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【Expert Report】Dr. Xingquan Ma: Medicinal Chemistry in Small Nucleic Acid Drug Discovery

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On March 24, 2023, Dr. Xingquan Ma, SVP of the Chemistry Department of Medicilon, gave a report titled "Medicinal Chemistry in Small Nucleic Acid Drugs Discovery" during the IDC2023 4th Chemical New Drug and Improved New Drug R&D Forum.

Xingquan Ma gave a report titled Medicinal Chemistry in Small Nucleic Acid Drugs Discovery.webp


History of Small Nucleic Acid Drugs

The development of small nucleic acid drugs is based on the discovery of DNA in 1940. In the past eighty years, there have been the following key nodes:

In 1998, the FDA approved the first ASO drug, Fomivirsen, for the treatment of cytomegalovirus (CMV) retinitis.

In 2004, Macugen, the world's first nucleic acid aptamer drug was approved, targeting VEGFR for the treatment of age-related wet macular degeneration.

In 2018, the world's first siRNA drug Onpattro (Patisiran) was approved by the FDA for the treatment of patients with polyneuropathy caused by hATTR.

Development History of Oligonucleotides.webp

It can be seen that the development history of small nucleic acid drugs can be regarded as a long history, and the popularity of small nucleic acid drugs has surpassed PROTAC and ADC recently, and has become the new trend of drug R&D.  Because the mechanism of small nucleic acid drugs is to solve the problems at the fundamental level of genetic material, this has a huge advantage over traditional small molecule drugs and antibody drugs.  Therefore, as the development of small nucleic acid drugs continues to deepen, more targets that were once considered undruggable will be overcome in the future.


The core of small nucleic acid drug development is medicinal chemistry

The drug development process is not as complicated as we imagined for small nucleic acid drugs including siRNA, miRNA, Antisense oligo, CpG oligo and Aptamer.  It is actually a very simple and intuitive chemical drug that can be synthesized by medicinal chemistry methods, therefore the drug development of small nucleic acid drugs mainly focuses on chemical synthesis and chemical modification.

So far, there are 16 small nucleic acid drugs, mainly ASO, have been approved, but the number of siRNAs has increased in recent years.  Among them, there are many small nucleic acid drugs targeting the liver, with the wider application range.

first small nucleic acid drug Fomivirsen.webp

The first small nucleic acid drug Fomivirsen, which is the first ASO drug in 1998, compared with natural nucleotides, the hydroxyl group at the 2 position is deoxygenated, and the oxygen on the phosphoric acid is replaced with sulfur.  This is the most basic chemical modification in small nucleic acid drugs, known as the first-generation chemically modified phosphate backbone modification.


In 2004, the first aptamer drug, Pegaptanib, added a methoxy group to the ribose.


In the PMO drug Eteplirsen approved in 2016, the phosphoric acid used for linkage was modified to phosphorus-nitrogen, and three similar PMOs have been approved too.


Patisiran, which was approved in 2018, is the eighth nucleic acid drug successfully marketed and it was the first siRNA.  Due to the double-stranded RNA structure, its chemical structure is obviously much more complicated than that of ASO and PMO.

During this time of the launch of siRNA, small nucleic acid drugs that use ethylene glycol to modify monomers are relatively concentrated in the market, which is also the second generation of chemical modification.


Givosiran in 2019 is the first siRNA to use the GalNAc delivery system, which has epoch-making significance for the development of the entire nucleic acid drug.  Different from the previous LNP liposome delivery system, the three galactosamines of GalNAc showed high selectivity and targeting, and had target specificity in the liver.

There is another kind of cEt Containing Gapmer under research, which has a relatively complex structure and is still in the stage of exploratory research.

Chemical modification of small nucleic acid drugs is beneficial to improve the stability and availability of drugs, and even improve the efficacy and pharmacokinetics of drugs.  With the upgrading of chemical modification technology, the drug activity of small nucleic acid drugs has also been continuously improved, mainly reflected in the frequency and dosage of administration.


Delivery Systems for Nucleic Acid Drugs

There are a lot different types of delivery systems. Here are 9 delivery systems:

Delivery Systems for Nucleic Acid Drugs.webp

Many types of delivery systems here have not been successfully tested in the market. For example, the third one that uses antibody bridging as an siRNA delivery system is not yet on the market, but many people are already studying it.  We are focusing on LNP and GalNAc as below.

LNP has immunogenicity in the human body and liposomes with altered properties cannot target drug delivery.  Cholesterol accounts for a large proportion in LNP, because from the perspective of microstructure, cholesterol has a large skeleton and relatively weak water solubility, which can support spherical and disc-shaped structures.

GalNAc does not exhibit immunogenicity, so it is much more safer.  GalNAc targets the ASGPR site on hepatocytes and can be absorbed through endocytosis with fewer side effects.


Medicilon nucleic acid drug research and development experience

The Medicilon nucleic acid drug R&D service platform is a platform integrated with drug discovery, production and preclinical research, which could complete the chemical synthesis and modification of nucleic acid drugs, and the detection of PK/PD in vivo and in vitro.  After nearly 4 years of research and development, the Medicilon nucleic acid drug research and development service platform has accumulated rich experience.  Currently, there are more than 10 ongoing projects, and a scientific research team with more than 50 chemists dedicated to the development of nucleic acid drugs, constructing more than 100 usable monomers.

Based on a rigorous scientific attitude, an open technology platform, extensive R&D experience and state-of-the-art equipment, we could meet the industry's R&D needs for cutting-edge innovative nucleic acid drugs.


Process of Chemical Synthesis of Oligonucleotides

Process of Chemical Synthesis of Oligonucleotides.webp

In general, each new base needs to go through 4 steps: DET, CPL, CAP, OXI.

1. Deprotection (DET):  Reaction of nucleotides with protected active groups pre-connected on the solid phase carrier CPG with trichloroacetic acid to remove the 5'-hydroxyl protecting group DMT to obtain free 5'-hydroxyl groups

2. Coupling (CPL):  Mixing the phosphoramidite-protected nucleotide monomer with the activator tetrazolium to obtain a nucleoside phosphorous acid activated intermediate, which undergoes a condensation reaction with the free 5'-hydroxyl in the solution

3. Capping (CAP):  In the condensation reaction, there may be a very small number of 5'-hydroxyl groups that do not participate in the reaction (less than 2%), and the synthesis can be terminated with acetic anhydride and 1-methylimidazole. This short fragment can be separated during purification.

4. Oxidation (OXI): Under the action of oxidants, the phosphorous acid form is transformed into a more stable phosphotriester, making the DNA phosphate backbone more stable.

Repeat the above cycle until all the bases to be synthesized are connected, and then enter the Ammonolysis reaction.

The ammonolysis reaction also has two reactions through different instruments, one is gas phase and the other is liquid phase.  After ammonolysis, it needs to be freeze-dried or ultracentrifuged, washed with deionized water, and then analyzed and purified.  Note that in these steps, the water content needs to be paid attention to.  It is because if the water content is too high, the side reactions are prone to occur, which will have a negative impact on the results.

For the analysis of oligonucleotides, Medicilon mainly uses two instruments, UPLC and HPLC.  The purity of HPLC test results can generally reach more than 80%, and the purity of UPLC and QTOF combined detection is also more than 80%. We could see the peak spectrum of the test results and generally the results less than 500 ppm are accurate.

Purification will use preparative HPLC, and the concentration of nucleic acid drug samples after purification could reach more than 98%.  The second method is to use AEX HPLC.  Oligonucleotides purified by anion-exchange HPLC must be free of unwanted salts, which is easily achieved by using desalting columns.

Here is an example of a synthetic siRNA:

example of a synthetic siRNA.webp

It can be seen that the purity of siRNA reached 92% after preparative HPLC purification and the peak shape on the spectrum is not too sharp.  In fact, this is related to the phosphorus-sulfur bond on the leftmost 5', and the possible chiral structure leads to imperfect results of the data.


Medicilon Nucleic Acid Drug R&D Service Platform

Medicilon can provide monomer synthesis and modification; oligonucleotide synthesis; delivery system synthesis and synthesis of oligonucleotide conjugates.  The siRNA library that has been built not only has a rich monomer inventory, but also has a huge monomer synthesis building block library, which can quickly complete the synthesis of various modified monomers.  Medicilon has a professional small nucleic acid drug R&D team that can provide efficient and fast R&D services.  There are several siRNA drug FTE projects have been completed and some projects are in progress.

On the biological activity evaluation service can be carried out:

Binding evaluation of siRNA-GALNAc and target liver cells (ELISA, SPR, FP, FACS, MSD, Confocal microscope)
Target mRNA/protein level reduction assessment (RT/Q-PCR, Western blot)
Evaluation of cell phenotype and functional interference (cell proliferation, migration, protein modification and interaction)
Off-target effect assessment



Nucleic acid drugs, as the third largest type of drugs after small molecule drugs and antibody drugs, have developed rapidly in the world, and have become the focus and hotspot of research and development by pharmaceutical companies.

The development of nucleic acid drugs is extremely fast and highly targeted, and it is expected to break through the "undruggable" targets that traditional drugs cannot solve.  In the R&D process, medicinal chemistry is the core and focal point.

The Medicilon nucleic acid drug R&D service platform has extensive experience in nucleic acid drug R&D.  Medicilon could complete the synthesis of various modified monomers, formulate development routes, fulfill the industry's R&D needs for cutting-edge innovative nucleic acid drugs, and provide nucleic acid drug discovery, screening and preclinical research services for pharmaceutical companies and scientific research institutions.

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