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DBL%20Hendrix%20small.png College chemistry, 1983

Derek Lowe The 2002 Model

Dbl%20new%20portrait%20B%26W.png After 10 years of blogging. . .

Derek Lowe, an Arkansan by birth, got his BA from Hendrix College and his PhD in organic chemistry from Duke before spending time in Germany on a Humboldt Fellowship on his post-doc. He's worked for several major pharmaceutical companies since 1989 on drug discovery projects against schizophrenia, Alzheimer's, diabetes, osteoporosis and other diseases. To contact Derek email him directly: Twitter: Dereklowe

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April 10, 2014

Encoded Libraries Versus a Protein-Protein Interaction

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Posted by Derek

So here's the GSK paper on applying the DNA-encoded library technology to a protein-protein target. I'm particularly interested in seeing the more exotic techniques applied to hard targets like these, because it looks like there are plenty of them where we're going to need all the help we can get. In this case, they're going after integrin LFA-1. That's a key signaling molecule in leukocyte migration during inflammation, and there was an antibody (Raptiva, efalizumab) on the market, until it was withdrawn for too many side effects. (It dialed down the immune system rather too well). But can you replace an antibody with a small molecule?

A lot of people have tried. This is a pretty well-precedented protein-protein interaction for drug discovery, although (as this paper mentions), most of the screens have been direct PPI ones, and most of the compounds found have been allosteric - they fit into another spot on LFA-1 and disrupt the equilibrium between a low-affinity form and the high-affinity one. In this case, though, the GSK folks used their encoded libraries to screen directly against the LFA-1 protein. As usual, the theoretical number of compounds in the collection was bizarre, about 4 billion compounds (it's the substituted triazine library that they've described before).

An indanyl amino acid in one position on the triazine seemed to be a key SAR point in the resulting screen, and there were at least four other substituents at the next triazine point that kept up its activity. Synthesizing these off the DNA tags gave double-digit nanomolar affinities (if they hadn't, we wouldn't be hearing about this work, I'm pretty sure). Developing the SAR from these seems to have gone in classic med-chem fashion, although a lot of classic med-chem programs would very much like to be able to start off with some 50 nM compounds. The compounds were also potent in cell adhesion assays, with an interesting twist - the team also used a mutated form of LFA-1 where a disulfide holds it fixed in the high-affinity state. The known small-molecule allosteric inhibitors work against wild-type in this cell assay, but wipe out against the locked mutant, as they should. These triazines showed the same behavior; they also target the allosteric site.

That probably shouldn't have come as a surprise. Most protein-protein interactions have limited opportunities for small molecules to affect them, and if there's a known friendly spot like the allosteric site here, you'd have to expect that most of your hits are going to be landing on it. You wonder what might happen if you ran the ELT screen against the high-affinity-locked mutant protein - if it's good enough to work in cells, it should be good enough to serve in a screen for non-allosteric compounds. The answer (most likely) is that you sure wouldn't find any 50 nM leads - I wonder what you'd find at all? Running four billion compounds across a protein surface and finding no real hits would be a sobering experience.

The paper finishes up by showing the synthesis of some fluorescently tagged derivatives, and showing that these also work in cell assay. The last sentence is : "The latter phenomena provided an opportunity for ELT selections against a desired target in its natural state on cell surface. We are currently exploring this technology development opportunity." I wonder if they are? For the same reasons given above, you'd expect to find mostly allosteric binders, and those already seem to be findable. And it's my impression that this is the early-stage ELT stuff (the triazine library), plus, when you look at the list of authors, there are several "Present address" footnotes. So this work was presumably done a while back and is just now coming into the light.

So the question of using this technique against PPI targets remains open, as far as I can tell. This one had already been shown to yield small-molecule hits, and it did so again, in the same binding pocket. What happens when you set out into the unknown? Presumably, GlaxoSmithKline (and the other groups pursuing encoded libraries) know a lot more about than the rest of us do. Surely some screens like this have been run. Either they came up empty - in which case we'll never hear about them - or they actually yielded something interesting, in which case we'll hear about them over the next few years. If you want to know the answer before then, you're going to have to run some yourself. Isn't that always the way?

Comments (17) + TrackBacks (0) | Category: Chemical Biology | Drug Assays


1. SAR screener on April 10, 2014 8:32 AM writes...

I haven't read the paper so they may discuss this, but I'm surprised they didn't run the locked version as a counterscreen.

That's one of the nice things about ELT - it's relativly easy to run multiple screens at once so you can screen your target of interest and also (for example) counterscreen against proteins you don't want to inhibit or mutants of the same enzyme and (hopefully) pick hits with the selectivity profile or MoA you desire.

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2. annon four on April 10, 2014 9:47 AM writes...

Lot's of recent papers on this for GSK. Any compounds in the clinic, or approved?

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3. again on April 10, 2014 9:57 AM writes...

@ annon four

Someone raised this same question the last time Derek posted on this topic. Was that you? I think GSK bought this technology in 2007. Its now 6 full years later. I would think some cmpds might be getting into the clinic. But this number would have to be compared with the number moving into the clinic from inhouse HTS and fragment based technologies (which would provide a baseline). The question of how many cmpds approved is snarky. 6 years from an initial screen to an approved drug? Is that how it the norm for GSK?!

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4. annon four on April 10, 2014 12:43 PM writes...

3, again: Nope, but still a valid question.

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5. again on April 10, 2014 1:16 PM writes...

I don't think so. Just my opinion. A company can be measured by drug approvals. A screening technology should be measured by hits found and the value of those hits. There's a lot that goes on between target selection and drug approval. The technology used to find a new chemical starting point is just a tiny tiny part of the process.

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6. Hap on April 10, 2014 1:38 PM writes...

Approvals or compounds in the clinic.

The method wouldn't be expected to give you approvals (and can't be judged on them), but if it works and were giving you leads more quickly, and you'd been using it for six years, then compounds generated from those leads probably should be entering the clinic, at least in Phase 1. Perhaps the learning curve is long, which might explain a "no" answer to the question, but if you include compounds in the clinic, asking the question doesn't seem ridiculous.

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7. Anonymous on April 10, 2014 1:48 PM writes...

You want a clinical trial? Like maybe this one on an ELT hit:

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8. again on April 10, 2014 2:33 PM writes...

After 6 years I think # cmpds in clinic may be a reasonable question. # approved is obviously not. But, there are many unknowns. Does GSK only use the technology for PPI's? Only for targets that have failed otherwise? How many make it into the clinic from other sources, like HTS? Again, a lot goes into getting a cmpd into phase I, and the screening methodology is a pretty small part of it. I see that #7 posted a link for an ELT hit in the clinic. But after years of seeing HTS hits and fast follower programs make it into the clinic, my opinion is that the target, the leadership team, and the med chem efforts play a much larger role then what screening technology the hit originated from. Its not that hard to judge the value of a hit. Chemists judge the merits of chemical starting points all the time, every time there's a hit to lead effort. So why wouldn't med chemists judge the technology by the hits generated?

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9. annon four on April 10, 2014 2:41 PM writes...

7: Thank you for the information on the sEH inhibitor in the clinic.

6: Yes, the purpose is to generate leads. But, if they are not suitable to develop into clinical candidates (minimally), then the exercise costs money and resources with not return. That is why such questions are VERY relevant in today's world of drug R&D....Pharma can no longer afford to be academics.

5: Yes, finding hits is just a small part of the process. (Try to convince the screening people of that one!) But if leads are not suitably workable, and companies want to see them worked on because of the source of the hits, then there is a real organizational problem.

I'm glad to have an answer to my question from #7, and do hope there are more successes coming, with some of the compounds ultimately getting approvals. The industry needs more successes, independent of the source of the "hit" or "lead".

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10. again on April 10, 2014 2:47 PM writes...

@annon four

Why would
"companies want to see them worked on because of the source of the hits" ?

I've never seen anyone much care where a hit came from. Does GSK have this problem?

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11. annon four on April 10, 2014 4:08 PM writes...

If a company (eg GSK) spends many millions to obtain a technology, some people in the organization can have a strong desire, motivation, (ego, career making/breaking) to demonstrate that the investment was smart/clever/insightful/brilliant/worthwhile.

Questions often asked in review of team progress can be:

1) did you use the technology in your screening?
2) if not, why not
3) if yes to 1, then were there compounds that showed up in the screen
4) if yes to 3, did you use them, or why not
5) if not, why didn't anything hit

Tough time for the person who has to defend not using the method, not following up on the "hit" to management knowing little about chemistry or optimization of leads, etc, etc.

Others can address if this might describe GSK, or other companies. But, I've seen in happen in Pharma, personally experienced such things, but don't feel the need to say what company(s).

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12. again on April 10, 2014 4:53 PM writes...

@annon four

There seems to be a major lack of trust/faith between yourself and those making decisions higher up.

You could modify your statement of management's goal to "determine if the investment was worthwhile?". This would also explain the questions. Someone wants to know if the money was well spent, and you have to follow up to determine this.

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13. sgcox on April 10, 2014 5:11 PM writes...

Never worked with integrins and hence a bit ignorant, sorry. Still, it really strikes me that the low digit nM compounds from AlphaLisa are employed at 50 uM in the cell assay (fig 4.).
It is about 1000 fold over and no dose-response data shown. Moreover, the target is extracellular and the experiment was done in a dish.
Can it hints on why the work is in the press but not in the clinical trials ?

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14. Anonymous on April 10, 2014 5:31 PM writes...

#12 again.
Unfortunately, the management charged "to determine if the investment was worthwhile?" is often exactly same person or part of the clique who spend millions to obtain the technology in the first place. "Now, answer the questions correctly or else..." And please do not say you work in a big pharma company it can't ever happen.

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15. annon four on April 10, 2014 5:43 PM writes...

14: Indeed.

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16. again on April 11, 2014 1:33 AM writes...


So the same people who spend the money then champion the program and are held personally responsible for its success or failure? That sounds pretty reasonable to me.

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17. Hap on April 11, 2014 7:52 AM writes...


1) they generally aren't held responsible for the failure (by the time anyone thinks it's a boondoggle, they will have been credited with its "success" and moved on). Was anyone held responsible for pharma's combi craze (well, other than the underlings)?

2) they are also in charging of making the judgment whether it failed or succeeded, and whether it actually works or doesn't is likely not relevant in that judgment. It's sort of like the prosecutor also being the judge - they are likely to have a tendency to throw out data that make them look bad (that doesn't fit the case they're making). Since they are in charge of the people doing the work with the tech they championed, they are likely to make sure that 1) it's used, whether it works or not and 2) it gives the answers they want.

This certainly does not seem like how a sane business or organization should operate; on the other hand, what in the last ten years would give one the impression that pharma has been behaving sanely?

If this technique works, it would be nice, but lots of techniques seems to be buzzward-laden bandwagons for someone to hitch a star to to make their career and not ways to find more drugs and make more money for something beyond this quarter. It's hard to tell between those outcomes, and J. Med. Chem. articles don't necessarily distinguish them, either.

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