21 January 2018

Linking fragments on DNA

DNA-encoded chemical libraries are one of the sexier new approaches for lead discovery. Typically, small molecules are synthesized while covalently linked to DNA and then screened for binding to a target. The structure of the molecule is encoded in the sequence of the DNA, and since incredibly tiny amounts of DNA can be sequenced (wooly mammoth genome, anyone?) you can fit massive libraries into a single Eppendorf tube. Indeed, some companies boast 100-billion compound libraries, nearly three orders of magnitude more than the number of molecules indexed by Chemical Abstract Service.

One might think this has no relevance for fragments. Indeed, the only mention of DNA-encoded libraries I recall on Practical Fragments was a comment by Teddy back in 2012 that the approach is “as opposite from FBDD as you can go”. A recent paper by Dario Neri, Filippo Sladojevich, and their collaborators at the ETH Z├╝rich and Philochem in ChemMedChem suggests otherwise.

The researchers have developed an approach called DNA-encoded self-assembling chemical (ESAC) libraries (see also their earlier paper in Nat. Chem.). Rather than synthesizing a single molecule on each strand of DNA, this approach involves assembling two separate sub-libraries of DNA-linked molecules, one attached to the 5’-end and the other attached to the 3’-end. These are then mixed together, allowed to hybridize in a combinatorial mixture, and screened against the target; if a specific combination of fragments is identified (through elegant PCR experiments), this indicates that the two fragments bind to the target in close proximity.

The researchers have focused on the protein alpha-1-acid glycoprotein (AGP), a prominent plasma protein whose function is poorly understood. In their Nat. Chem. paper, a library of 111,100 members (550 x 202 fragments) identified fragments A-117 and B-113. Neither of these fragments showed any binding themselves, but when linked together the resulting compound 1 bound with low micromolar affinity as assessed by isothermal titration calorimetry (ITC).


The linker connecting the two fragments is long, flexible, and not particularly drug-like; its improvement is the focus of the ChemMedChem paper. The researchers increased the size of their second fragment library from 202 to 428 elements, and an ESAC screen revealed that the pair of fragments A-117 and B-217 – both still attached to DNA – had a dissociation constant of 110 nM; B-217 itself (attached to DNA) was around 9900 nM.

To find out how these fragments could be productively linked, the researchers coupled them to 11 different scaffolds, each of which was attached to DNA. All of these bound to AGP, with dissociation constants ranging from 9.9 to 1300 nM. The moment of truth came when the researchers resynthesized some of the molecules no longer attached to DNA. Compound A117-L1-B217 bound with a Kd of 76 nM as assessed by SPR, while the weakest on-DNA binder (Kd = 1300 nM) showed no binding by itself. Although no explanation is provided for this discrepancy, it could be due to low solubility.

This is an interesting approach, though the molecules reported do tend towards molecular obesity (A117-L1-B217 weighs 765 Da and has a ClogP approaching 8). Indeed, this may be an inherent liability – the minimum allowable distance between two fragments that are each attached to DNA may be larger than desirable for most targets. Still, it will be fun to watch this develop.

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