Methacrylamide Covalent Ligand Directed Releasing Protein Binders

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Solution Overview

Problem

Current methods for selectively labeling endogenous proteins in live cells face challenges such as large domains disrupting native protein function, limited scope due to laborious genetic engineering, and chemical probes that often inhibit protein activity or have slow kinetics and low stability in cellular environments.

Innovation Solution

Development of α-substituted methacrylamides as Covalent Ligand Directed Releasing (CoLDR) compounds that form covalent bonds with specific nucleophiles, allowing for site-specific labeling and release of probes or drugs, maintaining protein activity and stability.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If traditional affinity labeling is used to selectively label endogenous proteins, then site-specific labeling is achieved, but the recognition moiety permanently occupies the ligand-binding pocket causing loss of native protein activity

Engineering Contradiction:

Improvesite-specific labelingVSAvoidnative protein activity

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The probe is divided into two functional parts: a recognition moiety (ligand) that binds to the target protein and a reactive functionality that forms a covalent bond. The recognition moiety binds reversibly to guide the reactive group to the target, while the reactive group forms the covalent bond with a nucleophilic residue. This segmentation allows the recognition moiety to dissociate after binding, preserving protein activity while achieving site-specific labeling.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The reversible binding of the recognition moiety acts as an intermediary step that guides the reactive functionality to the target protein's active site. This intermediary binding event enables precise positioning of the reactive group without permanent occupation of the binding pocket, as the recognition moiety can dissociate after facilitating covalent bond formation.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Manufacturing precision

If ligand-directed chemistries with activating groups are used, then covalent bond formation is achieved, but the size of activating groups and linkers is substantial precluding labeling of residues very close to the active site

Engineering Contradiction:

Improvecovalent bond formationVSAvoiddistance from active site

Core Design Contradiction:

Manufacturing precisionVSLength of moving object

Solution Approach 1:

The invention extracts and eliminates the need for large activating groups by using a straightforward electrophilic reactive functionality that directly forms covalent bonds with nucleophilic residues. This removal of bulky activating groups allows the probe to access and label residues very close to the active site that would be inaccessible to larger probe designs.

Inventive Principle:

Principle #2Taking out (Extraction)

3Productivity

If acrylamides are used as electrophiles for irreversible covalent inhibitors, then non-equilibrium kinetics and full target occupancy are achieved, but the intrinsic reactivity is significantly dictated by the nature of the amine precursor complicating structure modification

Engineering Contradiction:

Improvetarget occupancyVSAvoidstructure modification flexibility

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The invention applies local quality by using a methacrylamide electrophile with specific electronic properties (electron-withdrawing group at the α-position) that enhances reactivity and enables reversible covalent bonding. This localized electronic modification at the α-position allows independent optimization of reactivity and binding affinity without affecting other parts of the molecule, providing flexibility in structure modification while maintaining full target occupancy.

Inventive Principle:

Principle #3Local quality

4Productivity

If electron-withdrawing groups are added at the α-position to increase acrylamide reactivity, then reactivity is enhanced, but reversibility is introduced to the covalent bond formation

Engineering Contradiction:

ImprovereactivityVSAvoidcovalent bond stability

Core Design Contradiction:

ProductivityVSStability of the object's composition

Solution Approach 1:

The invention utilizes parameter changes by introducing an electron-withdrawing group at the α-position of the methacrylamide, which modifies the electronic properties of the electrophile. This parameter change increases the reactivity of the methacrylamide toward nucleophilic residues while simultaneously introducing reversibility to the covalent bond through formation of a stable imine or enamine product. The electron-withdrawing group stabilizes the transition state and facilitates both forward and reverse reactions, allowing dynamic equilibrium between bound and unbound states.

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

Enables efficient, selective labeling of proteins like BTK, K-RasG12C, and SARS-CoV-2 PLpro with preserved activity, and facilitates drug delivery or fluorescence/chemiluminescence probes, improving the toolbox for targeted covalent inhibitors and protein research.

Implementation Method 1

the ligand leaves the active site after forming a covalent bond with nucleophilic residue on the POI

Methodology Applied

Scientific EffectCovalent bonding: Chemical Bonding

Implementation Method 2

Fluorescent labeling of a protein of interest (POI) is a prominent example that can enable imaging, analysis of the structure, function, dynamics, and localization of a target protein

Methodology Applied

Scientific EffectFluorescence: Fluorescence