Cyclic peptide drugs are gaining renewed attention, but efficiently finding high-affinity, developable cyclic peptide ligands from vast chemical space remains a challenge. DNA-encoded chemical library (DEL) technology can build huge compound collections at relatively low cost, yet conventional DELs struggle with fine control over cyclic peptide conformational diversity. A recent study by Petrov et al. in Nature Communications tackled this problem using a dual-display ESAC library and a two-step cyclization strategy, achieving for the first time three levels of conformational restraint (open, semi-closed, fully closed) within a single library. This article breaks down the technical design and explores how specialized cyclic peptide discovery and physicochemical property optimization services can help translate such innovative strategies into real lead compounds.
DEL technology has proven its value in small-molecule discovery, but applying it to cyclic peptides brings two major headaches: poor library purity and limited conformational diversity. This paper offers systematic solutions to both problems.
Conventional DEL construction for cyclic peptides often performs four or more split-and-pool steps sequentially on a single DNA strand. Uneven reaction efficiency at each step leads to significantly reduced purity of the final library members. More importantly, cyclization is usually done in a single step, giving either overly flexible (linear precursors) or overly rigid (fully cyclized) peptides, with no intermediate states. Many protein targets actually prefer a moderately constrained cyclic peptide conformation. Missing those intermediate states means missing many potential ligands.
The Petrov team adopted a different approach using dual-display ESAC technology. Two sub-libraries were constructed on complementary DNA strands (HP5 and HP3), each undergoing two rounds of amino acid assembly and purification before hybridization into heteroduplexes. They introduced three levels of conformational constraint: open (DNA hybridization only), semi-closed (N-termini linked via click chemistry), and fully closed (additional C-terminal linkage via bis-electrophiles or disulfides). The resulting library contained 56 million members, with each conformational state individually encoded, enabling simultaneous evaluation of target preference for different peptide flexibilities in a single selection.
DP-DEL library architecture and synthesis1,4
Beyond describing the technology, the study validates it across three distinct targets. Each target exhibited a preference for a particular conformational state, offering practical guidance for the rational design of cyclic peptides.
Unlike conventional DELs, this study performed HPLC purification and mass spec confirmation on each sub-library member after the first amino acid coupling, ensuring high purity for the first two steps. Only then were the purified sub-libraries mixed for encoding and cyclization. This approach effectively avoided purity loss from cumulative inefficiencies. The molecular weight distribution of fully closed library members centered around 2000-4000 Da, matching the size range of typical cyclic peptide drugs.
Selections against thrombin showed that the most enriched cyclic peptides were mainly from the semi-closed version (version 2). Follow-up validation confirmed that the clicked format had stronger affinity than the open form, with compound 1 showing a KD of 609 nM in the clicked state versus 4.15 µM in the open state. When synthesized off-DNA and tested with different C-terminal linkers, a medium-flexibility linker (L1) gave the best IC50 of 314 nM, while both highly flexible and overly rigid linkers performed worse. Overall, the results indicate that thrombin favors N-terminal constraint while maintaining some flexibility at the C-terminus.
DP-DEL thrombin selections2,4
Selections against streptavidin also enriched semi-closed cyclic peptides. Related compounds showed strong affinity in the clicked format, while the open versions were significantly weaker. However, when tested off-DNA with different C-terminal linkers, only the C-terminally open form retained binding, whereas cyclized variants lost activity. This suggests that streptavidin prefers peptides with N-terminal constraint but requires a flexible, unclosed C-terminus. In addition, the peptides demonstrated selectivity for streptavidin over related proteins such as avidin and neutravidin, highlighting their potential for reagent development.
Selections against PLAP yielded an unexpected result: enrichment was dominated by the open version rather than any cyclized formats. Follow-up analysis suggested that an incomplete diazotransfer during library synthesis may have led to the selection of the free amine form instead of the intended azide-containing species. Resynthesis confirmed that amine-containing compounds were consistently more active, while the corresponding azide variants showed reduced affinity. Overall, the optimized hits remained in an open conformation, indicating that PLAP favors linear or only partially constrained peptides rather than fully cyclized ones.
DP-DEL PLAP selections3,4
The Petrov team's work demonstrates the value of fine-tuned conformational control in cyclic peptide screening. But for most researchers trying to apply similar strategies to their own projects, three practical bottlenecks often get in the way.
Dual display, two-step cyclization, multi-version encoding – this workflow requires expertise in DNA chemistry, peptide synthesis, and molecular biology all at once. For most medicinal chemistry teams, building such a platform from scratch is neither realistic nor economical. What they really need is access to well-designed, ready-to-use diversity libraries, or the ability to outsource custom library design based on their target.
Enriched sequences from DEL screening must be resynthesized, explored for SAR, and optimized for cyclization mode before they become credible leads. In this study alone, the thrombin work required multiple linker variants, and the PLAP work tested several dual-display partners. Such efforts demand efficient parallel synthesis and robust purification and characterization. The difficulty increases significantly when ring size, linker length, or non-natural amino acids need adjustment.
A cyclic peptide can look great in binding assays but still fail due to poor solubility, metabolic instability, or low permeability. The paper does not go deep into these properties, but in real projects they are the most common killers. Improving solubility and stability through residue substitution, N-methylation, or lipophilicity adjustment while maintaining affinity is a skill that requires both experience and data.
To address the bottlenecks above, Creative Peptides provides integrated services covering library design, hit identification, sequence optimization, and physicochemical property tuning. We don't just synthesize compounds – we help you build a complete evidence chain from screening to leads.
| Services | Contents |
| Cyclic Peptide Drug Discovery Services | For scenarios like DEL screening that require extensive downstream chemistry validation, our service modules offer precise support:
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| Solubility, Stability, and Permeability Optimization for Cyclic Peptides | Physicochemical optimization of cyclic peptides is often more complex than for linear peptides, because cyclization itself changes polar surface area and hydrogen-bonding patterns. Our optimization services provide tailored solutions to specific questions:
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The work by the Petrov team highlights the value of conformationally tunable cyclic peptide libraries, but the goal remains obtaining high-quality, well-characterized molecules that support real project decisions. Success across library design, hit validation, SAR, and optimization depends on strong chemistry and reliable characterization.
Creative Peptides supports this workflow:
Contact our scientific team today to discuss your project and turn screening results into validated lead compounds.
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