Open Source Antibiotics Series 1 - the Mur Ligases, 5 Oct 2021

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From YouTube: Open Source Antibiotics Series 1 Mur Ligase Update Oct 5th 2021

Description

Open project meeting for Open Source Antibiotics Series 1, the Mur Ligases.
Full Project: https://github.com/opensourceantibiotics/murligase
Relevant GitHub Issue: https://github.com/opensourceantibiotics/murligase/issues/51
On the call: Professor Matthew Todd, Dr Dana Klug, Yuhang Wang, Kato Leonard (UCL), Professor Chris Dowson, Laura Diaz Saez (University of Warwick), Lizbe Koekemoer (Diamond/Oxford), Dr Lori Ferrins, Dr Joe Eyermann (NEU), Peter Horanyi (UCB), Dr Jan Jensen (University of Copenhagen).

A

All right so welcome everyone. It's 5th of october um opens this antibiotics ligase meeting and we didn't have a meeting last month. So um we have lots to talk about. I think today, and there are a few key things I think, which of um which have come up uh which we can go through as the main part of the meeting. I don't really have. I don't really mind which we go through first actually, um but just because I'm biased, um I thought. Maybe we could start with.

A

uh You hang your compound, uh which bart was looking at or peter and peter, and about looking at at ssgcid, um where we think we have a nice structure of your compound bound to merge c.

A

um Now, as I understand that the crystal structure came back and there was a bit of disa a bit of misunderstanding about the structure of your compound, you had right. Did we resolve all of that? Yet in terms of getting the right small molecule bound.

B

I I think so uh previously I had a personal meeting with peter and he showed me uh he showed me a general view of compounds in the pocket. uh The problem is, uh he managed to overlay.

B

uh Let me let me show you.

A

Actually, you know what you hang if you can bring up that picture from in a minute when peter may be able to join us. Oh that might I'm sorry. I forgot it was actually peter who was who was doing this the structure. Let's do that in a minute when peter maybe had a chance to come yeah, I'm not.

C

Going to be able to comment on this, so you say that again sorry, I said I won't be able to comment at all. Okay.

A

Let's wait for peter sorry eugene. Can you unshare and we'll do this in a minute if peter comes along yeah, sure sure, okay, that'll be great, all right so um and we'll we'll see your chemistry update in a minute too, um all right, so we can skip to something else. Then, uh and I guess the people on the call who who are here will be able to comment on that. So laura. Did you want to talk about the thing you gave us a teaser on earlier about the atomized compounds.

D

Yeah, I can give you another teaser, because I'm not done so. I don't want to show all the interactions with the protein, but so I did. Let me try to share.

A

So the long last atomized combats have been examined.

D

Finally, yeah so finally, we desk on the china stars that we got uh that it was a mixture of compounds at the molecular weight of um fragments and the otherwise mini library that we got from ucl. They were tested at two different concentrations and I got different heats and one of them is in a crystal contact and the rest are all in the allosteric site, as you can see on the on the figure that I showed there.

D

So again, I'm not going to show all the details on the residues, because I need to do more refinement because there's a lot of residue movement in some of the cases, and so I want to have that done before I officially share it, but it'll hopefully be in the next couple of days.

D

um I did a lot today, so I think you know it should be done soon. um Yeah so, and we were thinking on the next steps after these kits because they look quite nice. The density looks quite nice, um except for one of them and but yeah. So we were thinking on the next steps.

D

So so we discussed- and we are thinking of making some activity essays and banning essays on the sbr against the three different mu li cases in the presence and absence of adb or amp pmp depends on how everything works out on the essay and see we get some binding themes. So I will do the sbr and becca will be doing the activity essays if yeah. If things go as planned and then this is the structures of the compounds.

E

I can can I'm sorry all right. Can you go back a slide? I'm just your nomenclature. If you could clarify your nomenclature here, a little bit so when you say something is allosteric. That means it's not in the adp site or not. It's quite clear what you.

D

Mean by alexa, yes, so that's the what we call the yellow static pocket or the pocket. So.

E

This is where the other earlier compounds uh and your e were binding. It's the same binding site.

D

So we had bind in three binding sites and one of them was the static site. So yeah, please.

F

D

On this, but we got hits on this side that inhibited, mu, e and mu d uh from becca's last results so yeah, hopefully that that's also continuing here.

E

And what's crystal contact me.

D

Crystal contact does the bottom compound here: can you see it it's white, unfortunately, but yeah that compound so is close to a crystallization contact, so I won't. I wouldn't consider this compound as um a binder like inhibitor.

E

Right so I'm discussing I'm just guessing here that that, when adam wise made their selections, they were targeting the atp site, or am I making a bad assumption? I want to make sure.

A

We need to go back and ask that question because.

E

I think this so if you can go to the next slide, thanks thanks thanks laura for clarifying what you mean and guys.

F

E

Allosteric again is the same binding site as the compounds that were previously solved and very correct. This is actually the same site.

D

Yeah, so it's the same site. We got data from already, but again we got heaps in other sites as well. This is a third type that we wanted to investigate. So I assume the atom wise compounds were targeting that site, because I'm quite surprised that everything is targeting that site and now the next slide is where you can't see all the structures.

A

You will eventually be able to get interaction maps here, obviously right so.

D

Yeah yeah yeah.

A

Right yeah to confirm the usefulness of the contacts exactly because, if you screen you can see, there's an overlay yeah.

D

So there's another like on the other one you can see, but we'll use this a bit difficult to see here with all of them, but yeah yeah. There are a few of them with some overlay, but yeah you will see and then this one here is close to the allosteric side and looking outside, uh but that one like we could consider that one as trying to get you know we could make a linger and make bigger, fragments or something like that. That kind of you know the I've seen on the fragrance yeah.

D

So that's another thing that could be investigated but again, like I'll finish their family, and I will upload all the contact things on the pdvs on on the shelf or shared website. So everyone can access it.

G

Great, what we did earlier like an hour ago is just a quick overlay between the same protein with adp band and no no inhibitors and the helix, that's just in the front of the binding site that moves significantly inward towards the binding site in the presence of adp.

G

So what was why we want to do the spr to see in the presence and absence of adp? Is that still an available pocket for for these? For these compounds.

D

Yeah, because yeah this protein is quite flexible, so yeah we were wondering how actually it will interfere into the essays or the inhibition. So yeah.

H

Laura can I just clarify so um when you tested all of them, you are you're not seeing any that bound in the substrate side or the adp side. You're. Only seeing these allosteric ones is that right, yeah, okay,.

E

So so, let's just follow up dana's question there. I guess um I mean looking at the structures now, I'm thinking it doesn't necessarily look like adp competitive ones. Does anyone actually know what adam wise did in terms of selecting these compounds? Were they actually using the muri structures previously solved that had this allosteric binding site? Is that what they were basing things on, or I mean, does anyone know actually what item-wise was actually how they were choosing their compounds? Is that documented anywhere.

A

Yeah, so I think we have the proposal. I think it's on the on github, but I can dig it out if it's not, um which describes what we asked them to do.

D

Yeah try I tried to find it, but uh I'm still getting used to there. Okay.

A

I'll make sure it's out there I'll link to it, so we can have a look because it is. I am a little surprised, yeah that that they are where they are, um however, that they are clustering so nicely in the same place makes me think that maybe it was intentional, but let's try and dig it out and find out what they did right. We are, of course, at liberty to go back to them. Ask for some clarification.

F

Another thing: if I can confuse it, is we only released the structures with that allosteric pocket enough last year. So that's after the compounds we designed, so it's not really likely that they had that information.

A

Oh that's interesting.

F

I

G

We could share this information with them in this slide.

A

G

The follow-up request saying this is what we've got yeah was that your intention.

A

Yeah yeah, okay, right I'll, follow up with and getting the original proposal and I'll I'll see who the person is. Who who is the right person to talk to about follow-up questions.

I

Good. Thank you.

A

But very nice- um and hopefully you have enough compound, obviously to.

D

A

To follow this up.

D

There should be enough for all of them.

A

Is the plan to do activity essays with the full set.

D

Er with the hits.

A

Only with the hits well.

D

That's what I was thinking but because I don't think we correct me, isn't if I'm wrong crease either we have the high throughput essay set up.

G

uh Yeah yeah yeah yeah.

D

So then we could do all of them. If you.

G

Know if there is hunt sometime and it'd be good to put the the ones that are not hit soon as theoretical, never negative controls when they're just yeah.

A

I mean there must be a a non-zero chance that the molecule inhibits the enzyme but doesn't bind here.

G

But it's not binding anywhere yeah yeah, so these are the ones um okay um yeah, so we're gonna have a go with the uh with the spr as well, because if we get a null readout in the assay, we need to get some idea as to what's happening, and maybe it's the impact of the structural change on adp. That's our problem or our friend. Maybe it tightens up the pocket.

G

Maybe we have to go back to atomwise saying this is great, but this is what this is. What it looks like in the presence of adp. You do some further modifications uh that might be worth going in for that request. At the same time, to avoid two emails, perhaps that's, let's see.

D

Yeah, I have already ordered the sensor for the sbr.

I

Oh thanks, laura.

D

Hopefully, we'll be here soon.

E

So I've forgotten what becca's experiments have been in terms of uh actual functional activity. So if you do the spr, that's not really telling you if something is a functional, inhibitor right! It's just telling you if it's binding, so I'm just trying to remember uh if this allosteric pocket has been shown to be a functional inhibitor beyond just a binder.

D

Yeah, so that's why I was saying we need to do the essay as well so they're compromising. I.

E

Was wondering what just the bigger question around? What with the earlier compounds that were found to bind in that pocket? Were they ever shown to be functional, functional inhibitors, as opposed to just binders.

D

So what I've seen on the hub and they were tested and they showed in inhibition, they were three showing innovation. um That's well, unless I got it wrong, but.

E

Yeah, it was a little unclear to me because I thought there was that was another thing was kind of unclear to me because some of becca's comments were it wasn't quite clear to me that she was saying they were actually I mean they showed some inhibition, but I mean this was all done at one one million more right and they were very high concentration, so it.

D

Was 0.5 million? I think so. Let me I'm opening anyway.

E

We can do this offline. I was just it's one of the questions I guess in terms of this particular site. Is it functional or not, and then you know what what at what level is actually functionally inhibiting. So.

D

This is what I saw understood right and you see it yeah, and then these are the results for the new e from becca and she had three hits a five eleven and eight that she followed up and tested them on community.

E

D

If you look at the bottom.

E

Yeah, if you look at the bottom, I think there are bottom comments, so just on this at the very bottom.

D

But there were no crystal structures.

F

Yeah yeah, so what happened was the ones that inhibited we didn't have crystal structures for the ones with crystal structures we didn't have inhibition and it wasn't that it wasn't really consistent. So it was, of course, the series, so it wasn't really predictable. So we kind of ended in a bit of a dead end there not feeling we've got enough evidence to prove that this is illustrated or not all right.

E

Yeah, that was kind of so.

F

That's so that's.

E

The last comment and that that was saying he didn't really look at the last comment there I mean, I think what he's trying to say here is that yes, it's a little inconclusive at this point. It's like, I think, at this very high concentration. If I understand that's how I was I was interpreting.

D

Yeah well, the constitution is thin, but yeah, but.

F

D

F

And also focus better compounds because our compounds are basically brick dust, so it was in any case difficult to do anything with them, except crystallography.

A

Share that sorry, could you show your screen again, just while you've got it in front of it, so we can have a look at the sites that we're talking about.

D

Yeah yeah, I want to stay calmed.

A

Yep so the the structure at the top of the page, which which site? Where, where are the atomized compounds, binding.

D

A

Right same place,.

D

A

Fine, okay just want to be sure it's consistent terminology.

F

Yes, okay, so you can explain it, but this actually, I don't know on the input structure, there's no gap there, and then we managed when the compounds bind it's as if they remove that helix the right thing, yeah exactly right and then it forms this little gap. That's why we call it a pocket. We just it forms this little pocket and our hypothesis is trying to see if it's allosteric or not, and we are inconclusive um on the side for the covalent people there's also cysteine in there which we manage to bind epsilon to.

F

So there is potential interactions, but we have no clue why? How or what our compounds even get to their you know form.

A

So on the face, if it's a little strange that the atomized compounds are.

F

A

F

A

Just a little bit.

F

Because I don't think atomize would have yeah postulated a potential pocket in the middle of an enzyme.

E

Right all right, gotcha.

F

Yeah well, no, it's very.

D

F

I mean it's very.

C

F

For my what we were trying to do that all that was compounded there.

D

Yeah, I wonder I I hope, he's relevant.

E

Actually, lisby, I'm sorry! This is this question a little bit off topic, but it's kind of interesting to me so under the x-cam you're, basically doing soaking experiments correct yes, so normally so normally, these are actually there's enough dynamics here to actually open that helix up and compounds to bind and trap.

F

E

So, actually, in this, in this kind of general x-chem experiments, you do see some of this dynamics still even in these soaking conditions. Yes,.

F

Now, especially for the mre that laura just mentioned, it there's a lot of helix movements in there. So when you actually start solving the structures, there's a lot of movements that you don't normally have with vapor, and that's also what takes so much time to solve these very crystal structures. Every time.

D

Yeah: okay, there's a lot of refinement. Even you know, on the pockets, because everything is moving.

F

And I don't know joe remember way in the beginning, when you sent your first, when you embarked at the library that I told you we were so that was two years ago, we were really having trouble because panda was failing, and that was because of those movements. So there was no consistent base model which it can could easily compare it to.

D

Yeah I had to make two models.

E

Yes, thanks thanks, laura thanks.

A

uh Lizzie, just while we're on the subject, I guess the um the question came up about those last results from becker and we were talking last time about about becca doing so.

F

Trying to validate.

A

Some of those hits with those response- curves- um I guess becca's not with us today chris, but do you have to know if that's the goals of the.

G

A

G

Yeah it's in progress but she's off at the moment. Okay, um yeah.

A

She sent me okay now, where is fema, because dana and kate and cato have been have been looking at some of these compounds, I'm looking at just they just posted earlier. um Some of the kind of you know the structures of the molecules that appear to be uh hitting both mercury and mercury, medium e, um dana and cathode.

A

Did you want to share that briefly, just to give people a sense of what you've been looking at, because we've been trying to make sense of which molecules have have been found to inhibit and which have not and whether there's any patterns.

J

Yeah, of course, I'll share my screen purple.

E

Using the word inhibit here, I think we ought to be careful with our nomenclature.

J

So yeah um maybe a little side. Note I'm not an organic. You see my screen right: yeah, okay, perfect, um I'm not an organic chemist by training. So it's new for me as well. I'm studying drug development, but little by little, I'm getting the hang of it. So please do not hesitate to correct me if I'm saying something wrong um I'll try to keep it short, but the aim was to visualize these.

J

I think it were 78 compounds or something uh to have a more clear view of what's going on with the mergie murdi percentage activity.

J

So I think it's from the fragment library and then these at compounds were tested against nerdy.

J

There were 12 compounds that had an inhibition greater than 80 percent and then six of them did pass the essay interference checks and these were tested against near e, um and so these are the six compounds that I was talking about, and this was my starting point to make the structure.

J

The um the cores that were mutual for the compounds- and then there were three main course structures.

J

So this is the first one um as you can see they, the cores differ um from each other here with the redeem, and you can see that the substituents, the r substituents here are quite small molecules.

J

um Interesting to see here is that if you change the methyl to methoxy that the activity, but the murder activity increases a lot, so it gets the inhibition activity, get gets worse and maybe important to say is that the compound compound numbers involved?

J

These are the ones that were also tested against near e, so they passed the inhibition and essay interference.

J

Checks then, in the next slides the substituents are quite bigger, so more the aromatic structures um seem to do better than just a cyclic structures with a mine. The compound really doesn't like the amine in that position.

J

um So yeah, the naphthalene here is doing real good, but, as you guys were saying, the concentration is quite high, so we need to dose response curves. I guess um to make something of that.

J

um Then this is the second core. It's actually the same, but with two substitutions.

J

Here, oh yeah, with the methoxy alone was quite it was just inactive. But if you add the bromine in the r2 position, it gets active.

J

um Yeah this was just to clarify what may be happening in the in the pocket, but this was the original fragment and it was assumed that this amine or the hyperzine nitrogen was necessary to interact with the glutamic acid, but as we can see in the next core, this nitrogen is not present, and it's here is one of the most innovatory active compounds of them all, but it was not tested against their e because of sa interference. So I'm not quite sure what that is. That means um what can I say about that yeah?

J

So again, the aromatic structures are doing better than others again the interactions with the amino acids, and then I thought it was interesting to see what's happening when you change the core but keep the substituent. And here you can see that from the iso guterramite to the cuisine it becomes inactive for bromine. um Unfortunately, for these aromatic structures, we can't compare them because of sa interference, um but maybe this one is interesting for the pyridine um here.

J

The the first score was really bad, but if you change it to the isobutyramide, it's get it's getting very active.

J

Then, if you compare these two, um the activity increases a little bit, but not as much as with the bromine. So I think aromatic adding aromatic structures is probably a good idea to get good inhibitors, um and this is the last slide. These two are the most common cores, so it would be interesting to see um how these activities change and for the aromatic structure they kind of preferred the vipridine ring here, while the bromide is rather preferring the isotope.

J

That was it, I'm not quite sure the organic chemistry behind it. I'm not an expert yet so I'll. Leave that to someone else.

A

Okay, thank you very much. That's great joe! Do you want to come in on your point about the inhibition thing, because the assay is trying to measure this and what's your what's your view.

E

Yeah I just yeah it's I think I I have to say I'm a little. I just we need to get. You know, ic50 data uh really to drive things. I mean this is great for, for I'm sure. Is it cato, I'm not sure how to say yeah, I'm sorry I mean I think it's just awesome. I know you're you're, you know just getting into this and so just going through this kind of analysis. This is just kind of a standard, qsr analysis.

E

So dana is helping you with that and it's that's just a great learning experience, irrespective of the data itself, um but yeah. I think it's at some point. You know we obviously would like to be getting. You know some real dose response data um so um and then again, I guess again. If you know with what the adam white's compounds you know now, you've brought in a lot more diversity. So now you can start thinking about. You know other kind of hotspot, analysis and other things about this.

E

You know this binding site, um but it would be great to figure out if it's actually a functional functional site. um Does it really inhibit? You know processing of a murray ligase, for example.

E

So but anyway no it's it's just more data right. So that's, I think, that's all a good thing, we're getting more data.

A

Any other comments on on this. You know we're trying to essentially spot patents for things that gave a uh some kind of you know: low percent activity versus those contents that did not, even though the compounds are small, obviously- and we have to be careful.

H

um So just to say one thing, which is that um so these are the data, for this would be um the fragments that fahimah and I made- and she also made some um with like a direct linkage between the sort of alkyl group and then the fennel ring, and basically none of those were really showing up a lot of activity um and then, of course, all of the sort of hits that becca investigated further against murray. Had this sort of amide structure.

H

So I think that that's probably important um and then just we can sort of see that from this data I think some further substitution on this ring is tolerated, because I think the original um hits that we were working off of had very small substituents out here. So I guess just sort of overall trends, the amide and then there's sort of room around that arrow ring to sort of get a bit bigger.

A

Okay, so I guess the the crucial dose of fun stuff is uh we're waiting on, and that will tell us much more yeah.

G

Yeah and then just thinking about following up, I mean historically, the failure of these compounds. Not these, but neoligase compounds is one is one big reason is and is getting into bacteria. So just have that front and center of any decision-making process as to what gets added.

A

G

A

Way, chris, thanks for you, hangs work um if we're happy to move on.

A

Okay, if you could just unshare and then eugene can take over the screen share. Okay, do you want to just talk about the chemistry you've been doing, and then we can? We can go from there to the crystal structure from peter, oh yeah, right.

B

It's my pleasure to share the results with you guys, yeah. I can see for the last two months uh so uh for the isa compound joe picked, uh I've managed to write it and sent out to sgc peter has to crystal over the data.

B

I just need to overlay this structure with az5495 in the pdb6, as well as the exit a074 in the 69f, so both of these two structures share some similarities with the product with the product made, but like they're different in some details, so I think we need to first overlay them with this product and to see if uh to see uh which pocket. uh This is the as part of the molecules occupying so yeah uh for the uh az5595.

B

uh So uh previously the uh h5x series did not give the right structure. I want a meso group uh pointing towards this direction instead of the other one, and this method was proven by the euro formation crystallography, showing that uh with this, under this condition, you can only make uh the five member core with the uh method pointing to towards this direction. So it's not the ideal structure. We want as we're expecting uh a key interaction here at the this nitrogen uh with uh with the music.

B

So uh so I changed the different routes I decided to protect. uh I did to change the starting material, uh protect this aiming first and then methylate methylated and then the protection, so it turned out to be working uh and uh it's pretty uh it's pretty promising. I managed to like destroy uh make this uh structure with the method pointing toward this area. uh Supposedly, I need to double check with the euro formation and crystallography just to see uh just to see uh the method group is actually pointing toward this direction.

B

uh So hopefully I could mic and ship the id595 uh in the next two weeks yeah. So uh the future focus we're just trying to make the aiming derivative of asic compound 595 and here's the uh synthetic route I planned just to see if any one of them worked. uh The short longer steps shot steps just to see yeah they're, all backups.

A

Yeah, that's all great! Thank you. um So the compound that you sent to ssgcid is: did you just go back to that again, yeah that guy okay? So um peter, are you able to say something about the crystallography for this campaign.

A

I can't hear you peter if you're talking.

K

Yep thanks uh double muted yeah. Now my uh little speaker had the extra mute button on it. Besides the one that's just on the screen, so um yeah we two-point, we were able to collect the 2.6 from set.

K

We can confirm that that is the compound that we received. um The methyl group is going to be, um it looks like it may be there, but I will give you a refined structure here in a couple weeks.

K

I just had some computer issues and so that's why it's not fully refined yet, but overall um we were very happy with what we received, as well as the feedback and the smile strings. So we were able to make the parameter files and so we're able to continue the data collection. I mean other the the refinement we're going to use the compound in the other systems in the acetobacter systems and so we'll be able to progress with that.

K

So we'll give you um information as those progress as well, both thermal shift as well as crystallization, and so we'll. Let you know how those will go and then um yeah we'll keep you updated and now that vacation season is back is over. I think that we should be back at it again, so providing you with some data.

E

So the s native factor is is near d,.

E

um Pardon so the s anita back. There is some your d.

L

uh Isis sign the mirror d, so we did the pseudomonas oregano's.

K

uh um Your c was the original one. We just reproduced the 6x9 f structure, right right of sorts, but we'll we'll continue in the acidobacter direction, because I know those are the interesting ones that you want to see whether these will bind, and so we can do mure c or muradi, whichever one you want, we can prioritize.

E

I think you know to show doing a dual binding and, and in your c and your d, so yeah. If your d would be my my preference anyway, um to try to get a structure and interview in an erd system makes.

K

Sense we'll we'll go in that direction, though.

E

So yeah the spr or I'm sorry, it's not the spr. What's the uh the technique you're using to detect binding or I'm sorry.

L

E

Differential scanning format.

C

Those three compounds uh from az, um so we did receive them on friday, um according to fedex I haven't. Actually it was working from home yesterday I'll go in today and make sure they're actually there on my bench, wow, congratulations! So, let's thanks to joe, because I had given up on them for sure.

C

What do you want me to do with those again, let's just pretend that I forgot everything that I've ever known and what would you like to do with these compounds.

E

So so I guess my thought would be if peter can do the this this nano dsf peter I mean that seems to be your go-to method right. um So would you use that to can you do that with a couple of the different isozymes, I mean again looking at we're trying to get some.

E

We have the mirror c structure right, your pseudomonas in your c structure and I guess the keys. The key thing we're looking for is to try to show that these compounds would also at least bind to mirror d, um and so that would be you know any of the mirror d isolines you have um so I don't know if the the most straightforward thing would be to test those three compounds in the nano dsf to see. If you see any interactions, would that be what you would try.

K

That could be one. So if you wanted us to to look and see whether these compounds are binding to either of those proteins that we have the mirror, these um spr will be a lot more sensitive and we could potentially do that, but we haven't done it before, so it would take some optimization so anyway, it would take a lot more time than that of the sf.

K

The nanodsf is just a good way for us to look and see whether or not there's thermal stabilization, because often thermal stabilization also suggests that we can get a structure.

D

K

D

Share the spr optimization whenever I have it and just to mention, we also got we just got this week, the e-coli new d and I'm gonna spread some pretty fine last week. So on our end they will something will be happening on e coli. Nearly.

E

So that goes to chris thousand, I mean so where's the status of your az negotiations with warwick for these same compounds.

D

Say again, sorry.

G

Yeah I have about contracts going on at the moment uh and I um I can't remember whether I'm happy or despairing on this one um I'll I'll, have a look and update people with a smiley or sad face.

E

Yeah, it's just, I mean basically the way these contracts are written. I mean you know uh the ssgcid gets, you know, got the three az compounds, but it has to be a separate contract for warwick to get the compounds, for example, for laura to do e coli, unfortunately, but yeah, hopefully, there's now now that at least you know, one group has been able to get the compounds. Hopefully um warwick will be able to also receive the compounds in the near future.

A

The structures of these three compounds must be somewhere, and I forget, where they are so to someone have the smile, strings and stuff that we can.

C

Well, yeah I mean I have we have them in the there's a publication and then it's also in the proposal which is on somewhere on the site, but I can send it to you again that.

A

Would be great yeah I mean I just want to because I mean I guess, the same experiments now that we've got the structure with you hangs compound. I guess we, you know we have that possibility too, that that that should also be looked at with multiple mers right and so there's four compounds which we can play with in terms of getting structures and binding data for for compounds across two enzymes correct. I want to make sure that the the you hangs, comet isn't one of those those air compounds.

A

C

So just to clarify, for me, I've got this box of compounds and I'm going to get it to my team member at ssgcid, the and they're going to do. The preferred thing to do with them is um spr against the mr um d.

E

Well, I mean, if you can do that, but you were saying the spr was a little more complicated, so I mean I guess, if there's the resource to do that, that would be interesting. I was thinking you know this. The first pass was going to be you know. If peter we can do crystallization trials with those compounds. You can smear d, any of this, your d isozymes and then you know I was just thinking he was.

E

You know talking about the nano dsf, that's just a kind of a filter or at least giving hope that if you, you know see some shift that you might have a better chance of getting a structure. um So I I don't know you know that that's up to peter.

E

What's the right process is, but the idea would be is mostly is just trying to get a structure of these in a mirror d, any of the four as as todd was saying, uh matt was saying that that basically the the four compounds you have four compounds in hand. Now right, you have what you hand made and then the three az compounds.

E

So that's four compounds to try to get a structure in a murder, a mere de-ice design. That would be, in my view, the goal.

A

Yeah, okay, that's it.

K

And you're agnostic, to which mere d, whatever works,.

E

A

E

Think you only.

A

Have you have that.

M

But you don't have.

E

You don't have e coli right, so um I mean maybe maybe at some point. um I don't think it really matters per se. If you can get a structure, I guess it becomes. You know which system might be more minimal.

E

There is already an e coli mirror d published structure, so someone's been able to get a you know an e coli or d structure or crystal structure, I'm not sure about acetobacter. I don't know if there's an estimator back from your d crystal structure, I I can't remember yeah, um we've got one, we do have one okay, so I think any of one of those ones you can try which material you have.

K

It sounds good merely from pseudomonas and acetobacter in hand right now. The other ones are not for us.

E

Oh okay, so actually, if you have mirror d, I mean obviously the the gold. The gold thing would be, probably you know, pseudomonas in your d right just to have the match pair in the same same system right. That would be obviously the preferred.

C

Well, you have the protein, but you don't have the structure. You're saying you have you have the struct? We have you have the structure, that's the needle back there right. There is a structure of e coli and- and I think laura is doing that one right, so we won't do that. We have the pseudomonas protein.

C

Do you have any idea if it's a decent construct or anything? I don't know.

K

We got some, we got some crystals that diffracted, but we can't solve it because it's got some funny business with uh some. We just can't figure out what it is. It looks like that we may be able to solve it, but it's been. We want more data, and so this we've been waiting for this compound to see if uh you know which direction to go. If we want to do april or if it's easier with the compound.

C

Do you say the data is weird? Is that but we'll well, you know my opinion on that. One change all the systems.

B

K

I get it, I think that you know overall we're we're close to the mere d structure, but we'll keep the group in the loop. It's just. um We were waiting on these some of these compounds and now that they've arrived, I think we're going to be re-energized and be able to give you more information.

E

That's great peter, thank you. This is this is really exciting times here now.

A

Yeah, it's really good um all right. We've got a few more minutes and I just wanted yan to have a chance to um that. Isn't any of the modeling that he was doing, because I I saw some activity but but didn't read it before the meeting yeah did you want to update people? Yes,.

M

uh Let me share my my screen, so we've been casper and I uh my let's see, can you see the github page.

A

Yeah: okay, it's a little small but ah okay.

M

Yeah, okay! Well, that's good, actually, because the bottom line is that these are wrong. um So in trying to get everything together, uh I made a mistake here and, and so there's a single precision and an extra precision and we trust the extra precision. These are the single positions. uh Precision so uh casper just sent me the data.

M

I need- and I need to make uh some changes here uh to yeah to to to show to you for and and then you can pick from them and once um we see what the top scores are, we'll also get the poses. So my understanding, where that you would like to see the docking poses before really zeroing in on on compounds, so so basically what watch this space watch this issue uh within the hopefully this week I'll be able to throw something up. That's that's correct.

A

Okay, awesome. Thank you for that.

A

That sounds great. Thank you very much. um I mean just on that. The uh the other, the only other may not reasonably agenda was that uh we uh so me, and chris and frank von delft, put in a proposal to antibiotics research uk for a little bit of bridging funding to um try and allow us to get the data that we're looking for, which is the preliminary data of one molecule, inhibiting two mers, and we got that grant um it's going to be announced in about a week.

A

So we've got a 12-month grant about 50k, which will go towards chemistry and supporting dana for a little bit more time on the projects. So um it means that we do finally have some. We have some money, it's absolutely amazing, so we we can buy molecules. We can buy some commercially available molecules because we put that in the budget.

A

So if, for example, you know the atomized compounds come good um and we're happy with those or yan, you predict some molecules that you're really confident about. We finally have some resources to get that which is great, so um yeah bridging funding uh nine months. You know probably nine to 12 months and that should allow us to get enough money to go for some proper funding for this project. So that's the aim in the next in the next nine 12 months. Obviously so, really nice, it's still under embargo.

A

It's a public meeting, it's under a burger, but we're hopefully happy to announce it in about a week. So really very grateful for that support, because that puts us in a strong position to get some chemistry done.

A

Okey dokey! um It's a few minutes to anyone got any aob that we we've messed down.

G

I can give you an update on the astrazeneca legals two weeks ago at people chasing their people and no one's responded so.

A

That's an awesome update chris. Thank you so much. Thank you.

E

That that's that that's very surprising chris I mean no! No! No! No! Don't don't don't laugh about this because kelly at az has reached back to at least to bart and so on and bart couldn't attest to this. That they've been very engaged in trying to help here. So I'm not sure when you say no one has responded, I'm not sure. What's going on there, um if you want anything, I can do to help whatever, but there's been.

E

A z has been over backwards here to help us. So it's it's that has really really not been az's fault here. Okay, take that as a former ac employee being you know, uh provincial protective whatever, but they have been very proactive and kelly wrote back several times trying.

M

E

What's going on so, if there's anything I can do to help. Let me know um so I don't know.

A

Go ahead, I think it's also worth saying that, with with a lot of these projects that are open, um where there's you know we're we're doing things that are not uh in something which is a commercial interest to the company. The fact that helping at all is really great right, so they don't need to help us if they don't want it, but they are, which is yeah really great right.

G

So, just as a bit of a timeline, uh you know warwick made some amendments 26th of july uh and and sent sent those off in july and then followed up september. I don't appear to have had a response, and so I just sent a so. These are big big lumps of time there.

C

So in my in my opinion, we were pretty dead in the water until joe and kelly got involved. um So if what I would whoever is at your place- email them together with kelly and connect them, because uh that we had a lot of, we have a lot of problems on our own side.

C

You know uh in our own institution it was also quite difficult um to identify the right person, but once once those two people could talk, it was actually quite easy right if you can figure out who your primary contact is at warwick and get kelly their email. I think that it will go much smoother.

G

Kelly's connected to all of these email trails, well,.

C

That was a theory. Sorry.

C

A

A

All good, okay, great, I will try and formulate you know the very brief summary of what we talked about and some actions just for next time, but lots of things happening almost all the fronts which is very exciting.

A

Okay, thanks everyone for your time and um see you all around soon. I hope take care.

J