The success or failure of small-molecule drug discovery efforts strongly depends on the “hit-finding” approaches that are applied at the inception of the drug discovery program (1). High-throughput screening of compound collections is still the main strategy (2), but several other approaches have shown promise. These include screening virtual libraries using three-dimensional protein structure or ligand information (3), de novo design of ligands (4), screening fragment (very small molecule) libraries (5), screening (cyclic) peptide libraries (6), repurposing existing compounds, and screening DNA-encoded libraries (DELs) (7). On page 939 of this issue, Rössler et al. (8) reveal a new hit-finding method that uses peptide-encoded libraries (PELs), which are similar to DELs.
In PELs, solid-phase peptide and small-molecule syntheses are used to readily generate large libraries of bifunctional molecules that each consist of a peptide tethered to a small molecule through a cleavable linker. After cleavage from the solid phase, these libraries are incubated with an immobilized therapeutic protein of interest for affinity selection. To identify those molecules that bind to the target protein, the peptide is cleaved from the bifunctional molecule and sequenced using mass spectrometry technologies that are normally applied in proteomics research (such as nanoscale liquid chromatography. tandem mass spectrometry). On the basis of the sequence of the peptide, the chemical structure of the smallmolecule ligand can be identified. This is because the single amino acids that are used for synthesis of the peptide directly encode the corresponding chemical building blocks that are used to synthesize the small molecules.
The advantages of PEL technology over DELs are manifold. Most notably, a PEL supports harsher and more diverse chemical reactions, including metal-catalyzed reactions and reactions that require strong acidic or basic conditions. This breadth enables the synthesis of a wider scope of drug-like molecules. Another advantage is the application of solid-phase synthesis for peptides and small molecules, which allows the use of excess reactants. This, in turn, supports a higher yield and purity of the final small molecules, which is expected to substantially improve the quality of the libraries. Changing the tagging moiety from four DNA bases to a peptide that contains 16 different amino acids enables a higher information capacity. Thus, in theory, even larger libraries of small molecules could be synthesized and encoded. If an eight-digit encoding string is used, then there are 16 amino acids (hexadecimal system) that can generate 4.3 billion possible codes. By contrast, there are only 56,535 possible codes using the four bases of DNA.
It is thought that the DNA tag of the DELs could interfere with targets that are per se DNA-binding, such as transcription factors or RNA. By contrast, libraries with peptide tags would potentially be better suited for screening against such targets because the amino acids used for the peptide synthesis are less likely to bind to those targets. To confirm that a hit identified in a DEL screen can actually bind to a target, the hit compound is synthesized without a DNA tag and then tested for its effect on biological activity. This can be tedious because, during DEL library synthesis, not every chemical reaction is successful. Occasionally, reaction byproducts are the biologically active compounds, and it takes several investigations to determine this. The hit resynthesis that stems from a PEL can still be performed by solid-phase synthesis using the same conditions that were used to construct the library. This allows a more rapid synthesis and makes the identification of potential by-products easier.
Some challenges need to be overcome to fully exploit PEL technology. Peptide concentrations must be present in at least a 10 fM range to be detected by mass spectrometry. This affects the size of a PEL because, in contrast to a DNA tag, the peptide tag cannot be amplified. The screens of Rössler et al. were performed at ∼1 nM concentration for each peptide-tagged compound. This means that a 100,000-membered library could be screened at a 100 µM library concentration. Because the peptide tags used by Rössler et al. were mostly hydrophobic, there is a certain risk of solubility problems and unspecific peptide aggregation of the library members, which could interfere with binding to a putative target and lead to screening artifacts.
The libraries generated by Rössler et al. were screened against the targets human carbonic anhydrase IX, the epigenetic reader bromodomain-containing protein 4 (BRD4), and the E3 ubiquitin ligase mouse double minute 2 homolog (MDM2). In all cases, several hits were identified that could serve as interesting starting points for further improvements of their potency and properties. PELs could potentially be enhanced by exploiting a wealth of already-established solid-phase organic chemistry reactions to generate new druglike molecules in a chemical space that is not accessible by the DEL technology. Such libraries would be of high interest to drug discovery groups for screening against therapeutic protein targets for which no small-molecule ligands are yet known.
REF ERENCES AND NOTES
1. D. G. Brown, J. Boström, J. Med. Chem. 61, 9442 (2018).
2. P. S. Dragovich, W. Haap, M. M. Mulvihill, J.-M. Plancher, A. F. Stepan, J. Med. Chem. 65, 3606 (2022).
3. F. Gentile et al., Nat. Protoc. 17, 672 (2022).
4. M. Skalic, J. Jiménez, D. Sabbadin, G. De Fabritiis, J. Chem. Inf. Model. 59, 1205 (2019).
5. D. A. Erlanson, S. W. Fesik, R. E. Hubbard, W. Jahnke, H. Jhoti, Nat. Rev. Drug Discov. 15, 605 (2016).
6. C. Sohrabi, A. Foster, A. Tavassoli, Nat. Rev. Chem. 4, 90 (2020).
7. R. A. Goodnow Jr., C. E. Dumelin, A. D. Keefe, Nat. Rev. Drug Discov. 16, 131 (2017).
8. S. L. Rössler, N. M. Grob, S. L. Buchwald, B. L. Pentelute, Science 379, 939 (2023)
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