Unlocking the Undruggable

How DNA-Encoded Macrocycle Libraries Are Revolutionizing Drug Discovery

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Introduction: The "Undruggable" Challenge

Protein structure

Imagine a complex lock with no key. For decades, many disease-causing proteins involved in cancer, neurodegeneration, and inflammation have been deemed "undruggable" by conventional small-molecule drugs or bulky biologics.

These elusive targets—often flat, featureless protein-protein interaction (PPI) interfaces—resist traditional drug binding. Enter macrocycles: versatile molecular middleweights bridging the gap between small molecules and antibodies. Cyclic peptides and their synthetic cousins offer the specificity of biologics with the cell-penetrating potential of small molecules 1 . But finding the right macrocycle for a target is like finding a needle in a haystack. DNA-encoded library (DEL) technology provides the solution, enabling researchers to screen billions of macrocycles in a single test tube. Recent breakthroughs are now turning undruggable targets into therapeutic opportunities 2 5 .

The Macrocycle Advantage: Beyond the Rule of Five

Macrocycles are cyclic molecules with 12+ atoms in their ring, typically weighing 500–2000 daltons. Their constrained structures offer unique pharmacological benefits:

Target Versatility

Their large surface area enables high-affinity binding to PPIs, which small molecules cannot disrupt 2 .

Preorganized Architecture

Cyclization reduces flexibility, minimizing entropy loss upon target binding 1 .

Enhanced Stability

Resistance to proteases and improved bioavailability vs. linear peptides 1 .

Cell Permeability

Shielded polar groups enable better membrane transit than antibodies 5 .

Table 1: Macrocycles vs. Traditional Drug Modalities
Property Small Molecules Macrocycles Antibodies
Molecular Weight <500 Da 500–2000 Da >150,000 Da
Target PPIs Poor Excellent Excellent
Oral Bioavailability High Moderate Low
Cell Permeability High Moderate Very Low
Synthesis Complexity Low High Very High

DNA-Encoding: The Billion-Molecule Library in a Drop

DEL technology links synthetic compounds to DNA "barcodes" that record their chemical identity. This enables:

  • Combinatorial Synthesis: Building blocks (e.g., amino acids) are iteratively added via split-and-pool synthesis, with DNA encoding each step 1 3 .
  • Ultra-High-Throughput Screening: Billions of DNA-tagged macrocycles are screened against a target protein in a single solution. Bound molecules are PCR-amplified and sequenced to reveal "hits" 3 5 .
Historical Milestones
2004

Liu's team synthesized the first macrocyclic DEL (65 members) using DNA-templated synthesis (DTS) 1 .

2008

A 13,000-member macrocycle DEL identified inhibitors with nanomolar affinity 1 .

2020s

Libraries now exceed 100 million members, incorporating non-peptidic scaffolds and novel cyclization chemistries 5 .

DNA sequencing

DNA sequencing enables ultra-high-throughput screening of macrocycle libraries

Breakthrough Experiment: Target-Guided Macrocycle Assembly

Lam et al. (2023) tackled a key DEL limitation: pre-cyclized libraries often contain linear impurities that mask true binders. Their solution? A "linear precursor" DEL that cyclizes only in the presence of the target protein 4 .

Methodology

  1. Library Construction:
    • Synthesized a 1-million-member DEL of linear peptides with terminal azide/alkyne groups and encoded building blocks.
    • Included cysteine residues for later cyclization via disulfide bridges.
  2. Target-Guided Selection:
    • Incubated the library with immobilized target proteins (e.g., Bcl-xL, Src kinase).
    • Target-bound peptides underwent in situ cyclization via CuAAC ("click" chemistry).
    • Unbound linear peptides were washed away.
  3. Hit Identification:
    • Bound macrocycles were eluted, DNA-sequenced, and decoded.
    • Top hits were resynthesized as cyclized and linear forms for validation.
Results & Analysis
Target Cyclized KD (nM) Linear KD (µM) Affinity Gain
Src 164 5.08 31-fold
Bcl-xL 210 4.20 20-fold
Key Insight

Cyclization on the target selected for conformations perfectly matched to the protein's binding site. The Src binder (164 nM) is among the strongest macrocycles for this target ever reported 4 .

Impact

This strategy eliminates synthetic cyclization inefficiencies and leverages the protein as a template to guide optimal macrocycle formation.

Laboratory equipment

Flexibility-Tuning: The Conformational Sweet Spot

Macrocycle rigidity profoundly impacts binding. Too flexible? Entropy penalizes affinity. Too rigid? The molecule can't adapt to the target. A 2025 study by Derda's group addressed this using a dual-display ESAC library with adjustable flexibility 5 :

Library Design

  • 56 million members split across three configurations:
    • Open: Two linear peptides hybridized via DNA (no covalent link).
    • Semi-closed: Peptides linked via N-terminal triazole ring.
    • Closed: Fully cyclized via N- and C-terminal bridges.
Screening Results
Target Optimal Configuration Top Affinity (nM)
Thrombin Closed 314
Streptavidin Semi-closed 22
Alkaline Phosphatase Open 480
Takeaway

Targets with deep pockets (e.g., thrombin) prefer rigid macrocycles, while shallow interfaces (e.g., streptavidin) require flexibility 5 .

The Scientist's Toolkit: Key Reagents & Technologies

Reagent/Technology Function Example
DNA-Compatible Building Blocks Encode diverse chemistries Unnatural amino acids, dipeptides 5
Click Chemistry Reagents Enable on-DNA cyclization Azides, alkynes, Cu(I) catalysts 4
Photocleavable Linkers Release compounds for validation o-Nitroveratryl (oNv) groups 5
LED Encoding System Streamline DNA recording of multi-step synthesis Large Encoding Design (LED) 5
Affinity Selection Matrices Immobilize target proteins Streptavidin beads, His-tag resins 4

Conclusion: A New Era for Undruggable Targets

DNA-encoded macrocycle libraries represent a paradigm shift in drug discovery. By marrying combinatorial chemistry with DNA barcoding, they unlock screening at unprecedented scale and speed.

Innovations like target-guided synthesis and flexibility-tuned libraries are solving historical hurdles in macrocycle development, yielding high-affinity binders for once-intractable targets. As DEL chemistry expands to include non-peptidic scaffolds and machine learning accelerates hit optimization , these molecular keys are poised to unlock a new generation of therapeutics for cancer, infectious diseases, and beyond. The undruggable may soon become the undone.

Further Reading: See PMC8729180 for historical advances, and Nature Comm 16, 3273 for flexibility-tuning.

References