DNA "modular" programmable strategies build bivalent ligands to achieve fine regulation of receptor activation

At present, a hot direction in drug design and development is the construction of bivalent ligand molecules
.
This class of molecules is formed by coupling two ligand molecules with clear efficacy through linkers; Among them, each ligand molecule recognizes the sites of two different protein receptors, or different sites of the same protein receptor (such as orthoallosteric or allosteric sites), and coordinates ligand-receptor interactions through linkers, thereby enhancing drug efficacy, improving drug selectivity, and overcoming drug resistance ^[1].

Bivalent ligand molecules can regulate many protein receptors, such as G protein-coupled receptors (GPCRs), kinases, ion channels, oxidases, and dimerin
.
Due to their advantages in drug research and development, bivalent ligand molecules have attracted more and more attention from major international research institutions, giving new drug potential to many targets that are not druggable in the traditional sense, and bivalent ligand molecules have entered clinical research ^[2], showing broad development prospects
.

The key to constructing bivalent ligand molecules is the screening and optimization of linkers, because it can affect pharmacological activity
by adjusting the spatial distance and spatial conformation of the two ligand molecules 。 The current method mainly uses polymers (such as polyethylene, polyethylene glycol, etc.
) as linkers to construct and screen bivalent ligand molecules, which faces the following problems: first, it is difficult to accurately control the polymerization degree of polymer linkers in synthesis, resulting in difficult to accurately control the spatial distance between pharmacophores; Secondly, polymer linkers are often composed of a single repeating unit, which is difficult to achieve fine adjustment of spatial orientation when ligand molecules bind to acceptors.
In addition, during the construction and screening process, polymer linkers of different lengths and structures need to be chemically coupled with pharmacophores one by one, and the synthesis and purification steps are cumbersome
.
In summary, the efficient construction and fine regulation of bivalent ligand molecules are the bottlenecks restricting the development of this field
.

Recently, Xiao Zeyu's research group of Shanghai Jiao Tong University School of Medicine cooperated with Zhang Jian's research group to publish a research paper entitled DNA-modularized construction of bivalent ligands precisely regulates receptor binding and activation in Chem, an important sub-journal of Cell.
A DNA modular programmable strategy was developed to construct bivalent ligand molecules to achieve fine regulation
of receptor recognition and activation conformation at the monodeoxynucleotide level.
This strategy draws on the programmable construction method of DNA molecules, modifies the two ligand molecules into "nucleotide-like" pharmacophore modules that can be used for DNA solid-phase synthesis, uses natural deoxynucleotides as linker modules, and uses DNA solid-phase synthesizer to automatically and efficiently construct a screening library
for bivalent ligand molecules 。 By programming the number of deoxynucleotides to finely adjust the length of the linker, the spatial distance between the two pharmacophores is regulated at the scale of 0.
33 nanometers, and the structural diversification of the linker is adjusted by programming the base arrangement of the deoxynucleotide to achieve fine regulation of the spatial orientation of the pharmacophore, so as to efficiently screen and obtain bivalent ligand molecules
with the highest affinity for receptor recognition and the strongest excitation efficiency.
This strategy provides a new idea for the design and development of bivalent ligand molecules, and has a wide range of application prospects
in drug design and biomedical research.

In order to confirm the feasibility of this design strategy, the team combined their respective research advantages to construct a "orthoallosteric-allosteric" bivalent ligand molecule for the positive and allosteric sites of the same protein receptor, that is, one ligand molecule binds to the positive site of the protein receptor, and the other ligand molecule binds
to the allosteric site.
Specifically, the protein receptor in this work was selected as M1 muscarinic acetylcholine receptor (M1Rs), which is a class of GPCR receptors
closely related to cognitive function and tumorigenesis and development.
Among the bivalent ligand molecules, one ligand molecule is the normal agonist Zhammelin (X), which recognizes the positive site of M1Rs; The other ligand molecule is the allosteric regulator BQCA (abbreviated as B), which recognizes the allosteric site of M1Rs
.
Through the co-regulation of such a "orthoallosteric-allosteric" bivalent ligand molecule, the recognition and agonistic selectivity of M1 receptor (instead of M2~M5 receptor) subtypes are realized, and side effects
are reduced.

The research team first constructed the pharmacophore modules of X and B by drawing on the modification strategy of natural deoxynucleotides, and confirmed that the activity
of the original drug was retained after modification.
Furthermore, by using DNA solid-phase synthesis technology, bivalent ligands containing 1-7 deoxynucleotides were designed between pharmacophores, and the screening found that when the linker was two deoxynucleotides, the bivalent ligand had the strongest
selectivity for M1 receptor.
On this basis, the base arrangement of the two deoxynucleotides was programmed to obtain 16 bivalent ligand libraries containing linkers with different structural compositions, and the screening found that the difference in the horizontal arrangement of monodeoxynucleotides made the titer intensity of the bivalent ligand to M1 receptor agitation show a minimum subtle change of ^10-3, and when the base arrangement of the linker is "AA", the bivalent ligand has the strongest ability to selectively stimulate the M1 receptor, which is nearly 30 times
higher than that of the traditional method.

Finally, the research team explored the molecular mechanism
of the interaction of this optimal bivalent ligand with the M1 receptor.
The amino acid site of the bivalent ligand interacting with the M1 receptor was predicted and experimentally confirmed by molecular docking
.
Based on molecular dynamics simulations, it was found that BAAX bivalent ligands can stabilize the M1 receptor conformation in the state closest to its activation conformation, thereby demonstrating the ability to
strongly activate the M1 receptor.

Professor Xiao Zeyu and Professor Zhang Jian of Shanghai Jiao Tong University School of Medicine are the corresponding authors
of the paper.
Dr.
Na Zhao, Dr.
Wenwei Wu and Master Ying Wang of Shanghai Jiao Tong University School of Medicine are the first authors
of the paper.
The work is guided and strongly supported
by Academician Tan Weihong of the Institute of Molecular Medicine of Shanghai Jiao Tong University School of Medicine and Professor Chen Hongzhuan of the Shanghai Biomedical Clinical Research and Translational Collaborative Innovation Center jointly established by the Ministry of Education.

Expert Reviews:

Chunhai Fan (Academician of Chinese Academy of Sciences, Expert in DNA Science and Technology)

As the main genetic molecule of the origin of all life, DNA has brought many enlightenments
to the progress and development of human civilization, whether in terms of biological properties, chemical structure, synthesis methods, etc.
For example, scientists inspired by the principle of complementary pairing of natural DNA molecular bases have developed DNA molecular logic gate computers to realize number operations in living ^{organisms [3]}; The adjacent nucleotide spacing inside the DNA is 3.
3Å to achieve precise control of chromophores, fluorophores and protein ^{equidistances [4]}; The DNA solid-phase synthesis technology, inspired by the ligation chemistry of natural DNA, enables precise control of the polymerization degree of this macropolymer [^5].

In recent years, using the accuracy and programmability of DNA, people have begun to expand the application of DNA engineering in biomedicine, such as designing DNA nanorobots for programmable drug delivery ^[6]; Manufacture of DNA-based molecular vaccines that achieve maximum B-cell responses by controlling antigen spacing and size ^[7]; and lipid transfer using DNA origami for precise control of distance ^[8].

However, so far, it has not been revealed whether DNA can act as a linker between bivalent ligand molecules to finely modulate ligand-receptor recognition conformation
.

Professor Xiao Zeyu and Professor Zhang Jian's team recently published Chem for the first time to clarify the new enlightenment
brought by DNA to the design of drug or probe molecules.
In this work, the research team focused on the potential of DNA as a linker in bivalent ligand molecules and used it to develop a molecular library
of bivalent ligands for "orthoallosteric-allosteric" interactions.
The bivalent ligand contains two pharmacophores and needs to be conjugated by linkers to form a molecule
.
Linkers of different lengths and chemical compositions have an impact
on the properties of the entire bivalent ligand molecule.
Traditional methods use polymers as linkers to achieve fine adjustment, and mainly rely on step-by-step liquid phase synthesis methods to achieve the construction of bivalent ligands, but linker coupling with different lengths and structures involves a more cumbersome process
.
This work cleverly solves this problem
by developing a "DNA modular" construction strategy for bivalent ligands, using DNA's building block deoxynucleotide as a linker.
By modifying the orthoallosteric and allosteric pharmacophores into raw material modules for solid-phase synthesis, a series of bivalent ligand molecules with precise differences in pharmacophore spatial distance and spatial orientation can be constructed with the help of DNA automatic synthesis technology.
In addition, the DNA modularization strategy greatly simplifies the steps of bivalent ligand molecule synthesis, facilitating the efficient construction of bivalent ligand candidate molecular pools
.

More uniquely, this strategy realizes the regulation
of receptor recognition by bivalent ligands at the single deoxynucleotide level.
Increasing or decreasing the number of deoxynucleotides can regulate the spatial distance between the two pharmacophores at the angstrom to nanometer scale; By replacing the base type of the deoxynucleotide, the spatial orientation of the pharmacophore can be finely adjusted, so as to truly realize the fine regulation
of the spacing and spatial orientation of the pharmacophore.

This research fully reflects the intersection of chemistry, biology, pharmacy and engineering, and is a pioneering work of DNA in the field of drug or probe molecular design, especially in the field of dual-recognition site molecular design, revealing the new possibilities and unique advantages of DNA as a linker between pharmacophores to finely regulate the recognition conformation of drugs and targets (or ligands and receptors), and provides a new perspective and construction strategy
for the development of new bivalent ligand probes or bivalent drug molecules 。 Compared with the traditional methods, this method has greatly improved the accuracy of structural function regulation and the simplicity of bivalent molecule synthesis, fully demonstrating the characteristics of precise and controllable DNA structure and its strong advantages
as a linker.
All in all, this research is an inspiring pioneering work in the field of drug or probe molecular design, as well as in the biomedical application of DNA nanotechnology, which is expected to provide a universal design platform for the development of bivalent ligand probes or bivalent drug molecules, and expand the application
of DNA as a functional element in the field of biomedicine.