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Hydrea 500 mg Hard Capsules - - (eMC) - Medicines
Eventual in vivo didanosine production may be realized via additional multiple parameter optimization: improved enzyme turnover and/or substrate selectivity of the pathway enzymes, continued retro-extension to enzymatically generate dideoxyribose, further engineering precursor supply, and optimizing efflux and resistance. Of note, the engineered RK/PNP system we describe may represent a specific solution for the generation of dideoxynucleosides drugs. Direct anomeric phosphorylation of 2- or 3-substituted ribosides (such as would be required for the biosynthesis of the 2’-difluoro nucleoside Gemcitabine, or AZT) is not favored by RK-Asp16Ala. These nucleosides therefore require the development of a PPM generalist activity, as we have discovered herein. Given the breadth of structural diversity of both the sugar and nucleobase moieties in nucleoside analog drugs, both PPM and RK variant enzymes may find complementary application in biocatalytic generation of a variety of compounds within this class of therapeutics.
The unexpectedly large increase in productivity in the pathway containing RK-Asp16Ala prompted further investigation of the improved RK. In particular, we considered the possibility of anomeric (C1) phosphorylation. To our knowledge, no enzyme has previously been reported to phosphorylate D-ribose at the 1-position in this or any other superfamily. Excluding PPM-4H11 and assaying RK-Asp16Ala in tandem with PNP-46D6 confirmed this unreported activity of RK. This two enzyme system has the highest didanosine production (44 μM), verified by HPLC/MS comparison to synthetic didanosine standard (). This 4.4% yield from dideoxyribose in this two enzyme system was 70-fold greater than the wild-type three enzyme pathway. Notably, the three enzyme pathway including PPM-4H11 demonstrated decreased yields (), likely due to conversion of ddR1P into dideoxyribose 5-phosphate by the engineered PPM. This has the effect of diverting ribose into the PPM-dependent pathway. Taken together these results suggests that the majority of the RK-Asp16Ala effect on activity in the full pathway was due to increased direct phosphorylation of the anomeric hydroxyl group rather than expected ribose 5-phosphorylation.
Search results for didanosine at Sigma-Aldrich ..
The primary mission of the Bachmann Lab is to apply knowledge of the design rules for secondary metabolism at the chemical, biochemical and genetic levels toward the biosynthesis of "non-natural" compounds of high value to biomedical research and the clinic. Key to this program in "synthetic biology" is the dissection of the mechanisms by which life makes bioactive molecules in vivo. The lab is organized according to three interlocking research areas: Biosynthesis, Synthetic Biology, and Discovery. These subgroups each have basic research and applied components and overlap with one another both thematically and methodologically.
The focus of the biosynthesis research subgroup centers on investigating the biosynthesis of pharmacophores by microorganisms from a genetic to a chemical basis. In all cases, we target non-trivial biotransformations that have little or no precedent in prior research. To date, pathways have been targeted in the bacterial Order Actinomycetales (also called actinomycetes), one of the richest microbial sources of secondary metabololites.
Preparation of Drug Crystals: Didanosine
Synthetic Biology is a burgeoning field, the ambitious aim of which is to use the machinery of biological systems (DNA, RNA, proteins) for the production of synthetic compounds and materials of high value to research, medicine and human life. For small molecule synthesis, these methods offer totally new avenues for the production of compounds and an alternative to petrochemical-based chemical synthesis. We believe that the global societal impacts of synthetic biology will be far reaching in this century. The primary mission of the Bachmann lab is to apply this knowledge of the design rules for secondary metabolism at the chemical, biochemical and genetic levels toward the biosynthesis of "non-natural" compounds of high value to bio research and the clinic.
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Solvothermal Preparation of Drug Crystals: Didanosine
To evaluate the improvements engendered by the separate pathway product selection-based directed evolution experiments, we measured changes in nucleoside production after replacing wild-type enzymes with the engineered counterparts in increasingly longer didanosine biosynthetic pathways. shows the selectivity and turnover improvements resulting from PNP evolution alone, totaling 140-fold change in substrate selectivity and 16-fold increased didanosine production. Extending in vitro nucleoside production to the two enzyme system, the combination of wild-type PPM and wild-type PNP showed a 1420-fold bias for ribose 5-phosphate over the dideoxy substrate (). Incorporating the evolved PNP variant into the pathway provided a 29-fold increase in didanosine production and a small loss in inosine formation. The effect on inosine biosynthesis was further compounded after pairing the two optimized enzymes, showing a 342-fold total change in substrate selectivity to create a tandem evolved biosynthetic pathway with 32.5-fold improved didanosine production and only a 4-fold preference for the natural substrates.
the crystal structure of didanosine ..
Implementation of a third retro-extension required identification of a progenitor enzyme capable of phosphorylating dideoxyribose to synthesize the non-natural substrate dideoxyribose 5-phosphate for PPM. To this end, we measured didanosine production from dideoxyribose by the evolved PPM and PNP variants in tandem with five candidate kinase progenitor enzymes: E. coli RK, S. aureus RK, B. subtilis fructokinase, E. casseliflavus glycerol kinase or B. subtilis hydroxyethylthiazole kinase. Although didanosine formation was detected in all five systems via tandem enzyme HPLC/MS assay, production was highest in the pathways containing the RK homologs. The E. coli variant slightly outperformed the S. aureus enzyme and is more thoroughly characterized and was therefore selected for use in the full pathway ().
Didanosine CAS# 69655-05-6 API Supplier Distributor, …
Following the structure-based mutagenesis, additional mutagenesis was required to improve PPM turnover for the target substrate. For this, we performed random mutagenesis by epPCR. Since both the Ser154Gly and the Val158Leu variants had large substrate selectivity changes through distinct and potentially competitive mechanisms, these were separately used as templates for random mutagenesis (). In the bioretrosynthetic coupled assay, the top two PPM variants from first generation epPCR libraries for each template provided 150-250% higher dideoxyribose 5-phosphate turnover in cell lysate than the respective progenitor (). These variants were 12D2 (Thr81Ile and Ile238Ile silent mutation) and 500F7 (Phe101Leu) from the Ser154Gly template and 650G11 (Thr81Asn) and 500F6 (Met190Lys and Pro361Pro) from the Val158Leu template (). Kinetic characterization of the top performing clone in each initial library screen (i.e. 12D2 and 650G11) revealed increased turnover of both substrates, as measured through increased production of nucleosides, accompanied by improvements in selectivity reflected in KM values (). While the Ser154Gly 12D2 variant showed 8.4-fold preference for ribose 5-phosphate, selectivity of the Val158Leu 650G11 variant was entirely reversed, actually favoring dideoxyribose 5-phosphate at 1.4-fold over the natural substrate after >1400-fold change in selectivity ().
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