Multienzyme cascades provide an efficient biocatalytic strategy for synthesizing nucleoside analogs from simple sugar donors and nucleobases. In pentose‐to‐nucleoside cascades, purine nucleoside phosphorylase (PNP) catalyzes the terminal N‐glycosylation step and strongly influences product formation and substrate scope. Here, seven putative PNP homologs from microorganisms isolated from low‐temperature environments were evaluated under Ec RK‐ Ec PPM‐PNP cascade conditions. A PNP from Colwellia sp. WH041, designated Col PNP, showed the highest arabinofuranosylation activity and was selected for further characterization. Col PNP displayed optimal activity at 45 °C and exhibited a 12.7‐fold higher catalytic efficiency than Escherichia coli PNP at the same temperature. Molecular dynamics simulations supported the favorable catalytic performance of Col PNP under moderate‐temperature conditions. After optimization of the Ec RK‐ Ec PPM‐ Col PNP cascade, vidarabine conversion reached 58.2% within 12 h. The optimized system was further applied to synthesize diverse nucleoside analogs with conversions of 45.8%–89.8%, and guanosine analogs were obtained through sequential Ec ADA‐mediated deamination. This study expands the PNP toolbox and demonstrates Col PNP as an efficient module for enzymatic nucleoside analog synthesis. A purine nucleoside phosphorylase from Colwellia sp. WH041, Col PNP, is identified as an efficient module for pentose‐to‐purine nucleoside synthesis. Integrated with Ec RK and Ec PPM, Col PNP enables direct conversion of unprotected D‐pentoses and purine bases into diverse nucleoside analogs with up to 89.9% conversion.
Xiaoxia Qiu,Yiyou Huang,Deyan Wu,Fei Mao,Jin Zhu,Wenzhong Yan,Hai-Bin Luo,Jian Li
Phosphodiesterase 2 (PDE2) has received much attention for the potential treatment of the central nervous system (CNS) disorders and pulmonary hypertension . Herein, we identified that clofarabine ( 4 ), an FDA-approved drug , displayed potential PDE2 inhibitory activity (IC 50 = 3.12 ± 0.67 μM) by structure-based virtual screening and bioassay. Considering the potential therapeutic benefit of PDE2, a series of purine nucleoside derivatives based on the structure and binding mode of 4 were designed, synthesized and evaluated, which led to the discovery of the best compound 14e with a significant improvement of inhibitory potency (IC 50 = 0.32 ± 0.04 μM). Further molecular docking and molecular dynamic (MD) simulations studies revealed that 5′-benzyl group of 14e could interact with the unique hydrophobic pocket of PDE2 by forming extra van der Waals interactions with hydrophobic residues such as Leu770, Thr768, Thr805 and Leu809, which might contribute to its enhancement of PDE2 inhibition. These potential compounds reported in this article and the valuable structure-activity relationships (SARs) might bring significant instruction for further development of potent PDE2 inhibitors.
Background and Purpose The concentrative nucleoside transporter 2 (CNT2) mediates the uptake of both natural nucleosides and nucleoside-derived drugs. Therefore, it is important both physiologically and pharmacologically. However, CNT2 expression is significantly repressed in colorectal cancer (CRC). Here, we have elucidated the mechanism(s) underlying CNT2 repression in CRC. Experimental Approach Repression of CNT2 in tumour samples from patients with CRC was identified using Western blot and RT-qPCR. The histone acetylation state at the CNT2 promoter region was then evaluated with chromatin immunoprecipitation and trichostatin A (TSA) treatment. To find the key enzyme responsible for hypoacetylation at the CNT2 promoter region, siRNA knockdown and RT-qPCR were used. Effects of combining HDAC inhibitors and cladribine were studied in HCT15 and HT29 cells. Key Results Histone deacetylase 7 was significantly up-regulated in CRC, leading to histone hypoacetylation at the CNT2 promoter region, especially at sites H3K9Ac, H3K18Ac and H4Ac. This hypoacetylation condensed the chromatin structure and reduced CNT2 expression. All these effects were reversed by treatment with TSA, a histone deacetylase inhibitor. In HCT15 and HT29 cells, inhibition of histone deacetylase increased cell uptake and decreased IC 50 for cladribine. Conclusions and Implications Histone hypoacetylation due to increased levels of histone deacetylase 7 results in CNT2 repression in CRC tumour tissue and could lead to decreased uptake of and consequent resistance to nucleoside anti-cancer agents. Such resistance could be overcome by combining inhibitors of histone deacetylase with the nucleoside anti-cancer agent.
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