Engineered PAL Polypeptides for Oral PKU Therapy

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Solution Overview

Problem

Current oral formulations of phenylalanine ammonia-lyase (PAL) for treating phenylketonuria face challenges such as enzyme degradation in acidic pH and proteolytic conditions of the digestive tract, leading to reduced effectiveness and short enzyme half-life.

Innovation Solution

Engineered PAL polypeptides with optimized amino acid sequences providing enhanced catalytic activity, reduced sensitivity to proteolysis, and increased tolerance to acidic pH levels, along with deimmunization for improved therapeutic and industrial applications.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If wild-type PAL enzyme is used for oral treatment, then the enzyme can catalyze phenylalanine conversion, but the enzyme is rapidly degraded by proteases and acidic pH in the digestive tract

Engineering Contradiction:

Improveenzyme stabilityVSAvoidenzyme half-life

Core Design Contradiction:

ReliabilityVSDuration of action of moving object

Solution Approach 1:

The patent applies parameter changes by systematically modifying amino acid residues at specific positions (e.g., Cys64Ser, Cys318Ser, Cys503Ser, Cys565Ser substitutions) to alter the enzyme's physical and chemical properties, thereby improving its resistance to proteolytic degradation and acidic pH while maintaining catalytic activity

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent employs pegylation (attachment of polyethylene glycol chains) to the PAL enzyme surface, creating a protective coating that extends the enzyme's half-life in the digestive tract without requiring the enzyme itself to be inherently stable, effectively using a disposable protective layer

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

2Productivity

If high doses of PAL are administered orally to overcome degradation, then enzyme activity can be maintained, but the formulation complexity and potential side effects increase

Engineering Contradiction:

Improvephenylalanine conversion efficiencyVSAvoidformulation complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

By changing the amino acid sequence parameters of PAL to create protease-resistant variants, the patent enables effective treatment at lower doses, simplifying the formulation and reducing the need for complex delivery systems or high-dose administration protocols

Inventive Principle:

Principle #35Parameter changes

3Duration of action of moving object

If PAL enzyme is engineered for protease resistance, then enzyme half-life is extended, but catalytic activity may be reduced

Engineering Contradiction:

Improveenzyme half-lifeVSAvoidcatalytic activity

Core Design Contradiction:

Duration of action of moving objectVSPower

Solution Approach 1:

The patent applies local quality by making site-specific amino acid substitutions only at positions critical for protease resistance (surface-exposed residues, cleavage site regions) while leaving the catalytic core and active site residues unchanged, thereby achieving protease resistance without compromising catalytic function

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent introduces multiple amino acid substitutions beyond what would be minimally required for protease resistance, creating a robust enzyme variant that maintains high stability even under varying digestive conditions, while carefully selecting substitutions that do not interfere with catalysis

Inventive Principle:

Principle #16Partial or excessive action

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

The engineered PAL polypeptides exhibit improved stability and activity in acidic environments, extended half-life, and reduced immunogenicity, enhancing their therapeutic efficacy and industrial utility, particularly in treating phenylketonuria and industrial synthesis of phenylalanine derivatives.

Implementation Method 1

Phenylalanine ammonia-lyase (PAL) along with histidine ammonia-lyase (HAL) and tyrosine ammonia-lyase (TAL) are members of the aromatic amino acid lyase family (EC 4.3.1.23-1.25 and 4.3.1.3). More specifically the enzymes having PAL activity (EC 4.3.1.23-1.25 and previously classified as EC4.3.1.5) catalyze the nonoxidative deamination of L-phenylalanine into (E)-cinnamic acid.

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 2

The engineered PAL polypeptides are optimized to provide enhanced catalytic activity, as well as reduced sensitivity to proteolysis and increased tolerance to acidic pH levels.

Methodology Applied

Scientific EffectProteolytic resistance:

Implementation Method 3

The engineered PAL polypeptides are optimized to provide enhanced catalytic activity, as well as reduced sensitivity to proteolysis and increased tolerance to acidic pH levels.

Methodology Applied

Scientific EffectpH tolerance:

Data Source

PatentUS11913045B2Engineered phenylalanine ammonia lyase polypeptides

Publication Date: 2024.02.27 CODEXIS INC

AI summary

The present invention provides engineered phenylalanine ammonia-lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia-lyase (PAL) polypeptides.