Open Source Antibiotics Series 1 - the Mur Ligases, 13 Apr 2021

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From YouTube: Open Source Antibiotics Series 2 Mur Ligase Update Apr 13th 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/42
On the call: Professor Matthew Todd, Dr Dana Klug, Yuhang Wang (UCL), Professor Chris Dowson, Laura Diaz Saez, Rebecca Steventon (University of Warwick), Lizbe Koekemoer (Diamond/Oxford), Bart Staker (SSGCID), Joe Eyermann (NEU), Peter Horanyi (UCB), Dr Chris Swain (Cambridge MedChem Consulting)

A

Okay, hopefully everything everyone's okay with the recording good I should have checked before I like. I clicked yes, um if there's anything confidential that needs to be talked about, we can talk about it at the end of the meeting. um So this is the ligase meeting on tuesday, the 13th of april, hello, everybody um hopefully on screen, is the issue to do with the meeting, um which has a few.

B

A

From last time that we can talk about in case um anyone's got any progress beyond what chris just said. um So shall I just run through these unless anyone wants to start with something of big importance.

A

um Otherwise, so the uh so you hang is looking at the synthesis of the az compound he's been um visually doing that yeah and do you want to say a few words about where you're up to on the synthesis of the uh the azad alcohol.

C

Yeah sure, thanks uh just let me share my screen.

C

Right, so uh this is uh what I've been up to. uh You guys see it clearly. What do I need? Yeah, it's good, okay, so this is the. uh This is the rationale I've finished recently and uh the euro was not good, because that was my first reaction, so I screwed up sorry about that, but I got the uh but I got the compounds which is around like 100 100 milligrams and the enema seems to be really pure, but the thing is, I can't be very sure.

C

I cannot be sure about the result like which one of the items I got or do they just uh can't can't be discerned via nmr at this stage or do I need to just go straight forward to maybe to the next step to maybe trying to uh separate them.

D

C

D

Annoy between the tb tile and the methyl.

C

Yeah, I was thinking about that, not sure if it's really worth doing so, okay testing, the testing fees uh is more expensive than just do a like quick, wear formation. uh You.

E

Could see it what's.

F

The expense in doing a uh an noe experiment.

A

So an noe experiment usually requires both isomers, so you can be sure of your result, a positive and a negative um in an ideal world.

A

um The alternative here is to do something which is, I think, has been done in the literature which is to react that primary amine with an isocyanate to make a urea which crystallizes easily and get a structure that way that could be.

A

That could be a simple way of doing it or the alternative is to go into the next step and try and figure out if the molecule we've got is identical to the literature compound. There's, no literature nmr for this.

B

Would a z have it.

A

I don't think it's in the paper, potentially okay.

G

um Did you make the beta keto nitrile yourself or did you is that commercial, the one on the left starting.

C

With your compound compound was the commercial, they are cheap and.

G

Have you checked that by nmr I so I've done quite a bit of this kind of chemistry and I found that that was actually unstable.

C

G

C

Okay, yeah, it's totally okay,.

G

Okay and then the other thing I found when I was doing it, um I actually got better yields. When I switched to the acetate salt, not the sulfate. I don't know why um it might be something you could consider, but I also was just using hydrazine. I wasn't using the n-methyl, which adds an extra layer of fun all right.

A

Right, okay, I think the idea is to repeat, and then try and structure a lucid date or maybe coming to the next step. But I mean it's early days right.

A

um Yeah the literature procedure in the asep paper doesn't have the the salt of methyl hydrogen. Then you use methyl hydrazine and you hangs um getting the free alien in situ, which is a slight difference. It shouldn't make that much difference.

C

Yeah, so I was thinking if the the compound five, the desired product was a little bit unstable, the more diamond dynamically than the then and the later one, because all I can find online like commercially was the compound file was all that easy to get a commercial code. uh They have to make a synthetic plan, so it will cost up like 1 000 pounds uh dollars for for this compound. Only for like a gram or several milligrams, I guess, but for this one which is available here commercially and has a cost code.

A

All right, thanks yang, I guess, keep pushing great thanks so in progress, um okay and then um the situation with the atomizers that we are still waiting for legal clearance for this. So we have clearance from diamonds for them to receive the compounds, we're waiting on clearly some warrant to receive contents, and once we've got those clearances, then ucl can say to atomize. Yes, we can go ahead, but we still we're still waiting on warwick then ucl to approve this.

A

Incredibly, even though we have these companies, I'm glad they're in the freezer, because time is ticking behind, there's nothing. We can do about it. Anything.

H

No, I mean I'm going to try again, I mean the more we keep pushing, but I I'm the system here, I think, is almost broken. um It just takes so long. There's such a hierarchy of sign-offs yeah.

A

H

uh You know I can list long a long, a long line of collaborations, where we're with we're the last ones sign off.

A

Yeah yeah, I know it's amazing. We've been shamed by the speed of diamond's response.

F

But then I thought brexit fixed this.

B

Or do we have to send the shipments? At the same time.

A

They we could yeah. So that's right. We could do that, so we could go back to atomwise and say that uh we've got approval for one. Can we go with one yeah? That may be a good idea, possibility.

B

H

Why why why not do that, and you know the you know the worst case is we could go down to diamond to do the asses essays without telling anybody.

A

We're recorded and we're open source. No, it's a suggestion. You're not going to do it.

H

And let's get the soaking bit done and.

F

So in that sense, um if you go to the diamond and you get some structures for some of the atomized compounds, so what's the what's the next step, I mean, is it the ability to at that point, you know what the compounds are. Can you actually at that point, are you free to operate or yeah so at that.

A

F

Could you actually just order? I mean these are all commercial compounds.

A

F

Commercially available, so at that point, could you actually be free to order those compounds and then just you know, send them to at that point, send them to work. I was wondering.

D

F

You know: what's the freedom to operate after uh you do something with the compounds. Yeah.

A

It's freedom to operate so absolutely if we found a compound that was good, then we could order it separately and then order around it and act.

H

Yeah, it's a good point, joe, let's just press on that as a as a worst case, probably the best case scenario. Actually.

A

Yeah great okay, um number three was becca about screen angle, visible, sgc, fragments and then daniel cook is a library frame. So I guess screening of warwick. Do you want to? Yes.

I

We've done lisby's screen. We've just got one final experiment to confirm the binding pocket. um Unfortunately, dana's are taking a while just because we're having problems producing the udp intermediate. Currently, it's really dirty and obviously we're going to need a lot to do beside the screens. We need to run so we're working on trying to clean that up, so we can get the levels we need, so that everything's, just one massive, goes through to try and make it as clear and coherent as we can for those experiments.

A

Sorry so yeah you're generating more protein, so was that.

I

Protein and the udp substrate we need, um but once done, we can just do a literal like just week of just screening at all and it'll get done in one massive block so that it's just as coherent as possible and as reliable as possible.

A

Okay, great fantastic, crucial stuff. Anyone got any questions about the screening. That's going on. Obviously, that's that's a key part of what we're doing.

F

Sorry generally, excuse me generating substrate in-house uh udp substrate in-house.

I

H

Yeah, okay, I mean the back story to that is joe's because of lockdown. uh We we haven't we, you know things have been left lying around and not being used for a long time, so we've just had to go through a root and branch of the whole synthesis facility with you know which all the co-factors you know it just it just ended up. There was a whole tier of things that needed to be changed and they were, they were all each.

H

You know 50 less active than they should normally be and by the time you've got a 50 50 50, you end up with stuff, not working, because it's a closely coupled recycled reaction and things weren't recycling properly and anyway, um julie who's.

H

One of the key facility technicians here has been running through all of that and I think pretty much got that sorted.

F

A

Wonderful um and then the the next one was um laura setting up crystallization drawers and mercedes any.

J

I have crystals uh for me or d they're still growing, because apparently it takes like about a month or more to grow them, so I'm parallelly I'm trying to get new crystals and optimizing those for s cam to see if I can get them faster, because it's nice too much waiting but yeah. I prefer like six plates. uh Yes, so we weigh and we have uh enough crystals for doing the experiment and I do have mercy. That's yeah just waiting there until I can go and do the screen which.

F

Is arms? Are these.

J

So is e coli me or c and saga murdy.

F

I'm sorry, which, which mere d.

J

F

Sorry, which I didn't quite oh.

J

H

F

From your date, from your day,.

J

Nearly an e-coli for mercy.

H

Yeah we're not doing any newer bees.

J

So so far yeah, if you want me to try anything else, let me.

J

A

Okay, great thanks um and then the next item was was jerry christian body pc he's a compliment shipment for crystallization trials. That was.

F

Someone done right, this mark clark can speak to.

A

D

Actually peter, I saw peter irani on earlier.

K

Yeah I'm here we received everything. So that's my update, I'm at the bottom of the page, but yeah we received everything, we're getting started ourselves with the uh the protein and the compounds that uh bart had shipped to us. So thanks for all that.

A

And somewhere we have which compounds those are somewhere. Do we, because I I can't remember, I can't remember off the top of my head.

F

Those are the ones I see.

D

Actually, I probably went through the anime.

F

Right yeah, so I yeah those are enemy compounds, but I guess I should somehow put the structures of those up on the the uh up on the up on the page yeah. I.

E

Don't think I don't.

F

Think I've actually shared those I may have not shared those with the team. Okay,.

K

That's an action item yeah right.

F

If you can share the smiles with.

K

Us that'd be great.

A

Sure, right: okay, yeah, I mean.

K

Any anywhere doing.

A

Like an excel sheet or a picture or anything just drag it onto the issue here.

K

um Smiles in an excel sheet would be ideal.

A

Yeah that'd be useful, no problem.

D

F

Didn't get the.

D

uh The az application submitted um yeah, I don't know yeah it got submitted.

D

There's a response to the last meeting notes.

A

Yeah, so that was a request for compound somebody said right yeah. I saw that so someone posted that called bartram. Who is that.

A

Sorry, mister, who is it me? Oh you right.

F

So is that some relationship, uh something relative to the simpsons or or what.

D

I don't know I was some github account I created like five years ago. For some reason I don't remember it, but now it's just my username like all right there. It is.

A

Great uh okay, um because I think there was a felicity bathroom. I worked at diamond right. That was, I was confused. Okay, um okay, great chris, and I were supposed to look at mrc schemes for funding. I guess we we keep on exchanging emails about funding, but we don't need to talk about that. Now. It's not totally relevant chris swain just found a scheme um for something uh chris. I don't know if you've got it to hand.

A

um I think I.

D

Just put the link in the email trail.

A

um Yeah, so I think everyone's got that right: yeah um as uh it's 50, 000 pounds potentially or 25 000 pounds right yeah from the.

H

Parity antibiotic.

A

Yeah, it's right. It's.

H

From uh you must know this source chris dalton, I'm a trustee and was chair of the science committee until december right.

A

um So I don't know if you I mean, is it is this? What do you think do you think this is a viable source of funding, for you know perhaps consumables kind of thing.

H

uh I think it's definitely worth a go. I mean there are some rubrics around the, so I'm not in charge of that fund anymore. uh uh There's some rubric around the focus of it to be thinking about combination therapies.

H

uh So if you go further down.

H

Actually yeah, I stopped there somewhere else. I was reading about it. It's it's it's things that are supportive of what the charity is doing, but you know this. This project fits nicely nicely into it.

A

Yeah, I mean, I guess it's a useful source of of consumables money right if a few of us went in for it. Yes,.

H

I think so um yeah yeah okay, but I think I think probably we just need to it's probably worth um having a conversation with the. Not me not me having the conversation as a trustee. Absolutely not me, but uh probably you matt, having a conversation with uh lloyd, payne who's. Now the head of the science committee, exiva tech. um I was just thinking david cameron and texts back into government there for a minute. Yes, definitely not me uh uh uh about about the fit, uh but it all too what to work with.

A

Okay, that sounds good. I mean uh I might just bring you after this meeting for for potential contact details in that case.

A

If that's uh that would work nicely, I was always used to the sort of support the expense of what we're doing um and then jan jensen can't be with us today. He has all the data he needs to try to work on the positives and negatives that we've got for in vitro data against the enzymes to try and do some predictive work uh he's still chewing on that he's been doing some work on the malaria project recently, but he'll eventually turn his attention to this project.

A

He just put a message on the website, um and this has already been done so uh peter already spoke about.

A

What's going on with those um in terms of all the rest of this, I think we've I mean we've pretty much covered everything um on the agenda there in dealing with those action items so separately from all of that, is there anything else that people wanted to mention or update everyone on new developments or anything.

H

uh Yes, please, um a little while ago I sent a google doc around for a matrix of what proteins we had and what we had available for them. I think it would be good to complete that and then agree what the ask might be to uh ssgcid to help around cloning and additional protein production to uh target the priority pathogens. So, for example, you know um d at the moment is you know, strep a galactic. I it's not hugely high in who priority list, but it's it was.

H

It was done for a particular situation with south africa and neonatal sepsis. So um you know, I think, there's a number of proteins. I think we really ought to have in our toolbox um structurally and assay-wise as well. So I think agreeing between us as a team what they ought to be and then putting that as an as a proposal to essa's gcid.

F

Yeah, so peter peter should speak up on this because I that was actually my question because I think peter didn't really specify which of the iso zones um because you're working on your d- and I don't remember which one I think it's e coli, maybe.

K

So we have pure uh mirrored cm or d and bar correct me if I'm wrong, but I think these are from pseudomonas oreganosa and uh acetobacter uh bomani.

D

Yeah yeah. Actually, then, that that um a document I posted um there's a table of all of our mercies and all of our murdees in it and also the ones that we tried and failed at. So you can see that we got the various stages. We didn't try it very hard. So if there was one we wanted to rescue out of those lists, we could work on those but primarily they're. The fascinator background pseudomonas.

K

But I think that what we, what ssgcid, has, is sort of figured out what construct works for crystallization, because some of those mirror c's are full length. Just doesn't work for crystallization, but the truncated versions do.

D

Right but we don't, we don't have a mer d, though that has worked yet yep.

F

Which is which is somewhat anyway, I mean it's it's in the literature right, so I guess it's a question of figuring out well,.

D

We don't know we don't try what's in the literature, because it's done already, so we were trying right one right, infectious diseases that hadn't been done so.

F

I'm just wondering if we you know, for uh I think I see colon your d just in the literature. um Is it worth getting that specific um clone um again when we're trying to get you know, uh structures and two, you know, and then your d and then your c.

F

um I don't know anyway just.

D

F

100 we're gonna.

D

We're we actually haven't ordered, started it yet, but we're the plan. Is that we're going to try the e coli and um as well as make a couple construct designs of what we have to improve crystallization.

A

But um christie you're you're talking about biology resources right so mostly what protein is available.

H

uh Yes, it was really yeah what you know. What what what constructs we've got, what protein can we have available et cetera, so I think it would just, I think, probably just a round of emails- probably quite quickly sort this out. uh Maybe there may be a couple of extras: we want to start from scratch.

H

I I I sorry. I've missed that table, but I'll go and have a look through with laura or if laura can have a look through to see.

H

You know what what uh what was tried and failed and what might be rescued, and we ought to do that so we'll we'll catch up on that, uh I'm just thinking, timing-wise and just thinking for the next 12 15 months. While we have now got people in post to do the biology, just you know, now's the time to strike to get the protein made and we can once the assays and reagents are there. We can rush through things pretty quickly.

F

So, along those lines, I guess, is there a any mtas or anything that needs to be done between say, warwick and ssgcid in terms of transferring protein, because I think you guys share you're willing to share it's just a matter. Is there any logistics with that? There's a.

D

There's a on our. We have a website ssgcid.org that has essentially a shopping cart. You can see like all of our 4 000 constructs. If you can find them and then you can, you basically put them in a shopping, cart and then there's an electronic mta, which is very basic that we do need for someone at the institute to kind of sign off on and then we keep that on record and then we will send whatever you request.

D

If it's on dry ice, um we do actually ask for you to pay for the shipping, because we don't have a budget for shipping and we're sending dry ice to all around the world. So we'll ask you to like your for your fedex number or something like that, and then we will ship you whatever you ask for clones, we will ship the protein if it's available or the expression clones.

H

um With the shopping cart, but if we do multiple orders, do we need to sign up multiple mtas or will the one do I'm just thinking of the pain here.

D

Yeah, so you can get multiple items in an or in an order, but if you come back, um you don't need to redo the mta. um So it's just one which case.

H

We'll we'll order something that you've already got now and start that mta process going now.

D

H

Laura we'll have a chat and go through that.

A

Mention that chris and I christensen- and I are- I think we have half an hour with the alpha fold team next week- to talk about the project to talk about whether they can get involved in doing some protein structural prediction.

A

For some of these proteins uh using the the approach they used to win the casp protein structure, prediction, competition and so we'll report back on on how that's looking.

D

So if alpha fault is actually was the winner, that's true, but we have a we as part of ssjcid. We have the the david baker lab at the university of washington and um we can have them predict any protein structures that are in our pipeline that'd, be.

K

H

Do they are they interested in in in trying to predict ligand binding as well or are they just protein structure.

D

Well, they are interested in it, but it's not something. That's publicly. It's not. As you know, it's not something they offer outsiders. I guess.

H

Could we become insiders.

D

H

I don't know I mean.

D

I'm not really an insider either, even though we have a collaboration with them. um So that would be a different conversation. We could try. Okay,.

H

Okay, I I for me with the conversation with our folders, to see how far we can push them as to whether we could get them to start thinking. Also about you know, beyond just the structures and ultimately the structures are you know, you know, you know a small part of what we finally need.

H

E

A

That's good to know thanks all right anything else, we're on the half hour.

A

Oh good, thank you thanks. So much um and I'll set up a time for in about a month or so, and I'll post some action items up but nice to see everybody. Okay, see you again next time right thanks very much.