Molecular Mimicry Redefined: Bridging Antibody Precision with Synthetic Macrocyclic Scaffolds

The Blueprint of Biomimetic Design

The adaptive immune system’s ability to generate antibodies with exquisite specificity for antigens has long inspired synthetic chemists. A recent breakthrough leverages a macrocyclic scaffold to display multiple chemical diversity elements (DEs), emulating the multivalent interactions of antibody-antigen binding. This scaffold, a rigid β-sheet macrocycle, serves as a programmable platform for combinatorial chemistry, enabling the systematic exploration of chemical space while maintaining structural integrity. DNA encoding ensures precise tracking of each synthetic combination, facilitating rapid identification of high-affinity binders post-selection.

The macrocycle’s design integrates three lysine side chains oriented on one face of the β-sheet, each modified with distinct chemical moieties during split-and-pool synthesis. A fourth site accommodates DNA tags or functional groups for downstream applications. This spatial arrangement mimics antibody paratopes, where hypervariable loops present residues critical for antigen engagement. NMR and crystallographic studies confirm the scaffold’s conformational rigidity, which minimizes entropy penalties upon target binding—a key advantage over flexible linear peptides.

Library construction employed iterative coupling of carboxylic acids, alkynes, and acylating agents under DNA-compatible conditions. Orthogonal protecting groups enabled sequential diversification at each lysine site, while enzymatic ligation and Klenow polymerization ensured accurate DNA tag incorporation. Quality control via LC-MS and gel electrophoresis validated coupling efficiencies exceeding 80%, ensuring library homogeneity. The final library, ETH-YL, comprised 35,393,112 unique macrocycles, each linked to a double-stranded DNA barcode for high-throughput sequencing (HTDS) analysis.

This approach circumvents limitations of traditional combinatorial libraries, where structural flexibility often obscures structure-activity relationships. By constraining DEs in a fixed orientation, the scaffold amplifies cooperative binding effects, akin to antibody avidity. The design also permits modular functionalization—photo-crosslinkers, fluorophores, or therapeutic payloads—enhancing utility in chemical biology and drug delivery.

The ETH-YL library’s success hinges on its balance of diversity and reproducibility. Each synthetic step was optimized for compatibility with aqueous conditions and enzymatic encoding, preserving DNA integrity. This methodological rigor contrasts with earlier DNA-encoded libraries (DELs), which often prioritized drug-like properties over functional versatility.

Decoding Selection Fingerprints

Affinity selections against nine target proteins revealed distinct 3D enrichment patterns, visualized as “fingerprints” in HTDS plots. Each axis represented a diversity element (DE-1, DE-2, DE-3), with dot size and color intensity correlating with sequencing counts. For carbonic anhydrase IX (CAIX), a plane of enriched combinations corresponded to acetazolamide, a known inhibitor, validating the library’s ability to recapitulate natural ligand interactions.

Horseradish peroxidase (HRP) selections highlighted phenolic DEs, consistent with HRP’s substrate preferences. Tankyrase 1 (TNKS 1) enrichments aligned with reported binders, underscoring the scaffold’s capacity to present DEs in bioactive conformations. Human serum albumin (HSA) selections yielded scattered patterns, reflecting its promiscuous binding pockets. Fluorescence polarization (FP) assays of synthesized HSA binders confirmed micromolar affinities, with linear analogs showing reduced potency—evidence of macrocycle rigidity enhancing binding.

Calmodulin (CaM) selections identified biphenyl DEs, with trivalent derivatives achieving submicromolar dissociation constants (Kd = 0.16 μM). This multivalency effect mirrors antibody avidity, where clustered interactions amplify affinity. Prostate-specific antigen (PSA) enrichments revealed DE-3 as a critical anchor, with progressive DE removal diminishing binding—a hierarchical interaction profile reminiscent of antibody paratope-epitope complementarity.

Tumor necrosis factor (TNF) selections using recombinant protein and antibody fusions (L19-TNF) identified overlapping DE combinations, confirming target-specific binding. One binder (TNF-1) exhibited cross-reactivity, suggesting DE redundancy akin to antibody cross-reactivity. These fingerprints demonstrate the scaffold’s adaptability to diverse targets, from enzymes to cytokines.

The scaffold’s structural tunability was probed via D-proline substitutions and lysine linker truncations. While backbone modifications had minimal impact, shortened linkers reduced affinity, highlighting the importance of DE presentation geometry. This tunability offers a path toward affinity maturation, analogous to antibody engineering.

Validation: From Microwell to Microscope

Selected binders were validated in biochemical and cellular assays, bridging synthetic chemistry and biological application. FITC-labeled PSA-1 localized to glandular structures in prostate tissue sections, matching anti-PSA antibody staining. This immunofluorescence-like performance underscores the scaffold’s diagnostic potential, offering an alternative to monoclonal antibodies with reduced immunogenicity risks. In vivo, a CAIX-targeting macrocycle conjugated to a near-infrared dye (CAIX-IRDye) accumulated selectively in tumors, visualized via fluorescence imaging. No uptake was observed with control scaffolds, confirming target-specific delivery. This proof-of-concept highlights the scaffold’s utility for theranostic applications, where DEs can be swapped for cytotoxic payloads.

Photo-crosslinking derivatives, such as CaM-PC, enabled covalent protein labeling. Incubation with calmodulin and UV irradiation yielded specific adducts, absent in controls. Mass spectrometry confirmed covalent modification, illustrating the scaffold’s adaptability for chemical probe development. Such probes could map protein interactomes or validate target engagement in drug discovery.

FP assays quantified binder affinities, with trivalent CaM-3 achieving submicromolar Kd—comparable to early-stage antibody fragments. Linear analogs exhibited reduced potency, emphasizing the macrocycle’s role in pre-organizing DEs. These findings parallel antibody affinity maturation, where loop rigidification enhances binding.

The scaffold’s fourth diversity site facilitated modular payload integration. For example, IRDye conjugation enabled in vivo imaging, while phenyl azide permitted photo-crosslinking. This plug-and-play functionality contrasts with antibodies, where conjugation often disrupts binding.

Benchmarking Against Biological Giants

The ETH-YL library’s performance was contextualized against phage display libraries, the gold standard for synthetic antibody generation. While phage libraries yield nanomolar binders, they rely on proteinogenic amino acids, limiting chemical diversity. The ETH-YL library accesses non-canonical DEs—carboxylic acids, alkynes, and heterocycles—enabling interactions inaccessible to antibodies, such as π-stacking with aromatic residues or coordination with metal ions.

Antibody affinity maturation typically requires iterative mutagenesis and screening. In contrast, ETH-YL’s selection fingerprints guide DE optimization via synthetic iteration—replacing low-affinity moieties with analogs, akin to medicinal chemistry campaigns. This approach could accelerate hit-to-lead progression, particularly for targets lacking crystal structures. The library’s micromolar affinities, while weaker than mature antibodies, align with early-stage antibody fragments (scFvs, Fabs). Larger libraries incorporating additional DEs or synthesis cycles may close this gap. Notably, the ETH-YL library’s 35 million members represent a fraction of phage display diversity (10^10–10^11 clones), suggesting scalability as a route to higher affinity.

Macrocycles’ pharmacokinetic challenges—poor oral bioavailability, proteolytic susceptibility—were not addressed in this study. However, the scaffold’s functionalization site offers a handle for PEGylation or prodrug strategies, potentially mitigating these limitations.

Beyond Binding: Therapeutic Horizons

The scaffold’s modularity extends beyond diagnostics. TNF-1, though micromolar in affinity, could be optimized for cytokine neutralization—a strategy employed by TNF inhibitors like adalimumab. Similarly, CAIX-targeting macrocycles might deliver cytotoxic agents to hypoxic tumors, exploiting CAIX’s tumor-specific overexpression. The photo-crosslinking probe CaM-PC exemplifies the scaffold’s utility in target deconvolution. By covalently labeling calmodulin in complex mixtures, it could elucidate CaM’s role in calcium signaling or identify off-target interactions in drug screens.

In vivo imaging with CAIX-IRDye demonstrates potential for intraoperative tumor margin delineation. Substituting IRDye for radionuclides or MRI contrast agents could expand clinical applications. The scaffold’s tumor accumulation, though preliminary, mirrors antibody-drug conjugate (ADC) tropism, with a critical advantage: smaller size enhancing tissue penetration.

Future iterations might integrate machine learning to predict DE combinations, leveraging HTDS data as training sets. Such AI-driven design could prioritize DEs with favorable physicochemical properties, accelerating library optimization.

Synthetic Biology Meets Precision Chemistry

The ETH-YL library bridges synthetic biology and medicinal chemistry. DNA encoding enables genetic-level reproducibility, while synthetic DEs access chemical space beyond biosynthetic constraints. This hybrid approach could yield “chemical antibodies”—non-immunogenic, tissue-penetrant molecules with antibody-like specificity. The scaffold’s rigidity pre-organizes DEs, reducing entropic costs upon binding—a principle borrowed from natural protein-protein interactions. This design philosophy contrasts with flexible DELs, where conformational heterogeneity dilutes binding energy.

Challenges remain in scaling library synthesis and decoding. Current HTDS methods, while powerful, require specialized infrastructure. Advances in portable sequencing (e.g., nanopore) could democratize DEL screening, enabling broader adoption. The fourth diversity site’s versatility invites exploration of quaternary interactions. Incorporating allosteric modulators or enzyme inhibitors could yield multifunctional macrocycles, targeting protein complexes or pathway nodes.

A New Paradigm in Molecular Recognition

This work reimagines antibody mimicry through synthetic macrocycles, offering a versatile platform for protein recognition. The ETH-YL library’s success against diverse targets—enzymes, carriers, cytokines—validates its design principles. Applications span diagnostics (fluorescence microscopy), targeted therapy (tumor imaging), and chemical biology (photo-crosslinking).

Future directions include affinity maturation via DE optimization, integration with AI-driven design, and exploration of in vivo therapeutic efficacy. As DEL technologies evolve, this scaffold-based approach could democratize access to precision recognition molecules, circumventing the immunogenicity and cost barriers of biologics. In an era of personalized medicine, such synthetically-encoded libraries offer a bridge between small-molecule agility and antibody precision—a toolset limited only by chemical creativity.

Study DOI: https://doi.org/10.1038/s41557-018-0017-8

Engr. Dex Marco Tiu Guibelondo, B.Sc. Pharm, R.Ph., B.Sc. CpE

Editor-in-Chief, PharmaFEATURES