB, other people can do experiments like this where we showed it versus another lipid kinase. We could actually use this tracer at at 8 nanomolar. So we're not at 2 micromolar anymore. We're very, very low concentrations. We show this, it works really well against one lipid kinase competes off with the parent and actually I'm, I'm telling you that lipid kinase 3 actually has the lowest threshold, the lowest assay window of the the three members of the family. We actually get much better signal when we versus 1-2 and three of the same family. And you know, we're interested with it being so potent in this versus this these lipid kinases, could we convert this tracer into a degrader. And so that's what we tried to do is we found an exit vector we can, we can attach a dye, can we recruit an E3? And so we actually do use the same Nanobra assay with that tracer to show binding of the various projects we've made Protect 5, although it's not submicromolar, it still binds an intact cells and actually the protects it potently inhibits the enzymes in an enzymatic assay. When we run the same nanobar assay in permeabilized cells, what we see is we do get a a shift showing that maybe our protects aren't very cell permeable and we are seeing a shift in the IC50 as well. And now we're at sub 100 nanomolar. This protect does engage with the lipid kinase and so now we're seeing does it degrade the lipid kinase? We'll see, stay tuned. So now I'm going to shift for the last little section about pick five and are working on with our Pick 5 pro. So when we got into the pick 5 field, there are there were other good molecules out there. It wasn't, it was not as understudied as some of the kinases we work on. Epilimod is the most advanced of the PIC 5 inhibitors. It's been in humans and in clinical trials for all sort of colitis for rheumatoid arthritis. It's very potent, very selective. It's a great chemical tool. This YM molecule, same. It's it's not as advanced clinically, but it's a really great tool. And there's this other compound you can see in the top that they all share this morpholine that is hinge binding. And you know, there's some good tools out there. And the Pillimod is, is not surprising that the kind of most used molecule. Well, what did we do when we made our molecule? You can see it looks different. It doesn't have that morphaline. It was in the height of the pandemic. So we were actually interested in, in pic 5 inhibition for an antiviral strategy. And so we teamed up with some researchers at UNC. We use the mouse hepatitis virus, which is a mouse coronavirus that's not infective to humans. So we can use BSL too and it's actually N luck labeled. So we're we're reading out viral inhibition of viral replication of this mouse coronavirus and we see that our molecule does really well comparative to a pillow mod and other analogs in the series. One thing that we discovered was there was really great correlation between the Nanobrat Pick 5, IC 50 or PIC 50 and the IC50 from this viral replication assay such that you could draw a straight line between our chemical series. And then some of those open circles are chemicals are inhibitors on that previous slide that are not chemically similar. They're from different scaffolds. So just pick 5 engagement and inhibition of viral replication is very correlative. So moving from the mouse coronavirus, we did, we tagged the SARS COV 2 with Nluck as well and showed a very similar thing and without associated several inhibition of cellular viability. So we saw that our molecule was a really potent inhibitor of viral replication. We also wanted to look at viral entry and we did so by looking at the just at the spike protein and inhibition of the spike protein in the presence of our compound was pretty similar to a pillow mod. We didn't inhibit viral entry in HEC 293TA2 cells and then we did it in Kalu 2 and Keiko 2, which the reviewers felt were more relevant to viral entry. And so we did show inhibition of viral replication and viral entry with our Pick 5 inhibitor. But this is our graphical abstract that we put out with our paper in 2022. And you can see we're showing inhibition of viral replication, viral entry, and actually viral transmission. And this was a, a paper that came out in 2023 from the Kita group and they showed that pick 5 was important in exosome release. So you can see some similarities. Either way, we wanted to, to, to read more about this. And what they showed is that they used a pillow mod and showed that in IPS CS and an animal models of ALS that it was mitigating disease if you inhibited pick 5. This the mechanism by which that it was efficacious was it clearing aggregation prone protein such as TDP 43 or dipeptide repeat proteins and that the end result was extension of survival. So a little bit more about what was in this paper. So for those not familiar with ALS, there's this non coding hexanucleotide expansion in a specific gene C9 or 72 that is the most common genetic cause of ALS when you impart. So when you get patients derived PS CS make them into noted neurons and they have this defect. You can see in the blue line that the viability is impacted the survival of those motor neurons is is is not the same as those that were that that variation has been corrected, which is the black line. So survival is is harmed by the C9 or mutation when you introduce a pellamod. Now we've switched the lines. The red line is the pellamod treated cells that have that, that C9 mutation, they are surviving better than those that don't that that have the mutation. So now we're extending survival by introducing 3 micromolar, a pellamod over 20 days. So we were interested to see, you know, does our chemical scaffold do the same thing? It's, it's chemically different. And sure enough, analogs from our library used the same concentration as a filamod also promote the survival of C9 or 72 ALS relative to controls that were treated with the MSO. So we have a way forward with ALS. We're moving into actually a zebrafish model to explore this a little bit more. One other thing we've done to show the utility of a chemical probe is we've identified a novel role of PIC 5 and pain. So we worked with the the Conna Lab, formerly NYU, now at the University of Florida and showed that we could decrease total sodium currents when with 10 micromolar PIC of our PIC 5 inhibitor and do dorsal root ganglion neurons. We had no impact on calcium or potassium currents. So it's a very specific sodium phenotype. We saw this really interesting and Vitra result and wanted to see if this translates in vivo. And so we had this kind of small model of using a male neuropathic pain model, a spared nerve injury model where we were looking at both tactile and cold allenia. And what we saw in B&C is that the mouse paw withdrawal with was reduced in the presence of our our molecule at 30 megs per gig. And then the bottom two we are showing that in presence of of cold acetone that the mouse paw withdrawals also reduced. So IP administration of our compound reduced, no susceptive behaviors in mice. Now final, I'll wrap up a little bit with our what we've been doing to date of just a few more slides. So we had this really great drug out there that's really potent in cells. I told you that we've made this chemical probe that's that's great. It's about tenfold less active than pick five in the Nanobra assay. It's got a similar half life and it's pretty selective, but we've actually made a second generation probe. Paper should be submitted next week where we've matched the, the, the efficacy of sorry the target engagement of pick five. We're now at the same, the same level of potency, same selectivity and it's got a really long half life. So we've, we've gotten an even better chemical probe and what we wanted to do to show for our paper that it's selective is we want to use that K 192 assay, the panel of Nanobra assays that are in a plate. And we can do this in our lab using the Promega kit. And so we did that and we showed that with 192 kinase assays that we only showed target engagement above 30% of 2 kinases. We set this compound to Matt and his team over in, in per mega and they repeated it and showed a very similar reproducible result. Cyclin, sorry, CDK 10 did come up as well. So we wanted to follow up on that. And then finally, they put, put it into a slightly larger panel of 240 assays. And what you see is really great agreement. We are adding a few lipid kinases because now they're part of the panel. And we would expect that given our our chemical scaffold that loves kinase, lipid kinases, but really good agreement across those these three runs. And this is just a summary of the percent occupancy in the middle. And then we followed up a full curve nanobrat essays to show the IC 50s. And what you're seeing is we actually have 500 fold selectivity in cells and a really good correlation between the percent occupancy that that K-182 or K240 cell panel gives you. And the IC 50 is really from top to bottom, it matches really well. So I've tried to tell you that a lot of important people helped from the start, but here's some of their names again, some of our partners, and for the sake of time, let's answer some questions. Thank you, Allison. Thank you, Matt. I would agree. And maybe in the sake of time, we might skip this polling question and just kick it over to the I'll try to advance this thoughts here. Oopsie, there we go. Try to kick it over here. So feel free to type in some more questions. We just have maybe a few minutes left here. I don't want to go too much past the hour, but maybe the first question to you, Allison. So in your opinion, I, I love the, the comparison now of the occupancy data for K-192 versus the IC 50. So what are what are some of the advantages and disadvantages of using K-192 selectivity profiling system? Versus the biochemical cell free like the the kinome scan that you previously showed, that's one of the questions we have. Yeah, sure. So there's definitely some pluses and minuses to each for sure. You can get more kinases right now because Permega's still building for if you use the cell free panel. But I think that a major disadvantage is the lack of ATP. It's it really makes a difference in the results that you get and the the absence of partner proteins. For example, we had a whole panel of CDKS come up in this, the K 192. They were absent in the Discover XL free panel. So those partner proteins were so essential for binding to those kinases that they would have been missed targets if we didn't have the intact cell with all of the partner proteins present. And actually it's more cost effective to run the pro mega assay than to run the Discover X assay. So it's a pro. Great. Well, thank you for that feedback. That's that's very interesting. Let's see here. Now we've got another question here for for Matt. Have you applied this technology to clinical kinase mutants and have you observed potency shifts or changes in a selectivity for mutant kinases, if you've done that? Yeah. We didn't really get a chance to touch on it today, but there are over 100 clinical mutants that we've developed with, with this technology. And a lot of that has kind of been based on the literature and what people are, you know, querying in terms of other formats that are cell free. And so it's really kind of easy actually to extend your nanobrit T assay into a clinical kinase mutant, provided that the, the tracer still works, right. So there are of course scenarios where the, the clinical mutant will actually impact, it'll be a, you know, let's say a, the drug resistance mutation that will change vulnerability that kinase. So we've had to actually do in some cases is switch tracers into the tracers that circumvent that that resistance mechanism. But we've had really good success. Like I said, it's, it's, it's a very simple thing to evaluate in terms of what we've seen for potency shifts. The it's pretty fascinating results. What we've seen is that in, in some cases, often times what you'll see is that an activating mutation will actually make the kinase more vulnerable to target engagement, likely through sort of stabilizing perhaps an active form of the kinase and relieving auto inhibition of the kinase domain. So we've seen this with Jack too. We've seen this with B RAF, We've seen this another a number of kinases where the activating mutation actually makes the the target more intrinsically vulnerable. And so we, we find that, you know, fascinating, More than happy to elaborate on that if anybody has any interest. Awesome. Maybe you would time for one more question or so let's see we we are interested in evaluating either novel or established in targets in a in a specific cell type of our interest. Is this an involved or time consuming process to optimize and validate ourselves? Is this something we should consider farming out as a fee for service instead? So this is a little bit more of a logistical question on developing either a novel nanobret or using a cell type specific as opposed to 29 threes. Totally. Yeah, that's a great question. We, we love the ability of this assay to be ported into, you know, the, the cell type of interest when that's going to more reflect this sort of pathophysiological state that you want to interrogate. And so we love, we love that question and we'd love to try to solve the problem. And we have some pretty really like very crystal clear examples where you see a massive change in pharmacology when you switch cell types. So the the laborious step, once you once you know that the cell, the new cell type of interest is transactable, I would say the labor of that step is revalidating the Nanobra assay in that new cell type to make sure you're using the tracer in a way that's still quantitative because the tracers characteristics may change in that new cell type. So depending on the number of targets you want to go after, if it's a very small number, chances are that we can guide you through that your own lab. So you can do that, you know, in a few days you can probably get revalidate a few targets in your selling of interest and then have the exact same performance, but now in your new cell type. If it's like a lot of targets or perhaps the cell type is difficult to engineer with an enelocluciferase tagged kinase, in that case, there might be worthy, it might be worth a discussion with our service group that can assist in that. But, you know, it really depends on the complexity that you're, you know, of the system you're trying to go after, how many targets and whether that cell type is easily, you know, you know, genetic, genetically modifiable. So, yeah, happy to happy to follow up on that question, Ward. Yeah. And we do have some, some teachings, existing teachings on how to do this because it's a it's a feature of our sort of our DIY target engagement platform. Is that fair to say, Matt? Yes, exactly that's. So yeah, we can definitely guide, guide more on this. So great. And I know we're a little bit over the hour now. So we what we can do is respond to any questions that have come in that we haven't been able to address yet. And I want to thank everyone for their time today and thank our speakers, Matt and Allison for the great presentations. It was really, really interesting to hear the the exciting sciences that you both have going on. So thank you both and thank you for attending today to the audience. Thanks for your time. Thank you. _1728025525845