We exist to lead people to become radiant disciples of Jesus Christ the gathering can be in your home You can have discipleship in your home. We've had baptisms and people's bathtubs We've had people give communion in their home people in this church Just don't stop like they find a way Radiant City vision is not an end. It's a beginning Well, hey radiant church so good to see you guys Everybody take your Bibles out if you have them and if you don't have it It's gonna come up on the screen if you're at home Let me invite you open your Bibles with this as well And I want you to turn to two scriptures today number one I want you to turn to Matthew 6 and Hebrews chapter 12 is the second scripture This is part four of our be radiant series all part of our radiant city vision Focus and strategy that we're all a part of how many of you were able to watch new rain last weekend or this week Raise your hand if you've watched it wasn't that inspiring man some of the stories in there just Jane and I watched all the way from Florida and We got choked up because I actually did not see it until it aired And I wanted to be surprised by it, but I was just so So moved so touched by it and if you're not registered to watch this week Make sure that you join us That you go and you can go to radiant dot church slash New rain and you can register there. I know it was on the update there But we want to make sure everybody gets an opportunity To see this and as part of that whole building a radiant city vision We're looking at the five core values of really what makes radiant radiant and last week Pastor Caleb brought an incredible message on being family oriented And it was just so powerful and today we're going to be looking at what it means to be kingdom focused Radiant church is a church that is kingdom focused look here at Matthew chapter six beginning in verse 31 Jesus said these words He said therefore do not be anxious saying What shall we eat or what shall we drink or what shall we wear for the gentiles seek after these things And your heavenly father knows that you need them all But seek first The kingdom of god and his righteousness and all of these things will be added to you Now turn over to heber's chapter 12 and look with me at verse 28 Hebrews chapter 12 verse number 28 says therefore Let us be grateful that we are receiving a kingdom That cannot be shaken and let us therefore offer to god acceptable worship with reverence and awe For our god is a consuming fire Our god is a consuming fire in both of these passages and there's a whole Whole sluice scriptures in the new testament where the emphasis and the focus is on this idea Of the kingdom of god This is the message jesus went forth proclaiming when he went from city to city village to village It said he went and he declared the kingdom of god is in your midst What does it mean when we use that idea of the kingdom of god? Well, the word kingdom comes from a greek word besalaia and here's what it means It means the reign or the rule or the realm Of a monarch of a king And so a king's boundaries of his and his borders of his kingdom that's called So and you know, we we would say like state borders. This is the kingdom of michigan Although it's a democracy, uh, it's different than a monarchy, but we understand boundaries Jesus went forth declaring the kingdom or the realm or the rightful rulership of god everywhere that he went And here's what we know. Jesus is the king. How many know that jesus is the ultimate king of kings And lord of lords. So here's what that means. It means wherever the king is That's where the kingdom is And wherever the kingdom is that's where you can find the king And wherever jesus went he went proclaiming the kingdom of god and how did he back that up? He preached it and then he exercised his rightful rule and authority. He cast out demons He healed the sick. He forgave the sinner. He gave hope to the hopeless He multiplied the bread and the loaves and what was he doing? Those were called signs and wonders What was he attesting to? He was declaring he had a rightful rulership over Even nature itself over the ability to forgive sins and the ability to cast out demons Why was that significant?  He walked into villages. He walked into towns He walked into situations where darkness was reigning and ruling And he exercised kingdom authority by saying i'm stronger than you demonic Spirit i'm stronger than you Sickness and infirmity i'm stronger than you death itself And he raised the dead healed the sick cast out demons and he declared i'm doing this to tell you that one Greater than the devil one brighter than darkness and one more alive than even death the grave or even human life itself Is in your midst and i am the messiah the king of kings and the lord of lords He ultimately proved it by going to the cross and then defeating death itself by being raised from the dead And because of that philippines chapter two says god has highly exalted him and given him a name above every name That at the name of jesus every knee will bow and every tongue will confess that jesus christ is lord That's my king So when we talk about the kingdom of god, we're talking about more than just you know god's god and he's over it All he's made it all but he's kind of given us our world now jesus came to declare that in the midst of this broken evil age The kingdom of god has broken in And he is exerting his authority and listen the church Is meant to be an extension of that kingdom The kingdom of god begins in us everybody take your index finger Raise it up turn it towards yourself and point it and say the kingdom of god is within me You got to say it like you actually believe it too. So take your let's try one more to rewind the tape Here we go. Take your index finger.  That's fire insurance So i pray the prayer now i want to live my life the way i want to do because if we have that mentality Here's what we're saying. I like jesus as my savior.  He's over it all i'm not Now this is really really pronounced especially in american christianity and let me tell you why It's because america is the great democratic experiment Now democracy is a great way to govern human governmental affairs. It's the best way to do it But it is not how god exercises his authority God is not a president Jesus is not a prime minister. Jesus is king And what that means is this is not the republic of god. This is the kingdom of god And here's why that's important. It's because you and i live in a democratic republic as a nation But if we're christians our citizenship first is in a kingdom And in our democracy, here's what we get to do. We get to vote on things We vote on who we want to be our leader. We vote on legislation. We vote on laws We vote on rules and if we don't like it then we change it That's democracy. It's power to the people But in a kingdom, that's not how it works In a kingdom, there's a king who's on the throne and it's his decree. It's his law. It's his power It's his rulership and we serve him now. I'm grateful that jesus is a good king He's the best king. He's a generous king, but he's not taking polls about which Legislation we're all excited about he's not finding out what's trending and he's not running for election Psalm chapter two god the father prophetically declared hundreds of years in advance before it ever happened He said i have already set my messiah my king on mount zion and that's jesus Jesus is king and yet we're used to having our say in things. Why is that a bad thing? It's because we if we take our democratic principles and we apply them to biblical christianity We end up with a me centered gospel instead of a jesus-centered gospel And we're called to be kingdom focused people We're called to bring our whole lives into focus around jesus christ and who he is That means We're gonna have to allow some other things to not become our focus If you've ever taken pictures with a camera with a lens that's adjustable that you have to Bring into focus some of you who are really, you know, like the old my so family pictures of my house Were a nightmare in fact i still have trauma and ptsd from them And i had to go to therapy for some of our family pictures because my stepdad had one of those old cameras with the lenses on it And so you had to stand there he had to get the lights and he's like hold still and he's doing all the adjustments It took like 10 hours and then all of a sudden he'd snap picture and he's like, okay Let's do it all over again.  It was terrible Because if it was back before you had previews where you could actually see the picture You were guessing what it was going to look like you had to learn to bring it into focus How many of you remember the days where you had those disposable cameras where you would like, you know Go on a vacation take pictures of everything right and you didn't know what was on it You had to take it to mires or to walgreens drop it off and then 24 hours later go back And then you paid 25 dollars to get it done quick and then you got and then you go through your pictures It was like i can't see a single thing. They're all out of focus Anybody had that experience so now you got all these negatives and you got all these pictures And it's like i think that's i that might beat you in there. It's out of focus Here's a problem What if we get to the end of our lives? And we look at The film of what we did with our life and we come to realize We were living our lives focused on the wrong things And the kingdom of god and the eternal things were out of focus And so therefore we weren't able to see them There wasn't any fruit in our lives There wasn't any glory unto god and it was because we Were distracted and focused on all the wrong things That in the moment we think are really important In the moment we think it's about me in a moment We think it's about the things of this world that are temporal and subject to change But in eternity it's only the kingdom of god that is going to remain That's what hebrus is talking about it says in hebrus chapter 12 Therefore everything god says yet once more in heaven and earth i'm going to shake everything that can be shaken How many know that we've been experiencing a shaking Of everything that we thought was stable everything that we put our trust in it has been shaken But god says i'm not doing it to destroy i'm doing it to unveil he says i'm shaking everything that can be shaken so that That which cannot be shaken remains What is that it's the kingdom of god So that when this whole thing is done when human history is done What is going to remain is the unshakable kingdom of god and the bible says that that actually is our inheritance that kingdom Because here's the good news not only is jesus king But he's made us royalty And he's given us an inheritance called the kingdom of god But in order to inherit it we have to be focused on it Jesus said seek first The kingdom of god The reign of god the way god does things The rule of god the power the presence of god seek that what does that mean? It means focus on it make it the primary focus Make it the main thing point your lens at Jesus at the kingdom of god and god's way of doing things and what he is doing in the earth From an eternal perspective make that your focus on your screen Tap that so that everything else around it might be blurry, but that is going to be crystal clear and obvious in your life And then he says and all these other things will be added under you You see god's god's a god that if we'll put him first And we'll focus our life on him. He's a good provider to take care of everything else But the world the way of the world is put our focus on everything else put our focus on our job Put our focus on our fitness put our focus on politics put our focus on money put our focus on You know, we're a selfie generation on our social media. So here's me I'm making another piece of avocado test and in here's it's like We have to put our focus on Jesus On the kingdom of god So How do we do that? How how do we bring our life into focus around Jesus? Here are some declarations. I believe that will help us Bring our life into focus and I'm making I put these in a form of declarations because I want us to be able to say these And to state these over our lives number one is this Declaration number one is Jesus is my king and I live for his approval alone Come on. I want somebody to say this with me say with me say Jesus is my king And I live for his approval alone. Doesn't that just feel good to say? My spirit jumps on the inside of me when I say that Because it's life think about these statements acts chapter 17 the early church in the formative Years and months where the church is beginning to move out and power and strength Here's what the world says about the church in acts 17 verse 7 it says these men who have turned the world upside down have now come here also And Jason has received them Jason was one of the leaders of the synagogue Jason has received them And look at this it says and they are all acting against the decrees of Caesar Saying that there is another king Jesus The church was accused Of worshiping Jesus not just as savior not just as a religious icon not just as an avatar They were going forth and saying no there is another kingdom that we are aligned with and there is a king and his name is Jesus You do not know how revolutionary that statement was You say Jesus is king and american it's like oh, yeah, that's really nice I mean, you know kanya came out with an album by that title as Jesus is king out composure You got this whole thing where Jesus is king and everybody's fine with that You said Jesus is king in rome and you got your head cut off Because in rome under the emperor Whether it was nero whether it was vespacean whether it was titus whether it was demission Whether it was caligula any of the roman emperors If you declared yourself to be another king or to be a servant of another king It was punishable by death and this is what christians were going around Declaring the kingdom of god is here just like jesus did just like the apostles did and they said no There's another king and his name is jesus. They tried to censor They tried to censor the the early church The jewish leaders tried to censor them rome tried to censor them. We would say they had cancel culture Where they were trying to tell them you can't preach in that name anymore But they did it boldly They said no you can't proclaim another king, but they did You know, we're living in a day and age cancel culture where it's like if you don't say the right thing Then we're gonna cancel you on social media. I'll tell you what I'd rather be canceled in history than canceled in eternity I do not like it here or there. I do not like it anywhere Let the hearer understand John's 18 jesus answered pilot when he asked him Are you a king and jesus said my kingdom is not of this world If my kingdom were of this world my servants would be fighting That I might not be delivered over to the jews, but my kingdom is not from this world Then pilot said to him, so you are a king and jesus answered you say that I am a king For this purpose I was born and for this purpose I've come into the world to bear witness to the truth Everyone who is of the truth listens to my voice the definition of jesus being your king Is that you listen to his voice? That when jesus speaks you hear him jesus said i'm the good shepherd my sheep hear my voice here's a question Who's approval are you living for?  Because this is what it comes down to Jesus said if you hear my truth Then you understand why i've come into the world i've come into the world to bear witness to the truth paul Understood this in galatians 110. This is a scripture that i've been thinking and chewing on and meditating on for about two years It's galatians 110 he says for am i now seeking the approval of man Think about that or of god Or am i trying to please man For if i were still trying to please man i could not Would not Be a servant of christ do you understand what paul's saying is you can't serve people And christ You can't do it In this culture what they're trying to train us to do is to self-censor Put a filter on you they're trying to break People's Mentalities, especially people of faith. They're trying to break our spirit like you break a horse You walk it around until it gives up and then it finally takes the bitten in the bridle and it will do whatever you tell it to do But it started off as a wild stallion who went where it wanted to go Ran it would not submit and till the spirit gets broken And then it will do whatever you tell to do that's what the spirit of this age is trying to do to the church It's trying to get christians to self-cancel Come on dial down your commitment to jesus dial down heaven and hell don't use language about repentance Don't over quote the bible just use a little bit more self-help make it more generic keep it to yourself Pray quietly in the corner and you were created to be domesticated The holy spirit on the inside of you was not created to be was not put on the inside of you for you to be Domesticated you are wild at heart.  Let me tell you something And that day we'll bow the knee to jesus.  I'm an ambassador here One of my favorite albums. I grew up into 80s Listened to a lot of christian contemporary music CCM music and one of my favorite bands was Petra anybody remember that Old band they had an album called not of this world We are strangers. We are aliens. We are not of this world And I remember even as a teenager 13 14 15 years old when that album came out just the revelation and the reality of that statement That I'm not from this world. It's not just a cute little metaphor. It's reality My citizenship is in heaven and just thinking and meditating on that that yes, I live on this planet, but I'm here on mission This is not my home Until jesus returns and makes it my home But right now I am actually an ambassador for christ a missionary if you will An ambassador is a representative of the head of the state of another kingdom And the bible says you as a born-again Child of god are an ambassador from the kingdom of heaven who's been sent to the earth to enforce And to legislate kingdom of god legislation Think about that's what jesus meant when he said when you pray pray your kingdom come and your Your will who's will god's will king jesus's will Philippians chapter 3 verse 20 paul's writing to believers at filipi and he says your citizenship Is in heaven and from it we await a savior the lord jesus christ This is interesting that paul uses this language in Philippians because filipians was a letter that paul wrote to the church at a town called filipi in asia minor filipi Was one of the wealthiest cities in all of asia minor and the reason for that is it was named after filip Who was the father of alexander the great how many have ever heard of alexander the gray? He conquered most of the known world established the macedonian greek empire His father's name was filip and filip was an incredible military general They named the city after him when realm became the most powerful empire They kept it in place because of the great debt to culture language and military Education that they learned from the greeks so they honored it by making this city filipi A city of high status so all retired military leaders all retired politicians and members of senate Had the retirement by moving to filipi where they were given tax-free status They were elevated to a very high wealthy place of honor in the city In fact, this was called the crown jewel of the roman empire And so paul's writing to believers who are living in this city that has great wealth has great status it has great luxuries it has Everything that you could possibly want and because if you were a citizen of filipi It was like you had it made and paul writes to christians there and he says i want to remind you Your citizenship is not in filipi It's in heaven your citizenship is in heaven you have a higher citizenship If paul were writing to the american church today, he would say to us your citizenship primarily Is in the kingdom of god Yes, you live in america you're a citizen there, but your eternal citizenship is in heaven Second carinthians 5 verse 20 paul writes therefore we are ambassadors for christ god Making his appeal through us. We implore you on behalf of christ be reconciled to god.   Did you notice that?    It says you are a chosen race Listen in the body of christ, there is neither male nor female. There is neither jew nor greek There is neither circumcised uncircumcised. There is not black. There is not white in jesus. We are all one There is one race. It is the new race in christ jesus the new humanity The new humanity if we want to see race and the issues of race that right now are destroying our culture That people are battling over it's not going to be this problem Is not going to be solved by the best fallen human minds coming together and building a better toolbox This is going to be solved by all of us coming to the foot of the cross Receiving grace and forgiveness and looking each other in the eye loving one another the way jesus has loving us Recognizing the beauty in the diversity and instead of fighting for our rights laying down our rights Taking up our cross and letting the world see unity in the name of jesus And he needs to start in the church We need to get over ourselves Oh, all right Number three declaration the gospel is my pledge and my power I'm a kingdom focused man. We're a kingdom focused church and the gospel of jesus christ is my pledge and my power paul writes in romans 1 verse 16 for i am not ashamed of the gospel For it is the power of god for salvation to everyone who believes For the jew first and also for the greek for in it the righteousness of god Is revealed from faith to faith as it is written the righteous shall live by faith paul says i am not ashamed of the gospel I am not ashamed of the gospel why because it is the power of god unto salvation You know what the gospel is mankind sinners We've sinned we've rebelled against god and if god were just a fair god god would have just And given us what we deserve But i'm grateful that we don't serve a god who's fair we serve a god who's merciful It always cracks me up when people say well, it's just not fair that bad things happen. I'm like, do you really want god to be fair? Do you really want god to give you what you deserve? No, you don't pop quiz Here's the answer. No, you don't know what we want is we want mercy from god And the gospel says you are a sinner All of us have sinned all of us have fallen short of the glory of god We were rebelled against a good god who made everything perfect We were deceived fell into sin god could have left it at that and he would have been perfectly just He doesn't need anything from us. He's eternally self-sufficient in triunity father son in holy spirit He's worshiped by angels, but yet god in his mercy Took on flesh Came in the form of a man Died on the cross for the sins of the world the one who knew no sin took on sin So that you and i who all we knew was sin could be made the righteousness of god and jesus christ He was raised from the dead you can't earn it He offers it as a free gift to whoever will believe on it and when you believe on it Your name is written in the lambs book of life You will live forever and eternally as one of his children co-heirs with christ Reigning and rule over all the cosmos in the universe someday you will judge angels and you get it all for free Jesus paid it all all to him i owe sin had left to crimson stain. He's washed it white as snow That's the power of god unto salvation And i'm not ashamed of that gospel because it doesn't mean god is an equal opportunity savior You want into the kingdom of god? It's absolutely free But it'll cost you everything Because you can't take your old life Through the narrow gate Jesus says repent And believe What does repent mean? It means i have to lay down all other allegiances I have to lay down all privileges I have to lay down all preferences I have to lay down all pleasure I have to lay down all identities I lay it all down and i die to myself And in that moment by his spirit he resurrects me And i'm born again by the spirit of god a child of god heaven lives on the inside of me And i'm wonderfully saved This is the gospel of jesus christ and it is my pledge And it is the power of god unto salvation This is what we're called to preach Here Around the corner and around the world Jesus sent us out for one purpose To preach the gospel to the whole world And when we say radiant is a kingdom focused church We say look we are focused on living our lives in submission to his lordship his kingship And we're saying lord we will give our lives For your approval whether anybody else ever approves of us or not And we will preach the gospel boldly courageously Strongly clearly without compromise and without watering it down Because it is the only hope that we have It's the only hope church it's the only hope we have it's the only hope for the world And as imperfect as we all are the gospel is perfect The word of god is perfect Jesus is perfect And he's calling all of us to put our lives into focus around him Do you stand with me even at home? Would you just stand with me?  There are a lot of things that we could easily put our focus on There are a lot of things individually we could put our focus on But in the end They're only mirages In the end The only thing that matters Is that jesus is king His kingdom has come And his kingdom is coming. It's here in part But jesus is coming back and when he comes his kingdom will fully come Do you know him today? Do you know jesus is king? I'm not talking about do you know jesus as the cartoon figure? I'm not talking about do you know jesus the religious icon? I'm not talking about have you ever said a prayer? I'm talking about have you You Submitted your life to the lordship kingship of jesus and all of the implications of that elliot Who was a missionary to ecuador? Some 50 years ago ended up as a martyr Made a statement. He said he is no fool Who would lose everything? He can't keep In order to gain what he could never lose This is the gospel of jesus christ This life is like sand That we hold in our hands and the tighter we squeeze it The more of it slips through the cracks of our finger till there's nothing left But if we put our hands To the plow of the kingdom If we offer our lives to the one who gave his life for us None Shall be lost Jesus said those that you've given me father none of them have been lost I want you to bow your heads with me right now if you would I believe in this room. The holy spirit is moving up and down rows up and down aisles and there are Individuals you know in your heart of hearts you have lived as a renegade against king jesus You've lived for yourself Jesus has been a good little add-on accessory to your already good life But you've never really bowed the knee and said jesus your way is my way Maybe you're here today and You know you're lost in your sins and you're an enemy of god You can't earn your way out of it today. He offers you Forgiveness today.  I repent. I'm no longer gonna live for myself.  I want you to be my lord Forgive me. I want to come into the kingdom of god I want to come in to your kingdom The only way is through jesus I'm gonna say a prayer in a moment But if you today are repenting of your sin and saying jesus forgive me. I want to get right with you I want to be forgiven.  Thank you.  Thank you god bless you If you not raise your hand raise it now Say jesus your lord and i'm submitting to you. I've never done it before but today i'm doing it Even online if that's you God sees it just raise your hand You can put your hands down. Thank you.  Can we just celebrate that?


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What's up everybody, Jason here for jazzy for jazzy's casebricks.com. We just sold out jazzy's for the collectors repack This is a one box break random teams number nine plus. We have a hit list promo guys So 30 to 37 total spots everybody gets two random teams From the list below remember this product is made by us here at jazzy's it includes 25 or 30 cards per box There's a lot of greater cards rookies, etc. Nothing too crazy, but a fun break for collectors who would like to add Some nice hits to their collection that include of course some greater cards as BGS PSA and SGC And again guys there is a hit list promo set at the end of every break including this one We'll take all the hit list customer names random mines them and the top two names each will win $50 So if you have three spots in the break and two go hit lists two spots We'll get you we'll get you'll still get two entries I should say and again since there is only 12 boxes produced guys most spots will only have a few hit possibilities hit possibilities This may run out for a team before the end of the box print run and again We go by the team on the card for any unlicensed or college cards We go by the label that we put on the back of the card and again guys We also have a promo too as well for customers that just buy into this specifically So if you go here, you'll read about it right here And again, we'll take all the customer names from 12 cases randomize them and the top Name gets the Josh Allen sign bills Jersey and then two through five get $75 and break credit And let me actually just paste in the promo right now for you guys so you guys can see it I'm gonna paste in number nine start right here and And here by Start number ten Perfect Alright, so again, we doubled up your names guys So you should have two random teams and then here's all the Names and teams here Let's roll it. We got ourselves a six and a five eleven times. Good. Look one two three four five six seven eight nine ten times Rosario down to Tom and Eleven times now here one two three four five six seven eight nine ten Eleven Mavericks down to the athletics All right, so again, it all matches up there What I'm gonna do guys is I'm gonna actually oftentimes my customer name I think it's a little bit easier for you guys to see all of your teams you guys got So let me do that really quick. Let me actually type in the Title of the sheet Hi, so Aaron You have Raven 76ers Adam Cumberman Cubs Padres Jaguars and deep acts Alfred last Balmogia the spurs and calves Barry Roberts you have the Kings Clippers Bear Steelers Corey Christian You have the Pistons Bucks Craig with the Giants magic and Daniel Katz with the Orioles heat tigers and Hawks David Duffy with the Bengals and other not listed Edwin hack with the Nationals Astros Ethan with the Reds and Packers Greg with the Braves and Buccaneers Howard with the Chargers Chief Suns Thunder John with the Brewers next Joshua with the Mets Hornets Keith with the Jets Royals Matt With the Lakers Rangers Michael Pacifico with the Mariners Red Sox the next over You have the Texans Saints watching football team Eagles Teebles Grizzlies all over the St. Louis Cardinals and Raiders PJ with the Rays and New Orleans Pelicans Rosario with the Mavericks and Twins Ryan H with the Giants Yankees Angels San Francisco Giants That is Yankees Angels Phillies Cowboys Bills Patriots Marlins Indians and Toronto Blue Jays Sarah with the Colts Dodgers Simon with the Seahawks Chicago White Sox tj with the Nuggets and Rockies And Tom with the Warriors Broncos Bulls and Athletics Hey, you never know man. There could be some nice stuff in here. Maybe some Jamal Murray rookies For the Nuggets some old-school Carmelo Anthony, maybe we'll see brother. You never know man All right, and I don't I don't think there's any trades at least not that I know of right no trades I don't think anybody's ever traded in this All right, thank you guys So now I'm gonna alphabetize it back to the teams And again, um, I'll be highlighting or you know, bolding the teams and hit that hit And then of course the others Uh, we'll we'll copy and paste for the hitless promo. We'll give away 50 bucks to two winners All right, I believe uh, two winners get 50 bucks It's kind of hard to see but you already seen your teams. This is just for the purpose of sorting and shipping All right, and again, we're just random cases. They're numbered one through 12 We just got case two just randomly chose it and a good luck guys. So again 25 to 30 hits So for the most part most Most teams should have something This one feels like it's all graded cards. All right guys, I think we got some good stuff 1998 ultra Peyton Manning PSA nine and again, that's gonna go to the Colts All right, nice Indianapolis Colts Sarah with that one We have a Eric Decker rookie nine five from bowman sterling 2010 for the Denver Broncos So Broncos That's tom We have for the Mets mark vientos bowman's first chrome refractor nine five ten numbered to 4.99 New york Mets josh with that one We have a Gavin Lux tops 2030 PSA 10 rookie for the Dodgers Sarah with that one We have Miko Ranton in upper deck young gun PSA nine So I don't think there was any hockey in this so that'd probably be under the other right So that'd be other not listed David Duffy Jacob eason Colts Colts for what is it called? Fort up. All right, I can't be desiring right there. That's probably like a Colts saying though Uh 15 out of 50 from encased football Jacob eason Indianapolis Colts We've got that one right there Shane Bieber the Biebs tops update 2018 Cleveland Indians PSA 10 Uh Ryan with that one Showtime Shohei Otani bowman first paper PSA 10 Tony's just been killing it Ryan with the angels We have a bowman chrome Bryce terrain mega box purple refractor numbered to 250 that's a PSA 10 Milwaukee Brewers John with that one We have oops. Sorry Kyle Lewis tops Uh rookie PSA 10 Seattle Mariners Michael Pacifico We have a tops update salute ronald akunya jr. Rookie PSA nine Braves going to greg PSA nine Juan soto rookie update Washington nationals Edwin hack We got an RJ Barrett donors optic graded rookie PSA nine for the Knicks John with that one We have drew breeze tops chrome 2007 PSA nine Saints going to nick Stover We have a blue velocity Rui hachimura donors optic rated rookie PSA 10 Washington wizards Are the wizards not on this list? All right, no wizards. So that'll go to the all other I'm about a ja marant PSA 10 contenders lottery ticket Memphis grizzlies It's been a Nick Stover the honors card stuck to this Oh, is this on purpose? I got stuck. That was weird Uh, Larry bird silver prism PSA 10 for the Celtics And I don't think the Celtics were part of this either Yeah, all other as well Then we have a two out of six for the Tampa Bay Buccaneers Mike Ulstad Looks like a recollection collection rookie from prestige That is Tampa Bay Buccaneers That is Greg We have a Paddy Mahomes field level base Chiefs can city chiefs Howard of that one Got a few more guys Mike Trout tops holiday jumping star PSA 10 for the Angels Ryan again 2018 or 19 tops Juan Soto PSA 10 Washington Nationals. We already got that one as well we have four Oakland Matt Chapman two-time platinum glove award winner 30 out of 50 Oakland athletics on a tom All right a few more guys We have a Jason Dominguez paper prospects PSA 10 2020 Bowman Yankees on the board going to Ryan Last couple right here guys We have Nick Modrigal for the White Sox that is a out of 50 Bowman's best autograph Chicago White Sox one of Simon We have a winter rookie Zion Williamson for the Pelicans PSA 10. That's a sweet one right there PJ there you go on the board And last but not least PSA 10 2019 tops rookie Fernando Tatis junior for the Padres Adam Cuperman So there you go. Boom majority of these were definitely great five and 26 So there you go guys appreciate it And again, if you went hit list unfortunately, I know it sucks But you should have a chance to win that jersey and some extra break credit and then in this break alone You're gonna have a chance to win backs of money 50 bucks two codes to customers hundred bucks So Let's quickly See who won hit list really quick Yeah All right, so there you go. I should be everybody there Roll the dice and it's a two and a four six times one two three four five All right, six times six times top two Joshua and Keith Kelly So I'll send you guys 50 bucks each and I appreciate you guys This was uh number nine number 10s in the store jazzy's case breaks.com


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I will bring you that in a subsequent bulletins, two persons have been arrested by the Commander-General of Special Intelligence Squad of the Nigeria Security and Civil Defence Corps NSCDC at a dump site used for storing adulterated petroleum products around Benin Agbo bypass area of the state. Spokesperson of NSCDC Edo Command said that the premises were uncovered following a top-off. The suspects, however, denied any involvement in the act and claimed that they were carrying out their legitimate business. His Special Intelligence Squad in conjunction with the anti-vander units of the NSCDC Edo State Command has uncovered this hideout where criminals hide or treat petroleum products to sell to citizens. As you have seen or as you can see, the NSCDC is not leaving any stone unturned to chase or to smoke out economic saboteurs out of the state and out of the country in general. I want to advise women and citizens to give us credible information so that this country will be a safe home for innocent citizens. Hello, hope you enjoyed the news. Please do subscribe to our YouTube channel and don't forget to hit the notification button so you get notified about fresh news updates.


So it is my real honor to introduce Professor Kevan Schochat as the 2015 David E. Green Memorial Lecturer. So David Green was a famous enzymologist and mitochondrial biologist at the Enzyme Institute here at UW Madison, and this lecture is endowed by his former postdocs and associates. So Professor Schochat comes to us in the Bay Area where he is a professor of chemistry at UC Berkeley and a professor and chair of cellular and molecular pharmacology at UCSF. So Kevan graduated from Reed College in Portland in 1986 and then did his graduate work with Peter Schultz at UC Berkeley where he studied new synthetic routes to catalytic antibodies. Then after a short postdoctoral stint in morph immunology with Christopher Goodnow then at Stanford, Kevan began his independent career as an assistant professor in the Department of Chemistry and Molecular Biology at Princeton in 1994. So it was really soon after he started his independent career that Kevan and his group made the observation that they could re-engineer protein kinases by making select mutations in the kinase active sites that allow these kinases to be able to accept unnatural nucleotides such as bulky ATP derivatives and that these same mutations could allow custom inhibition of these kinases with inhibitors that wouldn't inhibit normal wild type kinases. So these tools became very powerful means to identify the functions and the direct substrates of a wide range of protein and lipid kinases and his group has now done this for more than 100 kinases from a diverse array of organisms. So Kevan's group continues to use these tools and to develop a range of other chemical tools to study the specific functions of kinases and other signaling molecules like GTPases. With the goal of identifying which of these signaling molecules could be really effective targets for therapeutic intervention for diseases like cancer and other immune diseases. So Kevan's work really has been exceptionally influential in the field of signal transduction and for what she has been recognized with a wide range of honors and awards which include simultaneous, being elected simultaneously as both a Pew and a Searle scholar. So I didn't even think it was possible. A young investigator award from the Protein Society in Alfred P. Sloan Fellowship. In 2005 he was elected as an investigator of the Howard Hughes Medical Institute and in 2010 he was elected to the National Academy of Sciences. So with that please join me in welcoming Professor Kevan Shokat. That very nice introduction. I'm going to have to really improve my introductions because that was great with no notes. I usually have like page after page to try to remember. That was very, very kind. Very nice. Thank you for that. Well, it's honored to be here for the lecture and I'm looking forward to telling you about a number of projects in the lab. The central theme as David mentioned in the lab right now is to really understand how we can find the best drug targets to treat diseases like cancer and then develop tailor-made chemistry to exploit those drug targets to maximal therapeutic benefit. And what I want to do is sort of give you a very start simple with what the goal is in all therapeutics and how signal transaction pathways ideally work when they're simple, how the drugs work, and then sort of un-peel the layer of when things get more complex, how biology sort of thwarts some of those approaches we take and then how we can then come back with some new chemistry to try to deal with those challenges. So if I start with very, very simple premise that what we're looking for are targets that show themselves to be particularly important in the cancer cell but are dispensable in normal cells, the best way to find those are the sites of mutation that drive cancer, the driver oncogenes. And if we identify that they occur in a signal transaction protein like a kinase, one of the prototypes, being an example of a kinase called BCR-ABL translocated fusion that hyper-activates the kinase, then we know what to do chemically, we know to put a small molecule in the ATP site and compete for ATP in that kinase and we know that since the mutation drives this pathway in the absence of the normal input signals that when we deprive the cell of that signal that hopefully the cell is uniquely dependent upon that pathway but normal cells can tolerate inhibition of the proto-oncogene, the one that's not mutated, and the rest of the body and we can get a large therapeutic benefit. And many, many drugs that we make rely on that unique dependence that when you get the mutation the pathway becomes, the cells become addicted to this pathway and then starving them of that pathway with the small molecule drug will send them off into apoptosis and then we have a therapeutic benefit. So what I'll like to then introduce you to a little bit more of the complexity of the signal transduction pathways and what are the components I'll tell you about and that really comes from the importance of post-translational modifications and switching and really the importance of phosphate. Many, many of the signal transduction proteins that we know about rely on a unique phosphate group. The GTPases are sort of very poor enzymes and they're really switched on and off by the state of the nucleotide or the substrate. When they bind GTP, their conformation is capable of binding so-called effector proteins just capable of doing protein-protein interactions that then send downstream signals. When the intrinsic GTPase is active and that gamma phosphate is hydrolyzed, when bound to the GDP, the conformation of the GTPase adopts a state that's really incompetent for binding effectors and it's in the off state. And then the protein kinases are more traditional enzymes and we can see that they basically just transfer phosphate to serine 3 and inner tyrosine residues and then that creates an on state that sends the signal in its catalytic here so we can get normal enzyme turnover and signal transduction. What's great about signal transduction proteins is that evolution has used them over and over again and they can build very, very quickly modular signaling pathways. So I've chosen these two examples because the pathway that I'll focus on is the RAS, RAF-MEC-ERC pathway. So in one pathway, we have top of the cascade is a GTPase and then three different kinases that lie downstream and in that pathway, you can see that they are built up in a very simple way. When the GDP is bound to the GTPase, it's not competent to bind the cytosolic kinase, which has a domain on it that is capable of binding to the GTPase when it's bound to the GTP and then when the kinase gets recruited to the membrane, its activity increases by dimerization and then it phosphorylates a downstream substrate which itself is a kinase and starts the cascade. So before I get into the names of the proteins, I just wanted to introduce you to the biochemical functions and how they fit together and we'll get into that in a second but the other thing to remember and I don't have this sort of throughout the talk but one thing I'd like to remember is how fast these signal transduction pathways can modulate themselves and reorient and associate with the membrane and my favorite movie of that is a movie from the 1950s of a neutrophil chasing a Staph aureus bacteria through a sea of red blood cells and I'm sure many of you have seen this movie, I never get tired of it because it's just amazing to see this anthropomorphic sort of hunting and killing behavior of a neutrophil with the Staph aureus bacteria there in that black pivoting cell and then these are the red blood cells that get pushed aside and there's a tripeptide that emanates from the bacteria, there's a GPCR in the neutrophil that recognizes the tripeptide, setting up a whole cascade of kinases to turn left here, mobilize actin, degrade the bacteria with reactive oxygen species. So the dynamic characteristic of signaling is also something that is of course very, very important in signaling and probably why it's been so rapidly evolved and expanded in evolution. So coming now back to cancer and thinking about how this simple pathway that I told you about, how important it is in cancer, it's amazing that one third of all cancer is caused by mutations in the set of two proteins. At one position of K-RAS, G12, the glycine 12 position, and then in a valine 600 position in the immediate downstream kinase B-RAF, this causes about 8% of cancer, this is about 20% of cancer. So it's really remarkable, these two mutations never co-occur. So once you get one mutation, you never get the other mutually exclusive. So a very clean linear pathway. And over the last 10 years, we've had fantastic developments in drugs to treat this group of patients that have the B-RAF mutation. But the patients that have K-RAS mutations, even though it's in the same pathway and we've developed drugs that work at this point and below, the K-RAS patients do not really show much benefit when we use these drugs. So this is one of the first points I wanted to make, which is that these linear pathways give us the idea that they work like metabolic pathways, and that if we know the pathway and there's a mutation at any point in the pathway, we could make an inhibitor at any downstream point and get the same therapeutic benefit. But that has been over and over shown not to be true, which is very, very depressing because we have fantastic chemical tools, but they just don't seem to work. And it's probably because the drugs don't selectively inhibit just the mutant protein in the tumor cell. They also inhibit the proto-oncogene in the rest of the body, and we can't dose high enough to suppress the oncogenic signaling while still having the patient tolerate the therapeutic toxicity in the rest of the body. So we just don't have that therapeutic index, first of all because we don't have drugs that exclusively work on the mutant oncogene. And also the pathways are more complex, which I'm going to tell you about in a second than we first realized. So one of the most surprising demonstrations of this fact that even if we had a drug that worked downstream of a known oncogene and we knew that the linear pathway was present, the drug doesn't work is the following situation. Early on, people had developed inhibitors of RAF kinases, MEK kinases, and now ERC kinases, but these two had been put into many, many clinical trials for patients that had either the RAS mutations or RAF mutations. And the one drug that worked stunningly well is this drug, PLX-4032, or Vemerafinib, but it only worked in patients that had the mutant B-RAF-V600E mutation. A clue about why it wasn't working anywhere else came from an unexpected place, which is, well, let me show you that it works first. Very, very important. 81% of the patients on the first trial had very positive scans throughout their body of metastatic melanoma. After several cycles of the drug, you can see the scans show much less intense signaling after the drug has been given, so the tumors were going quiet, disappearing, and the simple cell readout was very obvious. If you start with the B-RAF-600 mutation, you had phosphomek and phospho-erc, and the downstream kinases were active, phosphorylation was present.  Everything's good. A very big surprise, though, came about when about 30% of the patients on the Phase 1 trial developed a second different kind of cancer while they were on the drug. The secondary squamous cell cancer was not life-threatening, so the patients stayed on the drug to treat their lethal melanoma that was growing. A very interesting observation came about that when patients that were taken off the drug for a different reason, those other tumors disappeared. So here's a tumor that is induced by the drug and its maintenance, it requires the drug. My collaborator on this project, Neil Rosen, immediately tried to develop a cell culture model of this phenomenon and found that there could be, so the idea is there's some paradoxical activation of the signaling pathway by this RAF kinase inhibitor. So he'd like to be able to have a cell line that shows that, so he found that if you had a BRAF wild-type cell, but one with a KRAS mutant in it, as soon as you started, there was a little bit of basal signaling at ERC and MEK because the KRAS is mutated, but then when you add the RAF inhibitor, there's a massive increase in phosphorylation. And this really intrigued us because we're always thinking about enzyme inhibitors inhibiting enzyme activity. That is like first day of class and yet here we're seeing that an enzyme inhibitor is activating enzyme activity. I was just stunned by this and so we cut a long story short and take you through all the logic that we use to get here. I just want to show you the model and it was supported by several other papers as well that came out around the same time, which is the following, that when an inhibitor binds to a kinase, it doesn't just inhibit the kinase activity, but it changes the confirmation of the kinase and that conformational change can have a paradoxical effect. So I told you that when RAS is active, it causes the dimerization and activation of the RAF kinase. It's not shown here as a dimer. Even though this is such an important pathway, we don't know exactly how dimerization happens, but we know it goes from a monomer to a dimer. And so when the first RAF inhibitor binds, it changes the confirmation to mimic the dimer and then pushes the equilibrium to the dimer and then in the half occupied dimeric state, one programmer has the drug in it, the other programmer is very active and that leads to increase in phosphorylation. We tested this with many ways, but suffice it to say that is basically a function of the kinase drug complex that just nobody ever anticipated. So that's the one example to sort of tell you that even though we might be operating just immediately downstream of where there's a mutation in this pathway, we can have a completely unanticipated effect. That's why it's much, much more important we know now, after 10, 15, 20 years, to target as close as we can to where the oncogenic mutation is. Okay, so all the frustrations, let me tell you now a couple of things that we've been working on and give you a little bit more background about targeting RAS tumors. So with all the frustration before I get there, with all the frustrations of targeting oncogenes and not having an approved drug, one thing that has always been true is that if we have a very good inhibitor of the mutant kinase, we always get an approved drug. We always have therapeutic benefit. And this has just proven over and over again. I still know of no example where this has failed. So that is one important guiding principle. These are all approved drugs. These are drugs that are in trials. You can see that the cumulative frequency of mutation, the first one that was approved, was very, very low frequency, but we're now applying this logic to more and more frequent mutations. But one that we don't have an inhibitor of in the clinic is the KRAS mutation, and it's the most frequent mutation and we just don't even have any drugs. So I'm going to get to strategies to treat KRAS mutant tumors. And this goes back, way back to the discovery of RAS and the oncogenes from viruses where in the 60s, people were cloning retroviruses, finding genes that caused transformation. Then sort of 15, 20 years later, people found mutations in human cancer cells that were in the exact same orthologs or the proto-oncogenes that were causing cancer. We had many, many very, very important biochemical mechanisms. They're bound to GTP. They hydrolyzed GTP. They have activators of their hydrolysis. And basically what I wanted to summarize here is just to show you how long people have been working on this and where one biochemical mechanism was found for the kinases, such as phosphorylation, that led to 25 now approved kinase inhibitors. So that is a very drug-able pocket and a place where mutations occur very frequently. Whereas the RAS proteins, we've had only one thing that has made it to the clinic in really these 30 or 40 years. And that is based on the farnesolation that's required of RAS. And so when people realize that RAS had to be the membrane and it got to the membrane by farnesolation, they block the farnesol transferase to try to prevent it getting from the membrane. Beautiful strategy. Worked in mice, and I'll show you some of that data. But ultimately it failed in phase three clinical trials for a reason I'll show you in a second. So this is, we were very close, but for one small detail, it has failed. So let me tell you about farnesolation of RAS and what was known about it. So K-RAS has two alternative splice forms, 4A and 4B. The oncogenic or transforming activity is 4B that has a tail with many, many lysines on it, near sea terminal cysteine. That positively charged tail promotes its localization up to the membrane, but it still needs a farnesol to be added to the cysteine and then several other trimming steps to get to the final membrane-localized carboxymethylated farnesol K-RAS form. And this is catalyzed by the enzyme farnesol transferase, which uses FPP, farnesol pyrophosphate, as a substrate. So this made for a fantastic drug target because this was imminently druggable. People made very good farnesol transferase inhibitors. At this point, people didn't really appreciate the difference between H-RAS and K-RAS. They were both RAS, and it seemed like they found... H-RAS was found first to be mutated. So it seemed many of the cell models that people used to validate their farnesol transferase inhibitors, they used H-RAS mutant cell lines. And when you add a farnesol transferase inhibitor to an H-RAS mutant cell line, they die. They don't get up to the membrane. Now, when the clinical trials were starting with the drugs, people realized there were more and more K-RAS mutant patients. So many of the patients, and just because of the sheer number of Farnesol transferase inhibitors in H, they got put into a lot of K-RAS mutant patients. And there... And so farnesol transferase inhibitors worked for H-RAS. When they got into the trials, and then they started to do parallel cell culture models, they realized that the farnesol transferase inhibitors didn't really prevent K-RAS localization to the membrane, and nor did they work in any of the K-RAS mutant patients. So very, very disappointing. Hundreds of millions of dollars. And the reason for this lack of efficacy was a bypass mechanism. When we inhibit farnesol transferase, we leave behind the cysteine and gerinal-gerinal transferase with one more isoprene unit as a substrate can phosphorylate at lower efficiency K-RAS-4B, and then it gets to the membrane with gerinal-gerinal there rather than farnesol. So this bypass, again, just shows you the adaptability of these very core important pathways and how some kinds of chemical strategies, the chemistry is beautiful, these are fantastic drugs. They just don't work in the patient population we wanted. So you might be thinking, well, what about all the H-RAS patients? Well, it took a long time for that failure to sort of wash through the industry. And very recently, we began to think, my colleague Frank McCormick, that maybe these farnesol transferase inhibitors should be tried in the H-RAS mutant patients. So we just began that. We took it out of, we licensed it from a company that had one that failed, and we're now trying it in H-RAS mutant patients. So we'll see if that idea works or if that's still yet another almost there idea or if it's not good enough. But this idea of a membrane localization being such an important thing for K-RAS activity, we began to think about more and more, and my colleague Frank McCormick had what I thought was a great idea. He said, well, if the cysteine is there, and the problem is that it can be gerenal-gerenalated, let's just put something else on there so now gerenal-gerenal can't do the backup system. So he had the idea of taking any small molecule with a cysteine reactive group and selecting for something that would bind to K-RAS-4B and then derivatize it in a way that would be cytosolic localized and not get to the membrane, and of course that would prevent bypass. And I like this idea very much, and like many conversations with biologists, their biology sounds great and the importance of it sounds great, but the chemical challenge of that seems huge because K-RAS is really like a bowling ball then with a flexible tail on it, and Frank wanted to get a drug that bound to the floppy tail, but I couldn't see why there would be any specificity. And so we were racking our heads thinking about how to exploit this idea but do it in something that seemed more chemically tractable. And a postdoc in my lab, Greg Hamilton, had I think a really nice idea which is to essentially use the enzyme in the cell that's already there to deliver something to that cysteine. And so he said, well, let's take and use farnesyl transferase, but now instead of FPP as the substrate, let's use Frank's idea and deliver, get an enzyme, get a substrate, what we call like a neosubstrate, for farnesyl transferase to deliver something to that K-RAS4B tail. And as a proof of principle to get us there more quickly, you'll see that what we did is we just used the known farnesyl tail to help drive it into farnesyl transferase to be a substrate. And now there are many, many reasons why this could fail. It has to compete with a lot of FPP. We have to get a new chemical reaction to happen in an enzyme that was never designed to sort of make a carbon, you know, cysteine attack this reactive group. So there are many things that failed, but we still went for it because we thought this was still an elegant strategy. So this would be the idea. Can we get K-RAS to be modified with something that won't let it be up at the membrane and will it depend on the activity of farnesyl transferase? So luckily, again, lots of chemistry was done in farnesyl transferase and there was one particular farnesyl competitive inhibitor that crystal structure was solved of the molecule bound with the C-terminal tail of K-RAS in gray as the farnesyl transferase. And this hydroxymate phosphonate is shown here and what Greg noticed is the nitrogen is at reasonable distance in this one co-complex to where the cysteine was. So we decided to attach electrophilic groups to a pared down version where we deleted the oxygen and kept the phosphonate and got rid of the other carbonyl, but then we modulated everything because we knew that we needed to get the precise alignment for the catalysis. So that was what I showed you up here. That design was here's the starting inhibitor. We pared it down and then we wanted several different vinyl sulfonamids, alkynamids, or epoxides, and tested each of these and you'll see that some of them have two carbons between the attachment of the electrophile and the phosphonate and some have one. And it was very, very sharp structure activity relationships, but when we give farnesyl transferase to K-RAS to each of these molecules, one of them modifies to high efficiency. Interestingly, we never really get to 100% modification because this product, essentially, is a bisubstrate inhibitor. So it starts to inhibit farnesyl transferase as we're delivering it, which is a caveat to what I'll tell you about, but it's one sign that we're getting productive binding. Okay. So then we... this just shows you that the modification is on one site of the cysteine and it has the expected mass and then we have a control molecule without the epoxide, but with everything else the same, there's 0% modification. So throughout the cell data, we'll use the negative control molecule as well as the active compound. So we went into cells where the bypass of a farnesyl transferase inhibitor is easy to see. And then we just treated the cells and then fractionated the cytosol or the membrane and then looked for K-RAS. So what you can see is without any drug, K-RAS is at the membrane and it's not in the cytosol. If we add a statin, which inhibits all FPP production, we then starve farnesyl transferase of its inhibitor and you don't get a localization and you can see that it's localized to the cytosol and the membrane. And there are a number of controls here, but here's a gerinal. Gerinal transferase inhibitor, there's no localization. Farnesyl transferase inhibitor, no localization. That right there is a whole observation why the drug never worked. If we combine them, however, you can see we get very, very good localization to the cytosol. So that shows that their gerinal-gerinal is the mechanism of the bypass. And then I'll skip over a couple of other controls, but here's the active compound. You can see we get a reasonable fraction localized to the cytosol, which is very exciting. And then when we just take off that one oxygen and leave the ethyl group instead of the epoxide, there's no localization. So it looks like the drug is going in there and it's getting modified on RAS, and RAS is not able to get to the membrane as efficiently. The nice thing is this time course, it looks like it takes about 12 to 24 hours to build up. That's because newly synthesized RAS is the only pool that we can actually get down from the membrane into the cytosol. So that was very reassuring. Then we want to know whether this modification is dependent upon pharmasal transferase inhibitors, so if we have pharmasal transferase. So if we add a pharmasal transferase inhibitor, we should be able to compete off this localization and let it be bypassed by gerinal-gerinal. And so if you look here, we see no drug treatment. We see no localization. Pharmasal transferase inhibitor on its own does not localize. And now when we don't add any pharmasal transferase inhibitor, we see cytosolic localization, but as we increase the dose of the pharmasal transferase inhibitor, we compete it and let it go to the membrane by gerinal-gerinalation. So with that, we were very gratified that we could get the new chemistry to be carried out by the pharmasal transferase inhibitor. We'd like to know how good it is at killing cells, and it's reasonable. It is, you can see that it's a sort of a 10, 20 micromolar inhibitor on its own, but if we use a dose of the statin that decreases the competition with FPP and gerinal-gerinal transferase, it can synergize and make this a much more effective drug, and it only synergizes, I left out the other one, it only synergizes with the active compound. The inactive lovastatin shows no extra effect. So everything, it looks consistent, like we can take this neo-substrate strategy and deliver this molecule to an enzyme that modifies a KRAS and begin to delocalize it and avoid this bypass mechanism. So with that, we're sort of just now optimizing, there's a lot more chemistry to do here. We don't know if the following steps of modification happen and there's a lot more to figure out, but it's sort of a work in progress on this project. So back to the timeline, many, many years of investment on the FPP, we then took a fresh look at the whole picture of RAS and asked, well, what would we really love to have with RAS? If we had this, then we would have a completely new RAS drug. Well, I showed you that we want to directly target the oncogene. FPP is sort of direct, but it's like one step away, but most importantly, for maximum therapeutic benefit, we want to inhibit just the oncogene and not the proto-oncogene. That way we can dose to kill the tumor and not worry about on-target toxicity on the non-mutant part of the patients. So how do we do that? So let me tell you a little bit more about RAS and why people couldn't directly target RAS on its own, which I sort of alluded to this. GTPases, they're really nucleotide state-dependent switches. They hold on to the nucleotide at super high affinity. So their KD for the nucleotide is 17 picomolar in comparison to kinases, which are in the micromolar range. So we would have to make a drug that would compete with GTP. We would have to be a million-fold better at drug discovery than we are now. That's supposed to be a joke. It's not possible to do that. So that's probably why we haven't been able to crack K-RAS in that simple just-go-for-the-substrate binding pocket reason, because it's built a completely different way. So, well, other parts of the RAS cycle could be drugged. And so I want to introduce those other components to tell you how this new drug we've made works, but also tell you about other parts of the RAS cycle. So I've told you that RAS in the GDP state is off. So two flexible loops in the GTPase switch one in dark blue and switch two in light blue have a confirmation that's incompatible with binding two effectors, like RAF or, I want to have it mentioned, PI3 kinase. But when RAS binds GTP, these two loops fold in, they're in formation, and then they're competent to bind the effectors. With such a high affinity for nucleotide, 17p-comolar for GTP or GDP, there has to be some way to get that off, and that's catalyzed by a guanine exchange factor that comes up and opens up the whole protein, the side chains of those loops, in order to let the nucleotide fall out. And then in that nucleotide-free state, the higher concentration in the cell of GTP will load in and put RAS always going through the cycle from the GDP to the GTP state to be on. Once it's in the GTP state, it can switch by intrinsic GTP hydrolysis, or it can be catalyzed, accelerated, that GTP hydrolysis by the GTPase-activating protein. It turns out the dominant mutations that cause this 20% of cancer interrupt this interaction. So now, sorry, with the zoom in, the colors are slightly different. In gray is still RAS. Here's the nucleotide. And then in blue is the GTPase-activating protein, or GAP. And GAP has an arginine, a so-called arginine finger, that brings a positive charge nearby the phosphates to accelerate hydrolysis. But the methylenes of arginine go right by two glycine residues in RAS. So if any cancer cell can mutate either of those two glycines to anything other than glycine or proline, it will push aside this arginine and decrease the ability of the GAP to hydrolyze. So it'll be stuck more in the GTP state. So we know then that sort of 20% of cancer is caused by mutation right here. We know why it's caused. What can we do to treat that? Well, if you look at this, the one pocket I've told you about is where the nucleotide is, but it's always occupied by nucleotide. And the rest of it is a sort of bowling ball. It's just not many pockets that are able to be drugged. So we needed some handle that could trump sort of any very, very shallow pocket and give us an interaction. And then, of course, for a chemical biologist, you can look through paper after paper, protein after protein, but if you ever see those three letters, CYS, that's what you start to look at because you just can't get distracted away from how powerful cysteine is in terms of the chemical modifiability. And people looked at the G12 mutations in RAS, and they're mostly aspartate and valine, and cysteine was the third most frequent residue, so sort of underappreciated, but in lung cancer, it's the most frequent residue that's mutated, and that's because there's a transversion that causes the cysteine mutation or the valine mutation, and that occurs in smokers. Never smokers get the aspartate because it's a milder mutation. So we thought, well, there's a cysteine there. What can we do to exploit that? And if we could exploit it, we would have something that would be good, which would be oncogene specificity. We would have something that's bad. It would only work for the cysteine, but there's a large fraction of patients that have this mutation. So we solved the crystal structure of the G12C mutation. Here's the GDP bound form. Here's the switch one and switch two. Switch two was very mobile, and in this particular crystal structure was unresolved. But the good thing is the cysteine was pointing out, and it looked like a target. So then we collaborated with my colleague Jim Wells to use some of his special cysteine fragment-based screening approach that he calls tethering to identify some lead compounds. Tethering is a very nice fragment-based drug discovery tool for several reasons. First is that if you have a cysteine you want to target, it's very cysteine-directed because of the disulfide. It makes a disulfide in a reversible manner, and you can add beta mercatoethanol to compete that away. So at very low doses of beta mercatoethanol, you can favor disulfide formation with very early, poor starting points. And if the molecule can survive BME, it's probably become some feature of the disulfide binds to a pocket that's next to the cysteine you want. And of course you can do controls without the cysteine and the like. So we did that screen, and we were very lucky we found two hit molecules that modified the G12C form of KRAS to 100%. We put the mutation in H-RAS. It also bound. That was encouraging. They're so highly conserved. Also negative control, very encouraging. It doesn't bind to the wild type, so it has three other cysteines, but it's not reacting with those. So all of that looked very good. Then we got a very, very depressing result. And that is that if we put the RAS into the GTP state and then asked that cysteine to get modified, we got zero modification effectively. And that's very depressing because we think that in the cells, the predominant active oncogenic form is the GTP state. So now our drug is not binding to the oncogenic form of the protein. So we went back and re-screened the GTP state against the whole collection and we got exactly zero. So we had no chemical starting point for the GTP state and we could have either stopped or we said, well, let's keep going. Let's just optimize. Let's see where it's binding and we'll go from there. Solve the crystal structure of the molecule bound, the hit molecule bound to the cysteine. You see here's the disulfide. And it's binding not into the GTP state, which presumably it could have because it's a covalent bond. It could compete with picomolar affinity. But it bound in a completely new pocket that nobody else had recognized because it is half folded in state of switch two. This is the pocket that switch two would occupy when GTP is bound. So that explained immediately why the molecule didn't like to bind to the GTP state because the pocket is absent there. But it also showed that this pocket is actually quite, you know, an attractive drug pocket, which is nice. We don't have to compete with nucleotide. But the other feature is it's in an important part of the protein. It's right at the heart of where the machinery is that switches between GTP and GDP. So that was a big, big encouraging result. We still got worried about the GTP state not binding. But we were sort of spurred on by this result that had been published a number of years before with a natural product that binds to another G protein, G alpha Q. And this depth of peptide binds behind switch one in the GDP state of this protein. So it looked like nature had found a very similar kind of pocket in a G protein, behind one of the switches in the GDP state. So we said this is sort of a good sign that this is a reasonable place to optimize for a drug. So then we needed to convert the disulfide into an electrophile to go into cells. And we basically just took the disulfide off and put various electrophiles, vinyl sulfonamides, acrylamides. And as you can imagine, sort of, if you have hotter electrophiles, you get higher percent modification. When you go down to weaker electrophiles, you get less modification. I think one of the more important sort of lessons on this slide was that subtle modifications to the hydrophobic element here, going from this dichloro to this dichloro phenol, we could get increases in modification even though the warhead was the same. So we wanted to know that the pocket was druggable and with SAR we could improve binding, and we did that. So in the last couple of minutes, let me just tell you what the protein, what the drug does to RAS. So it sits under switch two, and one of the first things we checked, and I won't show you the data for this, is that it looked like it would prevent the guanine exchange factor from catalyzing exchange, because it's sort of pushing right on the interface of where that GEP has to come. And absolutely, we don't let the guanine exchange factor load in, take off GDP and catalyze exchange. So that was fine, but people don't really know how much the GEP is really involved in the mutant RAS. It's a little bit a sign of debate that mutant is supposed to be sufficient on its own, so just blocking that wasn't necessarily enough. But what we started to look at more carefully is what is this doing to the machine of RAS? And the key switch that happens in RAS between the GDP and the GTP state is shown in this animation. So here's our drug complex in GDP. It's in the GTP structure, and then in animation, show you where on switch two and switch one the hydroxyl groups come to interact with the gamma phosphate. And those motions send glycine 60 right through where our drug is bound. So if the protein is in the GDP state with our drug and glycine 60 cannot come over and stabilize the gamma phosphate, and there are some subtle changes to 3N35 as well positioning in our crystal structures, we thought, well, maybe this gamma phosphate will be a little bit destabilized. So we can measure that by looking at the exchange and rebinding of GDP or GTP. And the prediction is that GDP binding won't be altered because the drug is bound away from that, and it's bound in the GDP state, but when our drug is there, the GTPase will not bind GTP as well. So here's the GDP titration with four proteins, the wild type, the G12C, or G12C with two different drugs bound. You see there's no difference in GDP affinity, and the reason this affinity is low is because it's the protein concentration that we used. It's in the micromolar range. So, but if we titrate GTP, you'll see that wild type and G12C are potently bound, but there's a tenfold shift when we put our drugs in. So we've destabilized the ability of the protein to bind GTP. And in the cell, that's very, very important because the cell allows GTP binding because it maintains the concentration higher than GDP. The intrinsic affinity for GDP and GTP is really the same. So we've shifted is the intrinsic affinity for RAS when our drug is bound. And what's great is I think is that we've used the cellular GDP as the inhibitor of the oncogene, which is something when we started, we thought that was the biggest problem we had is how do we overcome picomolar affinity. But when we found the drug binding pocket and this observation, I think it really tells us a mechanism that this approach will work. And so we would like to know that this works in a G12C-transformed cell-specific manner and these are lung cancer cell lines which have many, many mutations. But the four G12C cell lines, two or three of them are very sensitive and none of the non-G12C cell lines are sensitive at all. And then a more important control is to take the most sensitive cell line and put three different drugs that have different abilities to modify K-RAS. And this is the most active biochemically at modifying G12C. And then another one with the same warhead but with subtle changes in the hydrophobic element is 10-fold worse. And then a negative control compound that has the same warhead and a very... and the same warhead but a different hydrophobic element, you see that again the rank order biochemical potency is the same as it is in vitro. So we know now we're getting on target inhibition and we're now optimizing the compound. We've actually begun a bigger effort with the company to try to optimize the drug and then that's going on very, very extensively. In my lab, and this is just sort of a summary of what I told you that we've put a drug here, blocks guany exchange. So the oncogenic mutation blocks the gap. Our drug blocks the gap and prefers loading GDP rather than GDP, all of which tells us we're going to block P.F.Rikini's interaction and RAF interaction but at the level of the oncogene. And so now we're thinking of other alleles. We go from the easiest to drug, the cysteine to the most frequent mutation, which is the aspartite. So we're looking for really some novel chemistry to take advantage of the unique reactivity of the aspartite. So it was great to talk with Ron today and so I'm getting a shipment of all the diazo compounds you have tonight to take me back tomorrow. But I think that's a great... I was really great to hear about that. It was great. And so we're going to be working more on that. We're also exploiting the pocket, as you can imagine, and derivatizing with new compounds. So this is where the cysteine was modified. We've also recently solved crystal structures of hits that bind sort of outside of this pocket. And so we're thinking about merging these chemotypes to get more and more affinity. So we're just sort of in the midst of all of that right now, optimizing, exploiting this pocket more and more. Well, with that, I'll just tell you who did all the work. The first Farnesville transferase substrate idea, the idea really originally came from Frank and Greg sort of built in the Farnesville transferase delivery angle. And Chris Novotny was a graduate student that is finishing that project up as Greg has moved. And then the KRAS G12C project was done by two people in the lab, Postdoc Ulf Peters and a graduate student, John Ostrom. And the last new pocket I mentioned was from Danny Gentile, a graduate student in the lab. The tethering was done with Jim. And then E. Ping Dantroy have commercialized this and tried to see if we can get this compound optimized into the clinic. So again, thanks for the invitation. I'm happy to answer any questions. Oh, good question. I think there are actually, but I think the problem is there are multiple gaps. I think in the gaps in the GIF side, I put just one, but there are multiple. So there, oh, no. Well, what I said is true, but then what you said is also true. NF1, neurofibromahousis 1, which is a very famous tumor suppressor. That is a gap for RAS. So that's, there are, there are, I guess in those lymphocytes, that's the dedicated gap. And so that's why that's a very frequent one. Yeah, Kevin Shannon at UCSF. At least from a tool perspective, is that like a modifiable pocket dude? Yeah, great question. That's kind of what this is. So this is the cysteine with the G12C drug bound. But then exactly what you said is what we wanted to do and what we've put here is a cysteine in the case of a methionine. And this molecule is bound to that engineered cysteine. So that's exactly what we're doing now. So yeah, it would be great to be able to just put in any cysteine there and modify. We're checking now if that has a phenotype on its own. This, after we published this paper, another group was looking for inhibitors. I believe it's the RALG-GTPAs. And they did a computational screen to find drugs that bind. And they found a non-covalent drug that binds in the equivalent pocket in the RALG-GTPAs. There's no structure, so we don't know exactly how it works, but that is also further suggestion that we would be able to get a tool. Yeah. We don't know exactly. It's a good question. We were worried it was too close. It looks pretty close. It looks really close. The thing that stays on there, so yeah, it had to be a pro drug, but I skipped that. But what stays on is a single phosphate. So we hope that that negative charge would help keep it away from the membrane. But that probably, I think, may not be the final thing. I think it's what I referred to here. Not in, yeah, not in atras there is, but not in K-ras. And it's in K-ras, there's two other enzymes that have to clip here and then methylate. And you need both of those. So I thought that that data was saying that if you didn't methylate, you just had one negative charge, it didn't go to the membrane. So I thought that phosphate could be like that or we don't let these other two enzymes work when we put our thing on there. We haven't tested those RCE1, L-C-L-T-E-Y. I forget the two names of those. But yeah, it's probably because there's two more steps. Yeah, hopefully they wouldn't get recognized. Yeah, yeah. Say the last part. You're right. Yeah, yeah, the prediction is that it's a bell-shaped dose response curve that you activate and then a high dose you suppress. That's what you see in cell culture, but you can't get the drug at that high concentration in patients. No, but it's great. Yeah, people, it must be that there is a little bit of anti-cooperativity that the different RAF kinase inhibitors will induce, but then the shape of the bell-shaped curve is like wider or tighter depending on how cooperative. So some people are setting up screens. Very cool. To take a slow off-rate RAF inhibitor, put it on, wash it out of the cell so the cell gets stuck in the dimer, and then you screen for another drug, and then you can find what the state of that other one is. Other people are just screening for completely different things that don't let the dimer happen. What's fascinating is that the what people... So you would have thought the MEK inhibitor would be fantastic, right? Because it's downstream below these things, very little therapeutic index on its own. So, but because of this paradoxical activation, the company that made this, Plexicon, is making what they call a paradox breaker, which doesn't let the dimer happen, but the prediction is that that won't have as good of a therapeutic index, because in the rest of the body, this drug meets a cell that has just wild-type RAF and wild-type RAS, it just lets the signal go through, because it's kind of, you know, makes a little bit of dimer. So, but it doesn't inhibit. But anything that inhibits completely RAF in the tumor and the rest of the body will have the therapeutic index of MEK, which is almost nil. So it's really fascinating that cooperativity and then how it plays out, but it may be that these subtle differences, patients can tolerate better timing, something. Yeah, that's great. So either it's excess growth factor around that tissue that is sending a signal, and without a mutation, is just giving a little bit of a signal which the RAF inhibitor boosts by pushing the equilibrium over, or they have these cells that become the squamous cell carcinoma. They might have a KRAS mutation in them already, but it sent the cells to senescence. And then when the drug comes in and boosts the signal, they come out of that and make the tumor. There's multiple, multiple things. In one patient, they were given the drug. They had melanoma, BRAF mutation, gave them the drug, tumor was grown out, they sequenced, they had an NRAS mutation. So then what they did is they stopped the drug and the NRAS tumor disappeared. And then they kept cycling back and forth between the drug and on and off.