CD-NTase Nucleotidyltransferases for Diverse Cyclic Messenger Synthesis
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Solution Overview
Problem
The understanding of bacterial second messenger products and their functions in modulating immune responses is limited, restricting the development of effective therapeutics.
Innovation Solution
Elucidation of the diversity of products synthesized by microbial synthases related to Vibrio cholerae enzyme dinucleotide cyclase (DncV) and its metazoan ortholog cGAS, including the development of modified polypeptides with specific amino acid sequences and active sites that catalyze the production of diverse nucleotides, such as cyclic dipurines, cyclic dipyrimidines, and cyclic purine-pyrimidine hybrids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If bacterial second messenger products are studied using conventional methods, then limited understanding is achieved, but therapeutic development is restricted
Solution Approach 1:
The patent employs a universal enzymatic platform (CD-NTases) that can synthesize multiple types of cyclic dinucleotide second messengers (c-di-AMP, c-di-GMP, 2'3' cGAMP, 3'3' cGAMP) using the same enzyme family, enabling broad exploration of bacterial second messenger diversity and their immunomodulatory effects for various therapeutic applications
Solution Approach 2:
The patent utilizes parameter changes in enzyme substrate specificity and product diversity by engineering CD-NTases to accept different nucleotide substrates (ATP, GTP, CTP, UTP) and produce diverse cyclic dinucleotide products with different structures and biological activities, thereby expanding therapeutic options
2Ease of manufacture
If only purine nucleotides are used for CDN synthesis, then synthesis is simplified, but product diversity is limited
Solution Approach 1:
The patent demonstrates that CD-NTases function as universal enzymes capable of synthesizing CDNs from all four nucleotide types (purines ATP/GTP and pyrimidines CTP/UTP), breaking the limitation of purine-only synthesis while maintaining enzymatic simplicity
Solution Approach 2:
The patent achieves product diversity by changing the substrate parameter from purine-specific to all-nucleotide acceptance, enabling synthesis of dipurine (c-di-AMP, c-di-GMP), dipyrimidine (c-di-UMP), and purine-pyrimidine hybrid (2'3' cGAMP, 3'3' cGAMP) CDNs using the same enzyme family
3Loss of information
If bacterial CDN signaling pathways are extensively explored, then immune response understanding improves, but research complexity increases
Solution Approach 1:
The patent extracts and expresses bacterial CD-NTase enzymes in heterologous systems (E. coli, mammalian cells) to produce and study cyclic dinucleotide second messengers, simplifying the research approach by removing the need to study complex native bacterial signaling pathways while maintaining the ability to explore immune responses
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 the synthesis of diverse bacterial second messengers that can modulate immune responses, providing insights for better therapeutic design and immune modulation.
Implementation Method 1
a modified polypeptide that catalyzes production of nucleotides, wherein said polypeptide comprises an amino acid sequence having at least 70% identity to any one of CD-NTase amino acid sequences
Implementation Method 2
the active site comprises the amino acid sequence GSX1X2[ . . . ]XnA1Y1B1, wherein: A1, B1, and C1 independently represent amino acid residue D or E
Data Source
AI summary
The present invention is based, in part, on the discovery and characterization of the CD-NTase family of proteins, as well as compositions comprising CD-NTases, methods of producing nucleotide-based second messengers using such polypeptides, and methods of screening for modulators of the structure, expression, and/or activity of such polypeptides.