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Chemical Evolution Makes a Deal

Sanofi has signed a deal with a Stanford-born startup called DiCE Molecules, looking for small-molecule inhibitors of protein-protein interactions. So who are these folks and what route do they have into this perpetually promising-and-challenging area?

DiCE grew out of work at Pehr Harbury’s group at Stanford. The company’s web site makes some bold claims, but that’s what startup web sites are for:

DiCE Molecules’ technology selects and optimizes drug-like ligands to any given target, beginning with libraries containing billions of individual molecules. Unique among currently available options, DiCE Molecules’ technology restores the libraries to their original ligand concentration after each round of screening, revealing the full landscape of binding molecules and allowing them to easily be selected for enhanced potency, selectivity and drug-like properties through testing with proprietary assays. This novel approach may address long-standing chemistry issues and would enable monoclonal antibodies to be replaced by orally-administered medicines.

Unlike the older technology of DNA tagged libraries in which the nucleic acids serve merely to identify the compounds, with DiCE Molecules’ approach DNA is utilized in a manner to that analogous to biological directed evolution: to encode the synthesis of the compound using a physically linked replicable polymer (DNA), enabling one to amplify weak signals out of a vast background of irrelevant noise. It is impossible to overstate the importance of this distinction.​

I think one can be forgiven for not quite grasping the point of the first paragraph, but trying to unravel the scientific details from an “About Us” web page is almost always futile. The second paragraph is more parseable, at least to me, but also raises a thirst for more details. Some of those can doubtless be found in this 2004 paper, which is probably the first iteration of their technology (see also this 2007 review). It starts off with an excellent question which I’m sure has occurred to everyone who’s given some thought to chemical biology and evolution:

. . .Multiple generations of selective pressure and reproduction transform a diverse population into one consisting only of molecules fit to survive. Life on this planet thus emerged from a limited chemical palette, comprising proteins, nucleic acids, sugars, lipids, and metabolites. Over the last two decades, technologies that recapitulate this process in the test tube have been developed, and have produced an amazing collection of biopolymers with unprecedented recognition and catalytic properties (reviewed in Roberts and Ja 1999). At present, however, these in vitro selection techniques cannot be applied to compounds of nonbiological origin and have therefore not affected most areas of molecular discovery. The question arises: what would become possible if in vitro selection were applied to chemical populations of arbitrary composition?

Exactly. I’ve been kicking that thought around in my head for years as well. The evolutionary motor driving molecular biology is so relentlessly powerful that you can’t help but want to harness it for diverse small molecules as well, but that is no easy task. Much of the time these ideas lead you into coming up with conjugates of DNA and small molecules or proteins and small molecules, thus to hijack the molecular machinery as it does its thing. And there have been plenty of useful idea that have come out of these approaches: DNA-encoded libraries, yeast hybrid screening, antibody conjugates, phage and RNA display technologies, and others. But if you push this concept too far, you keep bumping into the fact that evolution has built this amazing heap of machinery to shuttle information from nucleic acid polymers into proteins, not to give you a platform for small-molecule diversity. All the tools are tuned up to work on DNA, RNA, amino acids, and proteins, not your library of drug-like small molecules. So most of the techniques in this area are indirect ones, bounce shots that try to harness the natural system somehow.

This paper details a DNA-encoded library, but with a twist: it’s not a static library of screening compounds, each with its own DNA bar code. In this case, the DNA tag starts off longer than usual, with several regions for hybridization, and it stays on through iterative rounds of screening as the small molecule on the end is elaborated. In this case, it’s a peptide, but there are many other chemistries that are available, as the conventional DNA-encoded library folks have shown.

In this case, they demonstrated making a ligand for a known antibody (3-E7) which has been used as a test bed before in these sorts of experiments. They start off by making pentapeptides on the end of their single-stranded DNA template, with ten amino acids to vary at each position. The N-terminus is either left as NH2 or is capped with one of nine small organic acids. The ssDNA is converted into double-stranded form, and the library is screened against the antibody, and the selected/amplified DNA coming out of that process is used as the input for the next round. What they found was that the process, after two rounds, converged on Leu-enkephalin, the known binding partner for the 3-E7 antibody. You can see it picking up amino acids along the way until it arrives at the actual pentapeptide. A second experiment started with every codon in the initial DNA sequence coding for a different amino acid than in the first run, but that one converged on Leu-enkephalin as well after round 2, so this wasn’t a lucky hit because of DNA coding bias.

The hope is that this process will zero in on promising binders without having to make them all up front:

Diversification between rounds of selection by recombination makes possible in vitro evolution of libraries with complexities exceeding the physical library size. Thus, a “best” molecule can be pinpointed without exhaustive testing of all potential species. Starting with a working population of compounds that sparsely sample a chemical space, molecules containing parts of an optimal molecular solution often have a selective advantage relative to siblings, and become enriched. Subsequent recombination processes splice together fragments from the numerous partially optimal molecules to form a globally optimal molecule. Thus, the best structure is found, even if the odds were negligible that it existed in the initial working population.

And that, I would have to guess, is the fundamental technology behind DiCE, although it’s no doubt gone through several refinements in recent years. I like it, but I can think of one possible pitfall (and there are surely others). This is, in a way, a sort of DNA-directed fragment optimization. But there are times when a good molecule cannot seem to be dissected into fragments – there’s no apparent path, because the binding doesn’t seem to add up piece by piece, but rather ends up being more synergistic. This is not a killer objection to DiCE, though, because it’s just a source of false negatives, and presumably there’s a reasonable universe of true positives to pick from. You’re looking for a technique that will give you some real hits, not all the real hits that could ever be.

It’s a long way from these ideas to replacing monoclonal antibodies with synthetic small molecules (although I’ll bet that the molecules will turn out to be only small-ish by the time they get optimized). But Sanofi has seen enough to be interested, and that should give DiCE some good funding (and good incentives) to see what they can do with it.

25 comments on “Chemical Evolution Makes a Deal”

  1. luysii says:

    The typical protein/protein interface has an area of 1,000 – 2000 square Angstroms — or circles of diameter between 34 and 50 Angstroms. [ Proc. Natl. Acad. Sci. vol. 101 pp. 16437 – 16441 ’04 ]. Think of the largest classical organic molecule you’ve ever made (not any polymer like a protein, polynucleotide, or polysaccharide). It isn’t anywhere close to this.

    Yet I’m convinced that drugs targeting these complexes, will be useful. Classical organic chemistry will be useless in designing them. We’ll have to forget our beloved SN1, SN2, nonclassical carbonium ions etc. etc. We need some new sort of physical organic chemistry, one not concerned with reaction mechanism, but with van der Waals interactions, electrostatic interactions. At least stereochemistry will still be important.

    For more along this line please see — https://luysii.wordpress.com/2015/10/12/the-next-big-drug-target-ii/

    1. Ash (Wavefunction) says:

      Well, ‘classical organic chemistry’ is still useful for designing such compounds. I have worked for two companies which were expressly trying to design molecules to inhibit protein-protein interactions, and 90% of the reactions used to make those molecules (especially macrocycles) were of the “classical” type – Wittig reactions, reductive aminations, simple peptide bond forming reactions etc. – along with some newer reactions like cross-couplings thrown in. And most of these molecules were pretty large: molecular weight > 700. So a lot of the organic chemistry is still old school and ‘classical’ and very useful for making large PPI inhibiting compounds.

      What *is* novel are better methods (like DICE is using) of making these compounds rapidly by the millions, separating them from each other or testing them as mixtures and decoding the identity of the molecules using advanced instrumental techniques like gene sequencing of tags. That’s what’s really changing the landscape here.

    2. anon says:

      And yet people have managed, as with Bcr-Abl tyrosine-kinase inhibitors. Strange world 🙂

  2. luysii says:

    Well what was their extent in Angstroms and how well did they work?

    1. anon says:

      https://en.wikipedia.org/wiki/Bcr-Abl_tyrosine-kinase_inhibitor

      And not all PPI surfaces are flat and featureless – and as Derek pointed out the other day: It does not help anybody to sit there and say “this will not work” to everything. While you will be right most of the time, there would be no progress at all. Unfortunately, failing is essential for science to be able to move forward. Wish it were easier.

    2. Ash says:

      In terms of sq A they can be small or big; in terms of affinity they can be µM or nM, just like regular drugs. As my graduate advisor used to say, think of PPIs as two doors closing on each other. All it takes is a tiny piece of iron at the right place in the hinge to stop them. Although it would certainly be easier with a bigger piece of iron.

  3. Calvin says:

    Kevin Judice is involved in this. He is a good guy so while there may be hype, in the background this will probably be properly run. Interesting ideas so I wish them well.

  4. Me says:

    Or you hit an allosteric site with a small molecule, induce a change in tertiary structure in the protein and inhibit it’s interaction with another protein that way…..

    1. partial agonist says:

      Yes, allosteric sites with druggable small-molecule-friendly pockets sometimes exist, and when filled they can impact the conformation of one of the interacting proteins. It’s probably a minority in terms of most protein-protein interactions, but it is surely a lower-hanging fruit than targeting vast shallow surfaces of proteins with molecules that would make Lipinski cringe.

  5. Me says:

    PS This reminds me of the technology that was used by Praecis, which was bought by GSK.

    1. morten G says:

      And Nuevolution and probably a bunch of companies more.

  6. shale says:

    Good luck to them! Sounds like a great deal. In vitro evolution has been around for many years and when applying the concepts to encoded chemical libraries one should not forget to appreciate the power of evolution. In a cycle of selection-pressure and library-amplification it is naive to believe the only pressure that is affecting the library population is target binding. During each amplification step many other pressures are being applied to the population (e.g. hybridization, amplification, chemical reaction rates) and each alter the direction of the “evolution”. Target binding must be the dominant pressure, but even so the population might not evolve as purely as one had hoped and believed.

  7. luysii says:

    Ashutosh: I certainly didn’t mean to imply that synthetic organic chemistry was useless, only that dissection of reaction mechanisms involving covalent bond formation wouldn’t help us much with protein/protein interactions. This is where a new sort of physical organic chemistry is needed.

    As elaborated in the link above — Nature got there first — one paper found a variety of mutations in oncoproteins which affected their interface with other proteins. Well, most mutations are single amino acid changes, so it is possible that small drugs will be found which can mimic them perturbing the PPI.

    It seems that a close study of these mutations will tell us a lot about PPIs.

    1. Ash says:

      Definitely agree with that! Harnessing weak interactions fruitfully is going to be the biggest challenge in drugging protein-protein interfaces, although we also shouldn’t discount the efficacy of covalent drugs (which have seen a real resurgence) in doing this.

      1. jbosch says:

        You can stabilize weak and transient interactions. Here’s just one example:
        http://malariajournal.biomedcentral.com/articles/10.1186/s12936-015-0834-9

  8. Slurpy says:

    “Life on this planet thus emerged from a limited chemical palette, comprising proteins, nucleic acids, sugars, lipids, and *metabolites.* ”

    Interesting, that the product of life is what apparently allowed life to emerge.

  9. Jonsy Bordas says:

    Folks, don’t be bamboozled by their small molecule evolution hogwash. It’s all VC-bait, it’s not what they’re doing. Evolutionary approaches only apply when the theoretical numerical diversity of the library exceeds the number of particles in the sample. That would only apply to libraries of immense theoretical size, which, by the arithmetic of split-and-pool, would have to be libraries with a high number of cycles. Likely they are peptides or peptoids, which incidentally are the only chemistries reported by Harbury to date.

    If DICE were open about pursuing peptides and peptoids as therapeutics, then I’d leave them be. But they’re claiming “drug-like ligands”. Therefore they must be making roughly Ro5 compliant small molecules. As anyone from GSK, X-Chem, or Nuev can tell you, Ro5 compliant libraries can only have 2 or maximum 3 cycles of chemistry. Given the numbers of commercial reagents available, these libraries would be small enough (in the 10^6 – 10^8 range) to be exhaustively explored without resorting to re-amplification (unless DICE’s selection method is particularly inefficient) and diversification.

    The bottom line is, unless they have a real interest in peptoidic drug candidates, Sanofi has yet again been sold a bill of goods.

    1. Mol Biologist says:

      Derek, I can not share your excitement about new chemical technology with the key for DiCE and Sanofi is unlocking protein-protein interfaces that oral drugs haven’t been able to reach inside cells.
      Jonsy Bordas, I tend to agree with you that Stanford-born startup called DiCE is misleading scientific community bu “extremely powerful technology” for several reasons. According to original paper published by Japanese scientists you may be able to regulate growth function in plants by inhibiting protein-protein interactions for protein called CLV3 (growth hormone). Authors suggested that synthetic analogs may be obtained by replacing the proline to get certain peptoids- so you can get antagonists for CLV3 and its receptor.
      Kondo T., Yokomine K., Nakagawa A., Sakagami Y. (2011). Analogs of the CLV3 peptide: synthesis and structure-activity relationships focused on proline residues. Plant Cell Physiol. 52, 30–36;
      They also claimed due to the features of proline structure peptoids can mimic proline residues in certain cases, and interfere with protein-protein interactions. And some peptoids may even increase the level of interaction.

      Hurber’s protein of interest is RIBOSOMAL PROTEIN L7AE which binds specifically to an RNA K-loop motif also have a conservative proline in the cis conformation in all reported structures of L7Ae and other homologous proteins.
      http://www.ncbi.nlm.nih.gov/pubmed/21708174

      I am wondering now what would be drug candidates against up to 12 targets selected by Sanofi 🙂

      1. Robot says:

        http://en.sanofi.com/NasdaQ_OMX/local/press_releases/sanofi_appoints_dr_yongjun_liu_1997708_29-03-2016!07_00_00.aspx
        Sanofi has named Yong-Jun Liu, chief of MedImmune unit, as its new head of research, global R&D. Dr Liu will lead all of early pipelines for new cancer treatments . Sanofi has had “pretty average financials for a few years now” due to factors like losing patent protection on valuable diabetes drug Lantus. Other medications like cholesterol drug Praluent have shown promise in clinical trials, but Sanofi doesn’t have a potent blockbuster medication to support falling sales.

    2. Mol Biologist says:

      Additional information on 3-E7 antibody and Prolyl Isomerization Governs Antibody Recognition of an Intrinsically Disordered Immunodominant Epitope*
      http://www.jbc.org/content/288/18/13110.full

    3. Mol Biologist says:

      The bottom line is, corporate development of Sanofi in the area of Cardiovascular Drugs since Christopher Viehbacher was fired by the company’s board looks very risky and rash. Lack of humility caused forced investment to MyoKardia and to the big deal of Chemical Evolution.
      Probably due to expiration of patents for clopidogrel and enoxaparin and the crisis in the new platform for cardiovascular drugs.
      Hippocrates: “Life is short, and art long; the crisis fleeting; experience perilous, and decision difficult.” Humility remains important in medical and pharmaceutical sciences and practice.

  10. anon the II says:

    These guys have a lot of high powered degrees and even a MacArthur prize amongst them, but it seems the labeling mechanism is going to preclude a lot of chemistries. Also, when someone says it gonna require a “new sort of physical organic chemistry”, you can be pretty sure it ain’t gonna work.

    I’m not sure I follow his reasoning but, I’m gonna go with Jonsy on this one.

  11. Kent Kemmish says:

    Let’s say I have a stealthy but well-equipped garage biotech lab and expertise with techniques like those used in Pehr Harbury’a group.

    If I want to be sold to Roche for a ton of money, and actually want to benefit humanity, AND actually want do something meaningful and not just get VC-bait results… what specific targets do you think I should pursue for orally deliverable PPI disruptors?

    1. Mol Biologist says:

      Let’s say humanity or public disclosure have nothing in common with the financial asset (i.e. patent) is regarded as freely alienable property that may be used as the owner deems fit.
      But I would say what it is not necessary to do. The area of prolyl hydroxylas inhibitor is insidious with a lot of players in the arena including Fibrogen, GSK, Sanofi,Vertex Pharmaceutical and many many others. Very limited success or say nothing was observed for Cardiovascular Disease. The new peptoid will be the ‘good’ SAR but unfortunately not able to resolve the paradox. You are always welcome to check out since Japanese technique does not look hard to reproduce.

  12. Barry H Levine says:

    what’s elided is the step from pentapeptide ligand to drug. No one has shown a general solution to that. If these were conformationally constrained (cyclic?) the problem would be much reduced, but not solved

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