Protein mutations create experimental negatives for binders

Transcript

Transcribed by machine (Whisper) from the episode’s audio, so a word may be off here and there.

1. 1:03:24

   The way that we get negatives is that we have a really good understanding of the distribution of the number of those molecules in a baseline condition. Okay. So, like, when we do the screen, we know, oh, okay, these bound a lot because now we're enriching for them when we pull the protein out. And now let's break the pocket and they are no longer enriched. Okay. They are no longer going. That one amino acid substitution that we made is making a bunch of these chemical series drop out.

2. 1:03:50

   But this is a one-off experiment. This is not a dough. No, this is Adele. Oh, no. So you're just, you're not saying you broke it. You're saying I find the case where it was broken. No, we break it. So we break the, so we take the, so let's say we screen a protein and the pocket is intact. Yeah. Sounds great. Yeah. Okay. We get a bunch of hits. Yeah. Let's go even further here. Let's say that we know someone else has already identified a chemical that goes in that pocket.

3. 1:04:14

   Let's buy it. Let's stick it in there and run the screen again. Yeah. That means all the chemicals that normally go in the pocket will be displaced by a high concentration of the one that we have. Oh, you have a competitive assay. Oh, we do it all the time. Okay, okay. We do this all the time. Okay, and then you say, okay, well, that looks pretty good. I believe that. And then you say, all right, now we know that this chemical that someone else has characterized goes in this pocket because maybe they made a crystal or some other thing.

4. 1:04:37

   Great. Let's look at the amino acids in that pocket. And then we will change the DNA construct that makes the protein for us. we will now mutate physically this one residue so now like this this you know this particular amino acid now we put a big bulky one and then the pockets full or like the different side chain or something different side chain all these things you can do yeah yeah and this is really easy now because we have alpha fold and you know bolts or all these structure models you can you can use this to design your proteins as you you can use this you can use that and for things that aren't as well characterized you know like we use conservation you know like we stare at we have a structural biologist on staff and kind of look at these things like oh yeah you know this guy Edward Kraft

5. 1:05:14

   which is amazing. And so, all right, so, you know, now we're going to make this one mutation. Let's do yet another screen. And, oh, look, all the chemicals that got booted out when we hit it with that one thing that's plucking the hole, they're also gone when we broke the pocket. So now we have really good evidence that they're all going into that pocket. Right. Right. And then here's the point where, you know, like the protein chemists come in and they say, well, if you made a mutation, how do you know that you didn't just break the whole thing?

6. 1:05:39

   Right? Reasonable question. You say, okay, well, we only lose the chemical series that got kicked out by that competitor. All the other ones that are binding everywhere else in the protein are intact. Yeah, so you didn't just break the protein. You didn't just break it, right? And then you think, okay, well, if you're going to be out screening 100 protein constructs a week, which is what we do on big weeks, like...

7. 1:05:59

   Wait, sorry. So you've broken the pocket somehow. Oh, yeah. And do you also find that there are other things that do, that start to connect, to bind to the protein? So when you change things around, you might change the shape in a way that does encourage other things to bind. We have definitely seen it. And is that an information source for you? It is. It is. And so another thing that's really nice about this and the nice part about DELs is you can imagine, if you're not thinking in large numbers, that, oh, I've got this one chemical that was binding and now I broke the pocket and now it's gone.

8. 1:06:31

   But because of the combinatorial nature of the DELs, maybe I have five different chemical series. It's like hundreds of molecules and there's another group that's the same hundreds of molecules with a single change in the building block. So like you have these repeated examples where it's like, oh, this one tiny thing shifted the population behavior enormously. And it's a big population. And so it's like, oh, I am now really convinced.

9. 1:06:54

   I can show you guys a plot that we, I don't know if you put pictures on the podcast or whatever. But it's very obvious like, oh, this one side group on this building block is clearly interacting with this one amino acid residue because we have like five independent examples of population behavior shifting because of it. It's beautiful. It's very, very clear. Yeah, go ahead. Tell us about this plot.

10. 1:07:14

    We pulled this thing up. And the reason that we pulled it up is we think it's really important for there to be a lot of variation in the proteins as well as the small molecules. And a reason that we think this is you want to be able to get a model trying to learn medicinal chemistry to really start to put together. You're having these hydrogen bond interactions between this part of the small molecule and this part of the protein.

11. 1:07:36

    We've seen other work that I talked about earlier about how you can, if you digitally break a pocket and you use the current state of the art models, that they have a really hard time understanding the physics and they'll just shove the molecule back in that thing anyway. So it makes this suggestive that they're memorizing. And so we thought one way to go around this is to experimentally engineer mutations in binding regions of these proteins and then re-screening.

12. 1:07:58

    And so what that looks like. Physically doing that. Physically doing that. You take the DNA of the wild-type protein that you've already screened, and then you use microbiology techniques to change one or more than one amino acids in that protein so that the functional part where the small molecules are binding is no longer the same. It's different in some way. We have different methods to try to identify which amino acids are the smart ones to break.

13. 1:08:20

    Sometimes you break ones that you're confident will do things. Sometimes you don't know. But we can do this at a pretty big scale, so it's not a big problem to us to take a couple wingers at each one. And so then you run the screen, and now when the pocket is broken, when the molecules go, what do the molecules do? Do they still go in there? Do some of them go in there? Is there something common among the ones that stick around?

14. 1:08:41

    Is there something common about the ones that get dislodged or pitched out? And then you have lots of different examples of these where you might have three to five-ish amino acids in a given pocket that are driving the binding behaviors. Maybe you try to break all of them independently. And then you do redo the screens each time and get a good feel for what part of the small molecule is interacting with the part that you have changed.

15. 1:09:02

    And then when you do this with a large Dell, then you'll have lots and lots of repeated examples of similar molecules. They're all following the same behavioral trajectories. And that way you get a really good feel for, oh, I've got lots of examples. They're all pretty similar. But the one thing that's super common is this one little bit on the small molecule. So I have a suspicion that it is interacting with the pocket that I just broke.

16. 1:09:24

    What are we looking at on this diagram?