Method for producing glycosylated estrogen receptor modulators using glycosyltransferase

Breast cancer drug with improved bioavailability, created by using an actinomycete-derived glycosyltransferase to attach sugars to tamoxifen analogs.

This technology utilizes MrGT2, a novel glycosyltransferase discovered in the actinomycete Micromonospora rhodorangea, and an enzymatic bioconversion method to attach sugars to selective estrogen receptor modulators (SERMs) such as afimoxifene, endoxifen, and toremifene. The resulting glycosylated SERM compounds are also included within the scope of the patent.

While tamoxifen is widely used to treat breast cancer, it acts as an agonist on estrogen receptors in the bones and endometrium, leading to reported risks of osteoporosis and uterine cancer with long-term use, which limits treatment to five years. There has been a need for new derivatives or prodrugs with higher bioavailability to achieve efficacy at lower doses, but there were no known cases of using enzymatic bioconversion to create glycosylated tamoxifen-family SERMs.

This technology involves inserting a polynucleotide encoding MrGT2 into an expression vector to produce the enzyme in recombinant microorganisms like E. coli. In the presence of this enzyme, SERMs are reacted with sugar donors such as UDP-glucose or UDP-galactose to attach glucose, galactose, or 2'-deoxyglucose. The products are recovered via reversed-phase medium-pressure liquid chromatography. The glycosylated compounds maintain estrogen receptor antagonist activity while exhibiting improved bioavailability. This can be applied to developing improved breast cancer drugs, tamoxifen-family prodrugs, and enzyme-based glycoside production processes, allowing for therapeutic efficacy at lower doses and reducing the risk of uterine cancer and osteoporosis associated with long-term use.

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Key Features:

• Glycosyltransferase MrGT2 derived from Micromonospora rhodorangea, represented by the amino acid sequence of SEQ ID NO: 4
• Recombinant E. coli microorganisms introduced with a polynucleotide encoding the glycosyltransferase or an expression vector containing it
• Steps of synthesizing and recovering a glycosylated modulator by reacting a selective estrogen receptor modulator with a sugar donor in the presence of the glycosyltransferase
• Glycosylated selective estrogen receptor modulator compounds where R1 is CH3, R2 is Cl, and R3 is glucose, galactose, or 2'-deoxy-glucose

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This invention was developed with support from the Rural Development Administration for the production of novel glycosylated antibiotics utilizing soil actinomycete genetic resources based on biosynthetic pathway engineering.

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