Hello and welcome to this presentation which provides references to documentation and URLs related to the implementation of TFM in the STM32U5. ST has released several documents that describe the implementation of TFM in the STM32U5. The application note 5156 presents the basics of security in STM32 microcontrollers. The application note 4995 supports the secure firmware install feature available on the STM32U5. The user manual number 2851 describes how to get started with the STM32U5 TFM application delivered as part of the STM32U5 MCU package. The user manual number 2852 describes how to prepare STM32U5 microcontrollers to make a secure system solution compliant with a CSIP profile for PSL level 3 using the STM32QFWU585 Security Certification V1.0.0 software package included in the STM32U5 MCU package. The BU585 IIoT02A board integrating the STM32U585 AI microcontroller is used as the hardware vehicle to implement and test a non-secure application using secure services but it doesn't bring any additional security mechanism. STM32QU5 TFM application and SBSFU application examples are provided for the BU585 IIoT02A board. The TFM-based application example consists of four main software components which can be configured by integrators according to their needs. TFM-SBSFU boot, secure boot and secure firmware update application TFM loader, application loader application based on Ymodem protocol over USART TFM-Apply secure, secure application providing secure services to the non-secure user application at runtime and TFM-Apply non-secure, non-secure user application. The SBSFU application is minimal with only the secure boot and secure firmware update services of the TFM. More general documentation can also be useful to gain knowledge on security standards such as the TFM user guide for V1.0 in all releases, the PAS developer APIs. Thank you for attending this presentation. You can now refer to the other presentations that detail how TFM works. TFM flash memory footprint and TFM offer in STM32U5.


Open Group has been the pillar of driving interoperability, boundaryless information flow and ensuring diverse entities come together for acceptable adoption of consumer and supplier across common standards. And Open Group is doing great noble work through its vision and the mission. And also in this current hyper-connected, hyper-digital, hyper-cloudified world is becoming very, very important to have a common understanding of how we could really build large enterprise systems and drive some common standards. HCL has been fortunate enough to be involved with Open Group as a platinum member and got active involvement with the board of Open Group. We contribute into the different work streams. We learn, promote and adopt the philosophy and the products of Open Group. Especially we've been very actively involved in the IT for IT work stream. And we've not only taken the IT for IT and started influencing and working with our customer to start seeing the value of what IT for IT delivers. But we're also taking some of the value streams of IT for IT and making sure some of the software products in HCL software, across value stream management, across our service management and ZAS, and in different areas, we are making sure that we are embodying the philosophy for IT for IT. Driving adoption of Open Standards is extremely important. As member and promoter, we take the common theme of Open Group and make sure that we are driving this across our customers, our communities, and the government, and the different subsystems that you're getting involved. And it's extremely important that this becomes a common minimum baseline for everyone to adopt and make sure that your building systems allows this whole philosophy of boundaryless information. We sent 2021 to make digital Bangladesh and that was to achieve middle-income country status and we have successfully done it. And we have used Open Source Yoga architecture to deliver the e-services. And we have also started the process to adapt yoga as the national standards. And we will be working with the Open Group for necessary collaboration and we look forward for the gold membership in the Open Group. We will be applying very shortly. We are in the process of, in the process, in the processing through the government approval. And I'm sure that the Open Standard will play an important role in our development process. I look forward for continued cooperation with the Open Group to help achieve our goal. I just want to thank Steve for such an extraordinary work in the Open Group. Brilliant, we love it. One of the reasons we applied for this is just that soy oppressor, how Open Group is doing things in the industry. This is Meenakshi, director, Unilever Global IT Operational Excellence and Control. Our framework is the Bible for us for the development of enterprise architecture for Indian railways, which we are doing. And we have named it as a star and that's why we have mentioned it in our... Very beginning from the conception of our company, innovation has been inside of our day-to-day operations but also our commitment to a very regulated industry, the commitment to governance and to follow standards. In that regard, not only we have been following the standards, but we have also created along with our peers a lot of standards for our industry and with the opportunity of the Open Group conception, we have also been part of the, as was mentioned, the OSDU community. On that, we have been committed to the contributions of our innovation as an open source for the better of the work, for the better of our energy industry.


Electronic components can be connected in a variety of configurations to form functional electrical circuits. The two simplest types of circuits are the series circuit and the parallel circuit. A circuit is said to be in series if all of its elements are connected along a single path with the current flowing in the same direction through each component. For instance, if the source current in a series circuit is 5 amps, then no matter where along the circuit you take an ammeter reading it will also register 5 amps. Although current is constant throughout a series circuit, voltage is not. Voltage in a series circuit is divided across the load resistors so that the sum of the individual voltages equals the total voltage applied. This relationship is known as Kirchhoff's law. By adding the voltage drop across resistors 1, 2, and 3, you can determine the total voltage applied at the source. In this case, the total voltage is 5 volts plus 4 volts plus 3 volts or 12 volts total voltage. The second basic circuit form is known as the parallel circuit. Like a series circuit, the parallel circuit has multiple components or loads. However, in a parallel circuit, the current has multiple paths along which to travel. Each of these paths is known as a branch. An important difference between a series and a parallel circuit is that the voltage in the parallel circuit is the same across all branches. The current along each branch will vary, but when added together these various branch currents will equal the current at the source. For example, look at these ammeter readings at the various branches and junctions of a parallel circuit with a source current of 10 amps. As the 10 amp current travels away from the source, it splits at junction 1 into 2 branches that have currents of 2 amps and 8 amps. The circuit branches again at junction 2 and the 8 amp current splits into 3 amps and 5 amps. As the current returns through junction 3, the 3 amp and 5 amp values recombine to give an amp value of 8. At junction 4, the 2 amps and 8 amps currents recombine to equal the 10 amps of the original source current. An important difference in a parallel circuit is that voltage is the same throughout the circuit, so using a voltmeter to measure volts at each load will yield voltage readings equal to that at the source. Total resistance in series and parallel circuits can be determined using basic equations. The total resistance of load resistors, R1, R2 and R3, shown here in series, can be determined by simply adding their ohms values together. So, if R1 has a resistance of 20 ohms, R2 a resistance of 30 ohms and R3 a resistance of 10 ohms, the total resistance of the circuit is 60 ohms. Calculating the total resistance of resistors in a parallel circuit requires the use of the product over sum equation. In this parallel circuit, if R1 is 50 and R2 is 80, then the combined or equivalent resistance of the two resistors would be 50 times 80 or 4000 divided by 130, which is 30.7 ohms. Determining the resistance of a more complex series parallel circuit requires reducing the overall circuit and adding together all the combined resistance values. In this more complex series parallel circuit example, let's begin by combining the resistance of the two resistors farthest from the source, R3 and R5. Since these resistors are in series, we can simply add their values, 50 ohms plus 30 ohms to get 80 ohms. Now, the R4 resistor and the equivalent resistance of the R3 and the R5 resistors are in parallel. Their combined resistance can be calculated by dividing their product by their sum. Thus, using the product over sum equation, we have 80 times 80 equals 6400 divided by 80 plus 80, which is 160, giving 40 ohms. Now, we have further reduced the circuit to two resistors in series, R2 and the equivalent resistance of the R3, R4 and R5 resistors. Again, because we have resistors in series, we can simply add the 60 ohms to the 40 ohms to get a combined value of 100 ohms. And finally, we are left with two resistance values in parallel, the R1 resistance of 200 ohms and the equivalent value of 100 ohms for resistors 2, 3, 4 and 5. Again, using the product over sum equation, we can determine the combined resistance. Multiplying 200 times 100 gives 20,000 divided by 200 plus 100, which is 300, resulting in an overall circuit resistance value of 66.67 ohms.


Whoops, sorry Sorry about that whoopsies. Sorry about that. Sorry about that Didn't realize whoops whoopsie daisies Hi, everybody. I hope you're doing well. I'm making a very quick thumbnail For YouTube because I'm also streaming on YouTube and I think that it's Important that I have The thumbnail, but the thought that I already had the thing up But didn't but that's okay. Yes Yes, here it is. Yes, here it is. Here it is BAM BAM When BAM Where? Help, what are you doing? Copy image post Paste. There it is. And then we go like this and we go Maybe not that bright. There we go, and then we export Thumbnail Sick. Hi. Welcome Sorry, I just had to make the thumbnail for this stream. Don't worry about it Thumbnail go thumbnail Now Yes There we go, baby. Hi everybody now. I'm reading chat. Hello. Hello. Hello. Um There's so much to talk about there's so much to go over Because last night Was the premiere of the documentary Which is crazy. That's crazy talk. Sorry. I just have to Open up YouTube chat because I did not have that open Did not have that open. Hi. Hey, hi Hello, hello, hello Hello, hello, hello. Did my message not get pinned? I thought I pinned a message. Oh No, it is. Okay. Good. Good. Good. Great Great great great Yeah, any who The premiere happened last night the premiere of the doc happened last night And it was so much fun. It was so surreal It was great, it was so it was so cool it was so wild like I did not I Did not anticipate the amount of people Like friends that showed up there were so many of my friends because like the the guest list for The guest list for the premiere was around 200 people Like 200 and no like 230 or something like that and so I was like, okay, that's a fuck ton of people Not everybody is going to do that And so I thought like a hundred people were gonna show up or like maybe a hundred and fifty, but there was easily.  I just I feel very like my heart is very full Because I Don't know it was just really nice having so many family and so many friends there And like obviously it was amazing having you guys there. I don't want to downplay you guys being there But but it was just really It was just it made my heart very happy seeing so many friends come out And it was so awesome seeing so many of you there and so many of you dressed up, which was awesome And so that was really cool like I wanted to give people a fun event and a fun excuse to dress up and everybody did So Yeah, it was very fun. It was really surreal Finally showing the movie Because like obviously I have seen the movie a bunch of times But it was it was really cool watching the movie in a room full of people who hadn't seen it and being like Going through it and being like, okay, this thing is about to happen. I want to see like what people's reaction is So yeah Remember to go to the show if you can well the physical one already happened But if you go to the pinned message in both twitch and youtube there's a pinned message So just click to join the premiere in an hour 50 minutes guys in 50 minutes the Digital premiere is going to start So get your tickets now Um, I should probably tweet that shouldn't I? I should I should less than an hour Digital release of doc If you guys could go and retweet tweet that I just tweeted that would be really nice That would be really cool Really cool and really nice Really cool and really nice Yes, we will go through the outfits in a second. I just um want to post that Can I buy tickets after the premiere so you Have up to you have seven days From today You have you have until next friday You have until next friday to get uh To get a thing Or you have till next friday to watch it. Um, and then it won't be available. Um, so yeah Um, you don't have to get it today. You can buy it at a different day, but you only have until next friday to watch it Sorry So yeah Um, and then it's not going to be going up on youtube. No It will not Seven days at ethan's documentary Um So where can we see it? You go to the link. It's moment.co slash ethan nester It's the pinned message in chat On both twitch and youtube if you're on youtube. You can also go in the description Um But yeah, I I feel really happy now that it's releasing. I'm really really excited I'm very excited too. So there's a live chat on moment. So you guys definitely Be uh, we bump in that chat Um So yeah Will it be on netflix or something? I don't know. I'm not sure. This is I'll let you know Currently, this is the only place it's gonna be Digitally um, also make sure to uh To tweet with hashtag i h t d t Uh and show me your uh show me your premiere Outfits I want to see everyone's outfits at home I want to see everybody's outfits at home Yes chat. I am hungover. Okay Yeah, all right. I had a little after party the last night. I had a couple drinks had a couple A couple too many shots of tequila. Okay Yeah, all right relax um, yeah, we can go through uh Let's go through the the official red carpet Here we go Here we go, baby So many people that were at this event Oh, it's gonna be all weird again. Hold on. Let me let me change the camera Actually, hold on Game big cam Is that what I want?  How can I Do this? Oh, it's gonna pull them up individually I don't want it to pull it up individually Um, it was interesting too because there's a lot of people that I like didn't know there So that first picture is justin who uh Who uh booked my tour? He's also like one of my best friends, but you'll learn about him in the thing You'll learn about him in the thing. That's julian who also works at really good towing. Uh This uh, hold on Uh, this is me My friend jess who is also the hair and makeup for uh anytime that we needed hair and makeup during the movie Here's me and justin Wow Wow Oh salmon non coming in with the fits baby coming in with the fits and me Coming in with my fit baby Wow, wow Non Looks so good I love the all-white I cannot believe I cannot believe he left without a stain Fucking wild Wild I want everybody in chat Because I'm not going to say who I think is like Best dressed. Well, maybe I will air it With the big old dress. It was sick.  It was sick Um, here's me and all of the boys. So this is zack and austin who um Who directed and edited the film?  It's like oof.   You can't you can kind of see them in this Sick like bat glasses They were so cool. It was so cool guys having everybody go in the red carpet and take photos and do the step and repeat Rosie looked incredible This is uh, this is ben so, um qcode did The sound for the film Um, and ben was the I Can't remember what is like what the actual title would be but he did all the all the sound for the movie So he went in and he mastered everything and he added any Um, any like sound design stuff that we needed to do and anything like that Um, ben did an absolutely incredible Incredible job Absolutely incredible job Oh, yeah, tiff and mario That looks so good It was so cool seeing everybody's different outfits.  Oh, it's happening In 40 minutes In 40 minutes, look at this. This dress is so cool This dress is so cool Everyone looks so great DJ's suit was so good DJ fought in uh in the first greater clash Mark was in there Mark was there Jesus Christ, you're all insufferable Ian Hi, Ian. Hi, Ian. How are you? Oh john look at him This shirt.  Is Eric's suit? Oh, it is like it's like greenish bluish. It's not just uh, it's not just uh white Adam looked so classic.  Whoa, just a little bit of a little bit of leg This is me and parker parkers my manager I had to lift the leg I had to Joko looking so good This is alessa. Alessa was the um, was the aeg rep. She was the promoter rep on uh on tour And then here's the here's a little tour family. This is pete pete filled in the first leg This is sarah sarah was the uh the bip manager and then jocelyn obviously Um, but look at all of us.  It was so pretty And then sam sam looked so pretty sam looked like a fairy Like a fairy princess And then laif her son looked so cute. It looks so so handsome Uh Uh My friend slone and matt. They just got engaged just got engaged sabrina Wow Here's matt and brandon you guys remember matt from uh the stream that we did with hyper a little bit ago And he really did not like the last maze in halloween horror nights Brie brie bud was there flew in For literally was here for like. I don't even know if she was here for 24 hours. She looked so amazing I was so like there were so many people that like Came to visit for this like brian morgan came for this And everyone was only here for like a couple of days if that Um, but it just like it made my heart so happy that people wanted to be a part of this and people wanted to come to this zack and melika looked so good melika's dress was so sick This is uh ana and ryan ryan has a part in the movie I don't want to say what it is though. You'll see Good ol adrew sigils his his suit jacket was also like kind of greenish Interesting Look how good brie looks uh Everyone is Oh On us after this really good Just getting some kisses from the boys, you know What's happening? We're looking at everybody's outfits from last night on the red carpet um And then i'm going to be sending you off To the premiere the digital premiere It's going to be great. It's going to be fun. And again, everybody use hashtag ihtt Send your uh red carpet fits or you can go on discord as well And the there's a discord channel for red carpet outfits shelby looks so good tyler looking good chris looking good Everybody's looking so good Making smoky smoky's dress was sick Pam Everyone looked Ravishing Everyone just pulled out all the stops All the stops for this It's justin justin came to the premiere as well Justin and i i I have a photo on my phone of justin and i playing pool and me kissing justin So funny me and joss Catherine There's a photo of me and katherine in here somewhere. I think katherine's dress was sweet There's my mom Here's my dad. Here's me and my brother. My brother did he composed the score Uh for the movie Here's me and my parents and me and my parents are my brother. Here's me and katherine And lots of here's Here's me and katherine It's very cute Very cute very cute everyone's looking dashing Everyone's just looking dashing aren't they? There's me mom Jordan and ellie and brian and morgan ooh right next to each other look at that everyone looked so good Everyone looked so good There you go guys. There's there's mark and amy. Now you now you can shut up and chat They did look so good though. They absolutely did And apparently amy didn't like mark's hat. She was like don't wear the hat and mark was like I want to wear the hat, but he pulled it off. I think he pulled off the hat very well I think he really pulled off the hat What a wonderful folk What wonderful A b and lina fellow is there Everyone looked so good. It's funny going through all of these because I Wasn't able there were like people that came that I didn't even really get to talk to at all um Hostel looks so good still picked me up at the uh at the after party There's quinn looking great. Ooh the shoe. I didn't even realize the shoes.  That's a good premiere fit Look at all these suits Hell yeah Hell yeah I'm so excited I'm so excited Uh uh I'm I'm very excited for you guys to finally see the movie Um It's been uh, it's been a long It's been a long time coming Um It's been a very long time coming Uh I want to see it you click the link Click the link that's pinned in chat. It's pinned in both It's pinned in both chats Hold on Oh, and you can actually see there's like a live chat already That's funny Um, also, I didn't even think about this until last night because I was talking to Some people on the red carpet and they were like, oh, I came to the show and blah blah blah And then I was thinking and I was like, oh People that watch the document like there's gonna be people that watch the doc that are able to see themselves in The thing Isn't that funny They're gonna be able to see It's out. It's about to be out in 30 minutes 30 minutes. Everybody click on the link moment.co slash Ethan nester Let's go, baby Let's go, baby Hope you're proud. I'm very proud. Um, I'm very excited For you guys to finally see it. I think it's going to be very different than what you're What you're thinking it's going to be Um, is there anybody in chat that was there last night? How long is it 82 minutes So it's an hour and an hour and 22 minutes I was hell. Yeah. Did you like it? Did people like the movie? I want to see if um I want to see uh I want to see if people started reviewing it on letterboxed from last night I would love not only myself, but zack and austin as well Um, who again directed it and edited it. Um, would love to see people's reviews on imdb or um letterboxed um So there bring my piss shirt from the tour tonight I'm glad if you can't wait to see the movie Just watch the fnaf movie you're moving it out That's fine That's fine Whatever Whatever that's fine No, it's fine. No Please it's fine Your suit was excellent. Thank you. Thank you. Thank you You're always singing the tune of brain stew. Oh, I was wasn't I? I'm so tired after last night. I am so tired and I am so hungover Do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do-do Will you be able to watch it next friday next friday is The last day that you will be able to watch it. Yeah. Yeah. Yeah. Yeah. Yeah Thank you for including us with the premiere event last night. It was so special Getting my light back to Tennessee in a few. Thank you so much for coming There were so many people that came from all over the place. There was uh a group of people that came from Alaska Which was wild just to come and watch a movie That's crazy so so crazy I took a shot before coming into the theater nice nice I wish I could have made it to the premiere, but I definitely couldn't have made it to LA. It's fair But you can make it to this for me, baby the digital premiere When can I watch the movie in 25 minutes Everybody click on the link the moment link moment.co. It's pinned in both chats Um and you can Um and you can uh join in And you can join in on the chat that's in there as well I literally just bought my ticket. Why would I be mad at you for just buying the ticket now? sitting here waiting for my door dash uh for For before the premiere And I'm getting anxious. Hell yeah You do have to buy a ticket to watch it.    Yes Okay, google Show me the front door Oh, then maybe Oh And then my friends delivered flowers But my friends got my flowers delivered to my house.  Sorry.  I think different. Ashley different.  I want to do a video where I like rate everybody's uh Where I rate everybody's outfit It would look so good so good So so good Uh guys, I'm I know that I've said it a billion times, but like I'm just so excited for you guys to finally see The doc Um, I'm excited for this to finally be out It's been um in the works for About a year and a half now um So I'm just uh I'm just excited. We'll happen to only fans now that this is done.  They are A price What are they? 20 bucks 20 buck arenas 20 dollars 20 dollars That's how much they are 20 dollars I need to I need to Sign in and get my I have to I have to get my ticket. I forgot Whoopsies Whoopsies forgot to get my ticket Whoopsies, and then I also Hold on God, I there's so many things that I completely forgot to do but it's fine It's fine Don't worry about it guys.  I'm very excited to see See see what you guys think about it Hmm. Mm-hmm. I'm very excited to see what you got what you guys think about it Tater dog get my ticket. I forgot Yes, enter the moment Yeah Yes, yes, it was great to meet you at twitcon and excited to see what you've been working on Thank you. Well, this is the thing that I've been working on If you want to click the thing you can turn the thing Why are you getting a ticket? You've already seen it. Yeah, but I want to watch chat react to it I want to be able to like see what you guys are See it does have Excuse me. It does have a letterboxed I will give I will give If you guys like letterboxed you can Add it to your watch list and uh Give it a good old review Running on empty To pay for your ticket. You deserve to see for the 426. That's time for free. That's really sweet. I didn't have to pay for it But thank you I was I was sent I I got a code That's super sweet. I feel bad now that you just gave me 20 dollars for the ticket Well, I didn't have to pay for it Really silly Really quite silly, isn't it?   I'm so ready. Yay. Yay. Yay Sean Mark and Aaron aren't it? Yep Yep, yep, yeah The reviews on letterbox are amazing already. They're really funny If there are a million fans of this movie, I am one if there are zero fans. I am dead. Do not mourn me Directors were distractingly hot Oh rose. Hi very excited to see this. Thank you Yeah, keep an eye out everybody if you went to the shows keep an eye out for yourself Because you might just see yourself You might just see yourself Made a movie, uh-huh Uh-huh made a movie Made a movie 11 minutes 11 minutes I might see myself Are you planning on going to Seattle for your next tour? I don't know when I'll tour next. I did go to Seattle though I got to see the tour in New York and I'm so excited to see the dock. Yay. Thank you Thank you How's my birthday? It was great When did the poster ship? I do not know I do not know How much of tour did you end up filming? There was a hundred and sixty hours of footage That was combed through So, yeah So Yeah Ten minutes folks Ooh The moment just changed That's fun The moment just changed guys Also, everybody make sure to change your settings On moment, um, to watch it in the highest, uh Highest resolution possible Look it changed Is Chrissy it changed? Look at that It's happening It's all really happening It's all really going down It's going down I wonder Is there music on it right now? Uh, see you can see the chat you can see the chat to post an instagram story It's happening People are in the moment chat The moment is happening the movie is coming out in eight minutes Click the link Come and join Wow Wa-wa-wee-wa Well, that's cool I'm gonna go join the moment chat. Bye. Bye. Bye. See you in there See you in there Do you see yourselves In the moment chat How do we change our setting slash resolution you hover over the thing I read something from moment chat and but then it's fine That's fun. That's fun. That's fine. That's fine The moon at the top is dark mode. Oh Good call Now it's like we're in the movies, you know, now it's like we're in the cinema Mm-hmm that nice I'm very confused. Why did you make a movie? Click on the link so you can watch the movie It's been being made over the past like Year and a half You're gonna have to go to eat this up and hopefully we could all talk about it after I What reviews I want reviews of the movies I want the review Legitimately though, we would love feedback. We would love a review We would love I would love to know Expectation versus reality with this. I would love to know what you thought the movie was going to be And what it is So there Oh man, I would love a video review. I would love that. I would love somebody to review this in the video Help I can't change the resolution.  It's because I am I have 30 seconds to pull my dinner out of the oven and run to the computer before it starts I think that you can you can like rewind and pause and stuff. I believe I'm almost positive so Don't worry. You just won't be caught up with chat or whatever, but Man, I can't wait to watch this movie for the 18th time this week five minutes Five minutes You can't rewind the live. Oh, you can't not on moment. I thought you could maybe you can't Can we watch it later if we get disturbed? Yeah Get the bow's headphones out for this one. Hell yeah Oh, yeah firetickets now everybody What's being premiered the documentary documentary about tor is coming in Why is it open elsewhere? What? Huh, okay, weird. Anyway What's happening in four minutes The documentary you people don't listen You just don't listen Currently the documentary will only be available on moment from now until next friday that will be the only place that you can watch it For the foreseeable future because we don't know what's happening with it after What's premiering? I missed it the documentary you can click on the Uh moment link that's in the description on youtube or that's pinned in both chats I'm gonna head over to moments. See you there. Hell. Yeah, maybe Two minutes I got the popcorn. Whoa. I think it's time that I end the stream And join everybody over on moment Um, again, thank you guys so much for supporting this documentary. I'm so excited to see what you think about it um I'm I'm just Frankly, I'm just thrilled. Um, so yeah, I'll see you guys over there See you in moment again moment.co slash ethanester um, please If you can share the hell out of the movie Um, get tickets for it. Um, and then let us know what you think we would really really love to know what you think Um, and I would love to know Uh, you know what your expectations were versus What it actually was


472 civil society organisations in the country have held the central bank governor, Godwin Amirfeli, for standing firm on the deadline for full implementation of the new Nira policy. They tasked him not to waver in his responsibility of enthroning a sound economic policy for the country. The groups under the age of the civil society's central coordinating council claim that Nigerians have started seeing the gains of the new Nira policy of the federal government. The national coordinator of the group Obed O'Kukwe said the hitches of unavailability of cash for small businesses were not unconnected to sabotage, noting that money moves in cycles. He said the scarcity of cash being witnessed by Nigerians was because state governors were mopping up the money and stopping it from circulation. The implementation of the new Nira policy, Nigerians and indeed the system have started seeing the gains, except for the hitches of the unavailability of cash, particularly for small businesses, which is caused by sabotage from those who do not want the policy so they can continue with their illicit trade. Other major indicators show that the impact of the policy on our economy is quite and 100% positive. On our electoral process, it is now obvious to Nigerians that it is only the vote by us that are complaining.


Hey what's up guys it's Josh here today I want to do a little review on Dr. Bowe's colon magic versus Dr. Bowe's colon fence the number one. For me I have tried the Dr. Bowe's the original product. It's one of my favorite like colon cleanse products to try like overall it's really aggressive very strong but it's like they pretty much like has the most evacuation let's say for those and so it's like it's been one of the more highly recommended ones for me it's for somebody who wants like something simple that's gonna work you know very effectively very quickly. I was in the little dilemma like ordering them on I was in a dilemma ordering them on Prime and I really needed the colon things quick in the situation so the colon magic which is another form of Dr. Bowe's happened to be cheaper you actually got it like that day versus like the next day after so I was getting really impatient decided to pull the trigger and I really like you know the original so I felt like it was going to be very similar to the original but unfortunately to me it was not basically I found this one to be pretty like lackluster pretty right down the middle on they use helium pus which is fiber which is like you know for that supplement to work you need several grams of it and it's like you don't know how much of a filler they're putting in the capitol so the original Dr. Bowe's is pretty much stacked with only great ingredients and it's just so effective but this colon magic for me just really wasn't as good so I want to put that my two cents out there just I would definitely go get there get the original cleanse and like there's a few other products like they're called like super cleanser ultimate cleanse I really like that products for somebody who something similar to this but much more affordable because like this doesn't do anything too special for me but at the same time it did work so I don't want to like really knock it I do like other products from Dr. Bowe so like I do want to recommend the original colon cleanse it's about $10 I believe it's like a copy of Dr. Tobias's colon cleanse pretty much exactly the same you actually get a few more capsules but every ingredient is exactly the same as that one and now again the two other ones I'd recommend I think are better than this that are very similar in like you know how mild they are but how effective they are super colon cleanse and that was only one of the original ones I tried and they have two different versions I find them both be pretty both be pretty good both to be pretty affordable they're about $20 but you get a shit ton of capsules you get like 200 or like 100 so like versus this product's where it's like you need to get 15 30 capsules like barely barely anything you know 15 serving so yeah so that product super colon cleanse and also it's called super cleanse I know it's funny they're very similar in how they're spelled but it's like completely different companies and there's a super cleanse and the ultimate cleanse I like both those the only problem I have with those is that they are hard tablets which means they're heat pressed so they're not as good as something like this capsule so but besides that I really really like both those products and if you're looking for the best Dr. Bowe's get the original and if you're looking for the strongest you know most effective colon cleanse that I know of right now I pretty much would go with Dr. Bowe's just be careful because it can hurt your stomach you take a lot and don't drink water so anyways guys thank you guys so much for watching the video I appreciate it so much let me know which favorite colon cleanse products are down below I'll see you on the next one peace


Hello, good afternoon, and welcome to the latest webinar in the BioXL webinar series. My name is Adam Carter, and I'm going to introduce today's webinar, which will be given by Andrew Proudfoot from Novartis. The title of the talk is High-Confidence Protein-Ligan Complex Modelling by NMR-Guided Docking, enables early hit optimization. So I'll hand over it shortly to Andrew to tell you all about that. But just while we get started, I'm going to give you a little very short overview of BioXL just for a couple of minutes.  It's likely to go on YouTube on the BioXL website after the webinar. So the chances are that you will be able to watch it again there afterwards. All right, so for those of you who are not so familiar with what BioXL is, we are a centre of excellence for computational biomolecular research, and we're kind of focused around three main areas. The first is biomolecular software. So we have three core codes that we work with in the project, and we actually have some of the lead developers of these codes involved as part of the project. So we have developers from Gromax and Haddock, and also people working on the QMM interface and CPMD. So the development of these codes, improving their performance, their efficiency and their scalability, that's one of the important aspects of what the centre does. Another important aspect is usability. So we're trying to make it not only possible to use these three codes, but also think about how they are used in wider workflows to make them easier to use by thinking about the whole process in which they are used. So the project is looking at certain different workflow environments and the building blocks for workflows so that these things can be used more easily. Finally, the project is also involved in consultancy and training, so we're trying to promote best practices and train end users into how to use these pieces of software and other aspects of biomolecular research using HPC and high-throughput computing. The project has a number of interest groups that you're welcome to sign up to. At the moment we have the following interest groups that you can see on this slide here. So if you're interested in any of these subjects, you can go to the BioExcel website and sign up there to be a part of these interest groups. The different interest groups work in slightly different ways, but in some cases they have forums, for example, and there's other services like code repositories and video channels that we can offer to interest groups if they want them. We will have time at the end of the session today to take questions for the speaker, so I hope you're free to ask some questions. We'll normally wait till the end. It just makes things flow more easily, so you can save up your questions until then. You can type them in at any time into the questions box in GoToWebinar, and I'll invite you to do that at the end as well so we know which questions we have for the speaker. If you have a microphone and you're able to talk, I can open your microphone and you can ask your question directly, otherwise I will read out your question to the speaker. If you're watching the recording of this later on YouTube and you do have any follow-up questions, you can send your questions to the forums at ask.bioxcel.eu. Now I'm going to hand over to Andrew shortly. Andrew completed his PhD in 2012 at the University of Sheffield, where he was working with Professor Michael Williamson there. He was using NMR for structural studies of certain enzymes, and then after his PhD he moved to the U.S., where he's been doing postdoc research in CURT. Futhrich's laboratory, apologies if I've pronounced that wrongly, at the Scripps Research Institute and at the Novartis Institute for Biomedical Research. So you can read more about Andrew here and on our website, but I'm now going to hand over to Andrew to allow him to go through the rest of his presentation and I'll be back at the end to take any questions. Alright Andrew, I will now make you the presenter and you can take it from here. You should be able to share your screen now. Thank you, Adam. That's good, great, thanks. Perfect. Well firstly, thank you very much for the kind introduction and thank you very much to everyone else who's logged in to listen to me today. So I would like to talk to you today about a common problem that we have in pharmaceutical work, in that we generally do not have problems identifying ligands that can interact with our targets of interest and these are usually identified using screening methods like NMR or SBR or DSF. But a problem that we have is that we can only generate structural information for about 20% of these binders and this is normally done through X-ray crystallography. X-ray is hindered to a certain extent when working with dynamic targets or when working with low affinity ligands. Normally you need to saturate the binding site to generate interpretable electron density. So on average you need to have roughly 10 times the KD, which means that generally only your more potent fragments will allow you to get co-crystal structures. This results in a deprioritization of compounds. If we have a structure then we will follow up with that scaffold rather than investigating the scaffolds that we cannot get co-structures for. So this leaves a problem. We have 80% roughly of fragments that we have identified interact with the target and we can do nothing with these and often these fragments have different features which may be beneficial for a drug-like molecule. So we then start to look at other methods to generate this structural information. There are NMR-based methods. We can do a complete structure determination. This takes time. It's fairly labor-intensive. There are other methods that can be used which use ambiguous distance restraints or chemical shift perturbations. There are other methods being developed like the NMR molecular replacement method or NMR squared as it's been called. One of the techniques which I decided to focus on and develop was to use haddock to generate docking models. That is what I would like to talk to you about today. So this is the workflow that I'm going to go through in my presentation and I'm going to address each one of these as they are all important parts in being able to generate the end result. So the first thing I'll talk to you about is the selective labeling of your protein of interest. When working with small soluble targets, uniform carbon or nitrogen labeling is often sufficient to analyze these proteins by NMR. But as your target gets larger, you need to think about possibly deuteration if you're working in the 20 to 30 kilodoton range. Or as you get to an even larger target, you need to start thinking about selective labeling of side chains. The target that I was using is an antibacterial target called CoAD, which is an enzyme. It binds ATP and for phosphopanethine and results in the production of dephosphor CoA and pyrophosphate. CoAD is a 115 kilodoton homohexamer. So it's not amenable to uniform labeling. So we need to start thinking about more elaborate labeling strategies. As you can see in the bottom panels here, CoAD is functional as a dimer and the enzyme active site is made up of two protein subunits. On the panel on the right, you can see that the enzymatic pocket is made up of two sub pockets, an ATP binding pocket and a for phosphopanethine binding pocket. And these pockets are surrounded beautifully by a number of methyl groups. In fact, methyl containing amino acids constitute about 44% of the polypeptide sequence of CoAD. So we decided to use methyl labeling. There are protocols in place for the selective labeling of each of the methyl groups individually. And here I have spectra of CoAD that have been selectively labeled for each individual amino acid. However, to maximize the amount of information that we could obtain from a single sample, I needed to develop a protocol that would allow the simultaneous labeling of all methyl groups. Here you can see on the left hand side a superposition of the six spectra that I previously showed you. And on the right, there is an NMR spectrum of a Milvap labeled sample. So this is where all six methyl containing amino acids have been simultaneously labeled. You can see that the spectra are very comparable. And the nice thing is with CoAD, there's very little overlap observed between the different methyl groups. There is always a concern that as you introduce more NMR active nuclei back into the protein that you're going to have adverse relaxation occurring. But as you can see in this case, the simultaneous labeling of all methyl groups does not adversely affect the spectral quality. So this is good. We can now use our Milvap labeled protein and we can start to analyze some of these small molecules that don't crystallize. Another consideration that needs to be taken into account is the type of carbon source that you use whilst expressing your protein. Here I have a 1D NMR spectrum of Milvap labeled protein, which has been expressed with carbon 12 protonated glucose. Now as you can see, some of the carbon 12 protonated glucose has been incorporated into the aliphatic side chains and the aromatic side chains of the amino acids. This is done primarily in a deuterated environment. So all of the protons that we have seen here either have come from the carbon source or from the selective introduction of the methyl groups. Now this can be problematic because as you can see, the signals for a small molecule come roughly around the regions where the aromatic bound protons are or the aliphatic bound protons are. So if you're trying to get into intermolecular NOEs between your labeled side chains and your unlabeled small molecule, you're also going to generate intermolecular NOEs between your labeled side chains and your unlabeled parts of your amino acids now too. When you express the protein with carbon 12 deuterated glucose, you can see that there is a significant reduction in the number of aliphatic or number of protons that are now incorporated into the aliphatic and the aromatic regions. So this will allow you to get much cleaner data and ensure that the only intermolecular NOEs that you observe are between your protein side chains and the small molecule of interest. So now we've identified the labeling scheme that we're going to use. The next thing we need to do is to assign the methyl peaks of the protein that we are interested in. A standard protocol for assigning methyl groups is to make point mutations. Here you mutate out the methyl containing amino acid of interest and you acquire a 2D spectrum. By comparing the apo to the mutant spectrum, you see peaks disappearing and you can start to assign methyl groups like that. As you can see on the panel on the left, it's a very nice clean data set where a single peak has disappeared after mutating 3 mean 10. So it's quite clear which peak assignment belongs to 3 mean 10. On the left hand side, however, we have another example where mutation of leucine 102 has resulted in global changes in the NMR spectrum, making it impossible to assign this peak. It's not just global changes in chemical shift which can inhibit this protocol. Often mutations can significantly reduce the amount of protein that can be expressed to the extent where you get no expression. So this method is highly variable and you can invest a lot of time with no guaranteed results. An alternative approach is to use what they call a methyl walk. In this case, you acquire a high-dimensional nosy data set. In the example shown here, we have acquired a four-dimensional HMQC nosy HMQC and this experiment allows you to obtain information about the spatial proximity of all methyl groups surrounding the methyl group that you are looking at. So in the panels shown here on the left hand side, we have focused in on all of the methyl groups which are observed by isoleucine 36 and based on comparisons then with a crystal structure by comparing the observed NOEs to the expected methyl NOE network, you can then start to make assignments and walk through your protein to assign a specific region. Because often when we're doing these studies, a full protein assignment is not required. You're only generally interested in reaching around an active site or in a specific part of the protein. So anything that we can do to reduce the amount of time that we spend assigning peaks is highly beneficial. Now this can be done in a manual way, but there are now also groups that have developed automated methods for the assignment of methyl groups using similar techniques, one of which is a group in Oxford who have developed a process called MAGMA and they will take the spectra and peak list that you have produced and they will automatically walk through and compare it to a crystal structure and will provide you with a peak list at the end. So if this is something that you're interested in looking into these automated techniques may be beneficial. So we've now labeled our protein, we've assigned our methyl peaks, now we need to start looking at using HADAC to dock these small molecules into the protein active site. So the docking protocol that I used was to titrate the small molecule into a milvat labeled protein sample and then monitor changes in chemical shift. Now this does two things, this allows me to transfer my assignments from apoprotein to a saturated compound bound form of the protein. I can also, if the peaks are in fast exchange, then fit the chemical shift later to generate a binding affinity. This is important because when I'm running my NMR experiments, I need to ensure that I have saturated my protein binding site with small molecules, so I have a concentration sufficient to do that. I then need to assign the protons on the small molecule. And once I've done this, I can conduct a 3D edited filtered NOSI experiment to identify the intermolecular NOEs between the selectively labeled methyl groups and the unlabeled protons on the small molecule. Now for my experiments, I used a mixing time of 120 milliseconds for all of my NOSI for my edited filtered NOSI experiments, but depending on the type of system that you are working with, you may need to adjust that parameter accordingly. So the benchmarking of the docking protocol was done with these three small molecules. Now these compounds varied by the number of rotatable bonds that they had and thus the total number of energy minimized conformations that they could achieve. The affinity that the small molecules had for CoAD and also the distribution of the NOEs that could be observed around the small molecule. The other key factor was that we had high-resolution crystallographic information for all of these targets. So we could compare our docking results to a crystal structure to validate how well Haddock was performing. The first fragment that we used was our optimal fragment. It was a high affinity fragment that had no rotatable bonds. This is important because it meant that there was only one confirmation that this small molecule could have in the bound state. When I titrated the small molecule in, you can see here that the peaks which exhibited a chemical shift perturbation all correspond to peaks that are in the phosphopandething pocket. So we were confident that the small molecule was interacting in that part of the pocket and not the ATP pocket. One assumption that we did make was that, as I previously said, the active site is made up of two protein subunits. Now in a Homohexma, it is not possible to differentiate between the different residues on the different subunits. So the assumption that we made was that peaks, for example in this case, valine 135 and lucine 131, these peaks came from the second subunit and not the original subunit. So this was important when we set up the docking runs because we needed to define where the NOEs came from. The edited filter nosy experiment that we ran was able to identify the intermolecular NOEs between the unlabeled ligand and specifically labeled methyl groups. And here I have a 2D projection of the edited filtered nosy and you can see that all the peaks that are present in the spectrum correspond to methyl groups that are surrounding the small molecule in the crystal structure. And each of these now contain information or intermolecular NOEs to that small molecule. So the small molecule needs to be assigned in the presence of the protein. And here I have a superposition of the 1D of the small molecule with the bound form of the small molecule in the milvat label protein. And as you can see, the peaks superimpose quite nicely. So there's not a significant change in the chemical shift of the peaks in the APO form, the bound form of the small molecule. Now if you're not comfortable with assigning small molecules, there is software out there that can help you with this. ChemDraw is a program where you can import a small molecule and produce a 1D proton NMR spectrum. While software may not get the exact chemical shifts correct, they will get the fingerprint of the small molecule correct. And normally the distribution of the peaks is pretty accurate. So you can use these programs to assist you with assignments if you're not comfortable with it. We then, I then looked at all of the intermolecular NOEs that I had generated with my edited filter NOESY and was then able to integrate these and group them into upper-distant bins for the docking experiment. Here, underneath the 1D spectrum, I've shown strips from the three-dimensional edited filtered NOESY. And as you can see, the chemical shift of the peaks in the indirect proton plane correspond nicely to the chemical shifts observed for the small molecule. And I have colored them to coincide with the assignments of the small molecule. So from this, we're able to build up a picture of how the small molecule is sitting in the enzymatic active site. So now, having acquired my edited filter NOESY, manually go through the data set and assign the intermolecular NOEs and integrate the peaks and also identify the peak intensities. From this, I generated a ratio of the peak intensity to peak height. And I used these to then place my NOEs into bins based on their ratios. During the first experiment, I correlated the ratios that I generated to the expected distances based on the crystal structure and then used this as a calibration curve for all other experiments. So the NOEs were placed into bins between three and a half angstroms and eight and a half angstroms based on their corresponding ratios. Now, this isn't a new technique. This has been done in the past and has been published. So, and we found this quite successful. Now, there's a lot of information on this slide, and I don't intend to go through it all, but these are important parameters that you need to be aware of and adjustments to the haddock docking protocol that need to be made in order to, in our experience, to successfully dock the small molecules into the active sites. There's a lot of conversation out there on haddock forums about how to generate ligand topology and parameter files. The methods that were suggested were not available to us at our company, so we used a different method called Explo2D. And when we were doing this, all partial charges of the ligand were set to zero. The ligand and the topology and the parameter files that were produced using this method were successful for all docking protocols. So that is another method which can be used to generate these files. The NOEs were defined as unambiguous restraints between the carbon atoms to the attached protons. And the NOEs which observed were placed into bends and contained a lower distance limit of 3.1 angstroms and upper boundaries of three and a half to eight and a half angstroms. As I previously said, the COAD active site is comprised of two protein subunits. So for the purposes of the docking, these protein subunits appeared as a single chain. That meant that haddock could not throw out one of the protein subunits during the docking process. I've also listed here a number of parameters which need to be taken into account when running haddock. I'm not going to go through all of these, but they are here for reference if you want to follow this process in the future. So how did this work? Well as you can see on the right hand side there's a superposition of the small molecule generated from the docking protocol in magenta with the actual crystal structure. Haddock was able to produce a cluster and the best representative of this cluster had a ligand RMSD of 0.84 angstroms to the crystal structure. Now this was a very good result and gave us confidence going forward that Haddock can use this experimental data to dock these small molecules. So the next part of this validation process was to start using ligands that had rotatable bonds. When you're working with such ligands you need to identify the confirmation of the small molecule in the bound state and this does not always correspond to the lowest energy confirmation that you have in solution. Now different groups who have worked with docking have made different assumptions. Some groups have assumed that you can use this lowest energy confirmation. Other groups have made other assumptions. What we decided to do was instead of assuming the state of the small molecule in the bound state we would dock a library of ligands. We would generate all possible energy minimized confirmations and then we would give all of these confirmations to Haddock and we would allow Haddock to use the experimental distance restraints that we generated to not only dock the small molecule but to then select the confirmation which best fit the data. Libraries of all possible low energy confirmations were generated using MOE and then each one of these confirmations was individually parameterized and used for the docking. Now there are two approaches that can be used for this. We ran independent docking experiments for each small molecule. However Haddock can work with libraries of small molecules where you provide it with all confirmations and then tell it to produce structures in its IT0 phase for all compounds. If you're going to use the latter technique then you need to make sure that you increase the number of structures that are calculated proportionally to the number of small molecules that you have in your library. So by default Haddock calculates a thousand small molecules per structure or per small molecule. So as you increase the number you need to multiply that number by a thousand in order to accurately select for the correct population during that initial docking phase. So the second fragment that I worked with showed weak affinity with CoAD and had a single rotatable bond. The NMRKD that we calculated was 1.3 millimolar for the small molecule and the small molecule had a single rotatable bond around the methoxy group. When I docked both of these small molecules comparison of the docking score showed that there was very little difference between the two small molecules docked. However, when I looked at the number of NOE violations there was a considerable difference. The structure 2a had four NOE violations while structure 2p had zero NOE violations. So it was clear from analysis of these two factors which of the small molecules best fit the experimental data, which of the conformation best fit the experimental data that we had. And when I superimpose the docking structure of 2b with the x-ray we can see that indeed is the correct conformation and we have a ligand RMSD of 2.4 angstroms. Now the final core fragment that we tested was the most challenging fragment. We had it was a high affinity compound which had multiple rotatable bonds, but it also had a sparse NOE distribution around the small molecule. Now in the compound structure on the left hand side I've highlighted the groups to which NOEs can be observed to and as you can see there is one side of this small molecule that does not have any groups to which NOEs can be observed to. There were 14 different confirmations that were calculated that I had to dock and as you can see from the scores in the table below there were two confirmations that gave very similar scores and had very similar NOE violations. Now based on this data alone it would be impossible for us to differentiate which of these is the correct binding mode. However if you look at the superposition of the two docking structures you can see that the groups to which we have NOEs observed to are located in very similar positions. It's only the groups to which we do not have NOEs to that are varying in position in the active site. In this case in order to confirm which is the correct binding mode we would need to go through and carefully select two or three analogs which would allow us to tease out the correct which one is the correct confirmation and I will show you examples of this later on but in this case 3n was the correct binding mode and as you can see there is superimposes with the X-ray crystal structure nicely and produced an accurate and reliable result. So based on these three core fragments I was now confident that this methodology was working and could be used for fragments for which we didn't have crystallographic information for. So here is a summary of the core fragments. As the molecules get more complicated the ligand RMSDs increase slightly when compared to the X-ray crystal structure but in all cases HADAC is able to dock the small molecule in the correct orientation in the correct part of the pocket and the small molecule is orientated in a way that we can select analogs for structure based drug design. The protocol can accommodate multiple different types of ligands with multiple different types of rotatable bonds and by using the experimental data to both drive the docking and analyze the docking results we can select the correct confirmation. So now we've developed the protocol the next thing to do is to use this in a prospective way and dock small molecules for which we have no crystallographic information for. So two hits were identified by NMR screening to bind to coAD. The goal was to take each of these small molecules dock them into coAD and then develop these fragments through analog selection based on the docking results and then if all goes well we can then generate a co-structure with a more potent hit to validate the original docking studies. So the first prospective core that I wanted to work with had two different confirmations it had a single rotatable bond again around methoxy group. I titrated in the small molecule was able to follow the chemical shift perturbations in the spectra and from this I was able to generate a kd of 2.8 millimolar so it was a very weak binding fragment. The edited filtered experiment to generate the intermolecular NOEs elucidated 29 different intermolecular NOEs to protons all around the small molecule and the docking results shown below showed that Haddock positioned the small molecule in the again the phosphopandetine pocket which corresponded to the chemical shift perturbations that we observed and the methoxy group was positioned deep into the pocket. Now if you look at the schematic of the residue surrounding the small molecule we can see that there is space to grow the small molecule at position R1 and R2. R1 can only accommodate a single methyl group but there is space to expand small molecule much further from position R2. To validate the model that I produced I also selected an analog that grew from position R3. Based on the if the docking results were correct there is very little space to expand in this region so any additional chemical matter that's positioned at this position should have an adverse effect on the KD and also the other biophysical and biochemical metrics that I was using to analyze these. So I selected a number of analogs but the three which I'm going to show you here have a phenol ring attached to the peridazine core and an additional methyl group. The second group or second analog has a chlorine substitution at the power position of the phenol ring and the negative control that I talked about is the same as the top compound but it has an alanine group positioned at R3 which will vary into the core and should have a negative effect and these are the results that we observed when working with the biophysical the DSF and also the biochemical and also when working with the IC50 data. As we increased the chemical matter that was positioned at R1 and R2 we could see that there is an increase in thermal stability of the protein up to when using compound 9 we could see that there was a 2.5 degree increase in thermal stability by DSF. The biochemical assay also showed that we went from an IC50 which was greater than 8 millimolar to a biochemical activity of 546 micromolar so that was an order of magnitude increase in biochemical activity which is quite significant. So at this stage we have gone from a compound that was completely intractable by crystallography to a very highly potent small molecule. The compound 10 which we selected for a negative control indeed had the adverse effects that we were expecting where it showed no increase in thermal stability and the IC50 was also greater than 2 millimolar. We suspect it would have been higher however 2 millimolar was the maximum concentration that we could measure with this small molecule and the Kd of that compound was also significantly higher than the other analogs which we had selected. So based on this we were confident that our docking model was indeed correct but to validate this I generated a crystal structure of Kd in the presence of compound 9 and through comparison with the docking model we could see that indeed the position of the peridazine core of compound 9 is in a very similar position to that of the docking model. The one difference which we observed was that the orientation of the methoxy group had flipped between the docking model and the analogs. However when we were generating the models of the analogs in Moe we discovered that when we added in the second methyl group to the peridazine core due to steric reasons that should flip the methoxy group. So this was a result that we were anticipating and it was indeed observed in the crystal structure. Below I show a superposition of the docking model produced with the x-ray crystal structure and also the predicted position of that small molecule that we generated in Moe based on the docking model and as you can see the predicted binding orientation and confirmation of compound 9 based on the docking model is very similar to what we actually observed in the x-ray structure and these superimpose with the ligand rmsd of 1.4 x-troms. So we were very pleased that based on the docking results alone we were able to prospectively grow, perform structure-based drug design and confirm this with the x-ray structure. So to summarize I was able to use experimental data to guide docking and I was able to dock a fragment for which I could not generate crystal graphic data for. Using Moe I could I generate analogs which I then screened using DSF and 2D protein observed NMR and confirmed that these were indeed hits and that they increased in potency as I expected. Compound 9 which was identified had a 100-fold increase in affinity and exhibited the largest DSF stabilization effect and had the highest biochemical IC-50 called the lowest in this case sorry. I was able to generate a crystal structure of compound 9 and this was determined at 1.8 angstroms and we were able to show that the crystal structure was highly similar to the docking result that we had previously generated. So to confirm that this wasn't just a one-hit wonder I then took the second perspective call and I docked this. Again in the top panel we can see the chemical shift perturbations induced. This fragment had a slightly what was slightly tighter binding it had a 1 millimolar affinity and it had a biochemical IC-50 of 4.7 millimolar. Similar to what I did previously I used Moe to grow the fragment to identify analogs and then I also selected a negative control to confirm the docking results. In this case the negative control was compound 13 which had which was a different stereoisima and it had the imidazole group flipped on the other side of the unsaturated ring. As you can see in the docking model and x-ray superposition on the top right hand side the docking model had the imidazole ring pointing into the pocket. If indeed this was correct the imidazole ring would now be facing out into the solvent and we should lose affinity and this is indeed what we observed. Again giving us confidence that our docking model was correct. Selected a number of analogs which grew from the imidazole ring. Compound 14 had the difluoromethyl group added on and Compound 15 had a vinyl group and each of these gave increases in KD, IC50 and also showed significant increases in thermal stability when measured by DSF. We were able to produce crystal structures with both of these and again the crystal structures matched up nicely with what we observed from the docking models with ligand armacities of 1.4 and 1.7 angstroms with Compound 14 and 15 respectively. So with that I'd like to conclude the talk and acknowledge Andreas Lingell and Dirk Buzier who were my advisors when I was doing this work and also multiple other people at sites in Emeryville, Switzerland and Cambridge who helped and answered questions on a regular basis I'd also like to acknowledge here Alexandra Bovin who helped with Haddock related questions and with the implementation of Haddock at our site. So with that I'd like to open it for questions. Thank you very much Andrew that's great. So yes as Andrew says we are now happy to take questions and so if you do have a question and you want to type it into the question area you can do so if you're one of the organisers and you have a question you can't type it into the question box you can maybe type something into chat or I can just open the mic or you can just open your mic and ask a question directly feel free to to cut in if you want to do that. So while I wait to see if there's any other questions I should firstly say although that I should firstly say this is not an area in which I'm an expert so apologies if my question is not not quite correctly formed but I was very interested in the part where you were talking about the selection of the parameters for Haddock because I think this is something that we've we've heard elsewhere and it's not just true Haddock but in general it can be quite difficult to choose some of the input parameters for for these simulations. I was wondering how how much time was involved in selecting these parameters whether you're able to tell how sensitive your results were to the parameters and how you know that you've got the right ones in the end you have to wait till your end result like you were talking on slide 34 to say that you you got good results in good agreement or were you able to perform some earlier tests to check that the parameters you've selected were the right ones if you see what I mean. Yeah so we're very we're very lucky here in sense that the the compounds that I was benchmarking with all that high resolution crystal structures so we had a definitive endpoint that we knew was the a correct solution. Now again what is observed in crystals and what is observed in solution could be two slightly different things but generally they're in agreement with each other. So we have that checkpoint at the end of each run I could take the best cluster I could take the best structure from that cluster superimpose and I could see how far off I knew from the NOE data that I had based on the crystal structure that if haddock was doing its job properly it should put the small molecule in the correct place. Now with regards to how long it took there was a lot of reading on forums a lot of people have done work with small molecules it's not so extensive so there is a lot of information already out there about parameters that that haddock that works well with haddock and this is also where talking with Alexander came in very beneficial because he was able to also help us discern which of these were important for what we wanted to do and which were not so important but to answer your question yes it is just a lot of trial and error it's a case of trying these different parameters seeing which ones produce the best models and then ensuring that it's not just a case by case specific set of parameters if you go on to the next small molecule and you find that you need to adjust a different parameter will you then need to go back to the start and need to re-run all of the controlled experiments to ensure that that one change that you've made has not affected results elsewhere and through the iterative process of trial and error you can start to formulate a global set of parameters which that work yeah yeah good okay well um you mentioned Alexander we have him uh watching as well I think he has some questions so um Alexander do you want to uh unmute your microphone I think you can ask your question directly yes hi Andrew hi Alexander how are you yeah nice story uh I'm of course a bit biased here and so and so um so I have a few questions for you uh first the NMR part of it so you measure I know he's up to 8.5 angstrom yes because you have a deterioration and uh VTI labeling only that you can exactly so there are reports out there that uh in these highly deteriorated environments the amount of relaxation always this significantly reduces the amount of relaxation and you can observe distances up to 10 angstroms away now the reports of 10 angstroms come when you're working generally with stereo specific labeled leucine and valine sidechains uh these are the cases where only the pro r or the pro s have been uh selectively labeled and the the other uh methyl group is deuterated um in our experience when we were assigning our methyl groups using the metal walk we were quite confident that we could see distances up to 8.5 angstroms uh in our um in our milvat labeled deuterated system so yes we were confident up to 8.5 angstroms but you can certainly observe distances more than the 5.5 angstroms that you you generally measure as your upper distance limit in a uniformly carbon nitrogen labeled protein okay thanks and then related to the NOEs as well so you show different example first the benchmarking and then the the real application to a blind case which you get crystal structure at the end yes so do you have a feeling of and you reported different RMSDs for the ligands for different cases so do you have feeling of these correlates with the number of NOEs that you observe so whether NOEs were only on one side so yeah so it's correlate you you picked up the two factors there it's both the number of NOEs and it's the distribution of the NOEs around the small molecule and these are the two important things that determine how well haddock docks that small molecule in the in the test case where there were no NOEs on uh uh one of the sides of the small molecule in our results from haddock we could see that there was just significant variation of the position of that small molecule or that side of the small molecule um in in the docking results the the regions to which NOEs were observed to they were fixed well in position but everything else there's a lot of variation so it's if I was to say what was the more important factor I would say that distribution of the NOEs around the small molecule was more significant than the number so you can know a priori basically uh what to expect based on what you observe in terms of NOEs so that that's a good point yeah and I guess that that you have a large distribution of poses if you have a bad distribution of NOEs also comes exactly because of the lack of electrostatic exactly charges yeah which brings me to a question about the ligands so you more to generate the the minimized conformations yes compare those to the crystal structure so are those conformations really close to what you see in a crystal or and uh those haddock deform the conformations for example so related to that will be should you freeze the conformation between docking so this is this is an important point that I I neglected to mention during the presentation was that the benefit of so in each case one of the energy minimized conformations produced by MOE was the correct conformation during the docking process the small molecule was fixed it was not allowed to be uh rotate there was no conformational freedom allowed to the small molecule so the assumption was that in the in the library of fragments produced one of these is the low energy conformation found in the bound form and and haddock was able to select from the library based on the experimental data which of those conformations was indeed correct so but that's a very important point the small molecule was was not defined as flexible during the docking protocol yes and so more must be giving you also some partial charges or not so the MOE uh I don't recall if MOE gave partial charges but when I produced the topology in the parameter files using x2d all partial charges were removed from the small molecule okay and I have one question Adam yeah go ahead yeah no we have one other question from the floor um you okay can I take one from Andrea first and then we can come back to yours Alexander just in case uh Andrea needs to go um Andrea are you able to um open in fact I'll let me allow you to speak um if you've got a microphone um Andrea would you like to ask ask your question uh yes can you hear me I can yes yes that's good yeah thank you so I have a couple of uh technical questions um I'm from the University of Hanover um so one is how does your assignment strategy um obtain or deal with obtaining stereo specific information do you get it from the methyl walk in the nosy or do you or do you use an OR statement in the in the docking and then second oh sorry yeah uh so to answer that question uh the during the methyl walk we're able to identify the partner pairs of the leucine and the valine uh methyl groups however um correlating those to the uh crystal structure is a different thing because even though you can uh generate samples where the pro art or the pro s are selectively labeled so you can identify in your um you can identify in your spectra which is correspond to which when crystallographers um assign or when they they they they fit the the the residues into the electron density often you can't you can't tell the difference between flipping the site in around if it's poor electron density so what's labeled as pro r and pro s in your crystal structure may not necessarily correspond to what is actually pro r or pro s in your nmr spectrum it's again similar to what we were talking about before um it's the case of trial and error you you see when you run your docking runs you set them up with one permutation if um and but you need to make sure then that once you have connected pro r peak in the spectrum to the corresponding methyl group in the um structure that you use that same nomenclature each time yes that's what we did so we identified we identified the correlations and then i annotated it accordingly and made sure that i used the same um the same nomenclature each time yes and the second question is whether you have tried using glycerol as a deuterated glycerol as a carbon source for your milvat labeling whether if glucose is absolutely necessary because this makes a difference in the in the cost it does it does and this was something that we did not investigate we did not look to see whether deuterated glycerol could work i see no reason why it shouldn't deuterated glycerol can replace glucose in a normal expression so there's no reason why it wouldn't work but i would assume that there would be similar caveats that this it would be slightly slower growing and it may impact your your total cell growth or total cell mass at the end but i see no reason why it shouldn't work okay thank you okay thanks very much for your question andrea um so alexander i think you had one last question that you wanted to ask i think we've still got time for one more yes out of curiosity did you try to use some of our small molecule docking software more is capable of docking as well i would think so so we tried looking at um other uh software that we had uh access to at novartis um but in all honesty haddock was superior when working with the uh experimental enemy restraints because haddock would use this data to actually drive the docking other programs appeared to want to use other parameters to actually drive the docking and then use the distance restraints that we put in to filter the results um which wasn't what we wanted we wanted to use the experimental data to to actually drive the docking so we realized very quickly that if we wanted to do this that haddock was pretty much our only option so we looked at mo we looked at gold we looked at um icm focus also uh but i said haddock with regards to setting experiments up and producing data um we found that it was um far superior these other programs were quicker to run their um their their docking runs but like i said they they didn't run them in a way that we wanted to run our experiments we couldn't we didn't find a way of running them in a way that we wanted to run our experiments okay thanks general thank you thank you very much everyone for your questions and thank you andrew for your answers i think we're going to wrap it up there for today but if anyone is watching later and they have any other questions do come over to the bio excel forums at ask bio excel and you can post your question there and we can pass it on to to andrew or andrew can connect directly to to answer that um so we'll hopefully see you again soon at another bio excel webinar and until then thanks very much for coming along and we will uh speak to you soon bye


The energy challenges of Japan and the world and Asia go far beyond just China and just Japan. Today I'd like to talk first about the energy challenges, four of them. First of all, geology, geography, growth and geopolitics, and how in their inter-relationship they create some rather counter-intuitive responses that are going to have major long-term implications for the broader world. First of all, as we know in terms of the geology, the energy resources of the world are certainly finite. There are many ways they can be satisfied, but finite. In terms of energy, Asia is particularly short. 2% of world oil reserves and only 8% of world gas. The geography is also difficult. As you can see, the reserves most importantly are in the Middle East. The growth, the demand is in Northeast Asia as we know, beginning with China, Japan and Korea. And so the imports have to come long distances. Japan for example, 85% dependent on the Persian Gulf for its oil imports. Korea 87, India 62 and China has been diversifying more toward Africa also as we will hear. But still 42% of all of China's oil coming from the Middle East. Growth has intensified this problem. We know very well about the rapid growth of the East Asian nations and also their adaptability since the layman's shocks. They've recovered rapidly, more rapidly than other parts of the world, which has intensified the energy problems. Oil demand in general has been growing faster in Asia despite tremendous improvements in energy efficiency that it has in many parts of the world. And as a consequence, oil imports have also steadily risen both through the sea lanes and also increasingly overland, with China being the most important. Finally, geopolitically, Asia in the world of course stands in an unusual position. Increasingly dependent in the Middle East in a world where the United States position has traditionally been stronger. Also the multinational, the so-called Seven Sisters and others have been crucial in the energy world. I'd like to look now at the responses that are possible for Asia. Of course they're the sea lanes that I'll talk more about, but I do want to dwell on the concept of energy continentalism. The major producers in dark and in light, the large consumers beginning with China, are right next to each other geographically as you can see. Major political changes, the collapse of the Soviet Union, China's modernizations, the Indian reforms in particular have, those critical junctures have created a much more interdependent Asia whose importance I think is just beginning to understood. This is a geopolitical as well as an economic phenomenon. And the growth process will deepen the interdependencies. 18 million barrels of oil roughly a day from Hormuz to Northeast Asia. That will probably rise to around 30 million. We've heard about shale gas, certainly that will be important, but the levels of demand are rising also. Conversely from the Middle East itself, from land rovers to land cruisers as I put it, the dependence on Europe has been declining, dependence on Asia in the Middle East for autos, for textiles, for electronics has been rising. This has major geopolitical implications all over through the sea lanes. There are various dynamics in the sea lanes, particularly deep in the Indian Ocean, I would stress, where the Blue Water Navy capabilities of the United States are particularly important, Diego, Garcia and so on. What is the response for China? One increasing possibility is continentalism. China's energy demand is rising rapidly. President Xi, the last 10 days, as you know, has been in four Central Asian countries in Russia. His second trip to Russia within this year. Pipelines are increasingly important. The ASPO for oil has been completed. As you can see from this map, the western reaches of China are considerably closer to the straits of Hormuz than they are to Beijing. Energy demand is moving further to the west. The alternative one, as Professor Lampton suggested, could well be nuclear. The other is deeper geopolitically related ties, be they natural gas, be they oil, further to the west and a deepening interdependence with the Middle East. So where is that continentalism likely to lead and how fast will it emerge? Thank you.