Screening of small molecule inhibitors targeting DNA polymerase <i>η</i>

doi:10.3785/j.issn.1008-9209.2023.02.221

Journal of Zhejiang University (Agriculture and Life Sciences)

2024, Vol. 50

Issue (1): 35-41 DOI: 10.3785/j.issn.1008-9209.2023.02.221

Biological Sciences & Biotechnologies

Screening of small molecule inhibitors targeting DNA polymerase η

Jiajia CAO(

),Shumai YE,Ye ZHAO(

)

College of Life Sciences, Zhejiang University, Hangzhou 310058, Zhejiang, China

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Abstract

The repair of DNA damage and maintenance of genomic stability are essential for the normal growth and adverse defense of plants and animals. In view of the genomic instability caused by the misincorporation of DNA polymerase, this study took DNA polymerase η as the research object and screened its possible small molecule inhibitors by computational simulated molecular docking and detected their enzyme kinetic parameters. The results showed that deoxyadenosine triphosphate (dATP) had an inhibitory effect on the activity of DNA polymerase η, resulting in a relative extension efficiency of 36% to 42%. Simulated molecular docking and in vitro experimental results showed that cyclic GMP-AMP (cGAMP) had a lower binding energy (with an affinity of －35.1 kJ/mol) than dATP (with an affinity of －26.7 kJ/mol) to DNA polymerase η. Enzyme kinetic experiments also showed that cGAMP had a stronger inhibitory ability than dATP and achieved the maximum effect at the concentration of 0.5 mmol/L (with a relative extension efficiency of 13%). Therefore, a potential small molecule inhibitor targeting DNA polymerase η was screened out in this study. At the same time, in view of the tolerance to antitumor drug (DNA damage agent) caused by high expression of this protein, these results provide a basis for the development of new drugs.

Key words： repair of DNA damage DNA polymerase enzyme kinetics computational biology cyclic GMP-AMP (cGAMP)

Received: 22 February 2023 Published: 01 March 2024

CLC:

Q513

Corresponding Authors: Ye ZHAO E-mail: 22016014@zju.edu.cn;yezhao@zju.edu.cn

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Cite this article:

Jiajia CAO,Shumai YE,Ye ZHAO. Screening of small molecule inhibitors targeting DNA polymerase η. Journal of Zhejiang University (Agriculture and Life Sciences), 2024, 50(1): 35-41.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2023.02.221 OR https://www.zjujournals.com/agr/Y2024/V50/I1/35

DNA聚合酶η小分子抑制剂筛选

DNA损伤修复以及基因组稳定性维持对于动植物正常生长和防御逆境至关重要。针对DNA聚合酶错误掺入导致的基因组不稳定性，本研究以DNA聚合酶η为研究对象，通过计算分子模拟对接的方式，对其可能的小分子抑制剂进行筛选，并对其酶动力学参数进行测定。结果显示：脱氧腺苷三磷酸（deoxyadenosine triphosphate, dATP）对DNA聚合酶η的活性具有抑制效果，使其延伸的相对效率为36%～42%。分子模拟对接和体外实验结果表明，相较于dATP（亲和力为－26.7 kJ/mol），环鸟苷酸-腺苷酸（cyclic GMP-AMP, cGAMP）与DNA聚合酶η具有更低的结合能（亲和力为－35.1 kJ/mol）。酶动力学参数测定结果也表明，相较于dATP，cGAMP具有更强的抑制能力且在浓度为0.5 mmol/L时达到最强（相对延伸效率为13%）。因此，本研究筛选获得了针对DNA聚合酶η的一种潜在的小分子抑制剂。同时，鉴于该蛋白质高表达导致细胞对抗肿瘤药物（DNA损伤剂）的耐受性，这为新型药物的开发提供了依据。

关键词： DNA损伤修复, DNA聚合酶, 酶动力学, 计算生物学, 环鸟苷酸-腺苷酸

Fig. 1 Simulated molecular docking results of dATP (A) and cGAMP (B) small molecules with DNA polymerase ηDNA polymerase η (blue), DNA molecule (pink), Trp297 (green), and dATP or cGAMP small molecules (yellow) are highlighted in different colors, respectively.

小分子 Small molecule	亲和力 Affinity/ (kJ/mol)	结构偏差 Structural deviation
小分子 Small molecule	亲和力 Affinity/ (kJ/mol)	RMSD l. b.	RMSD u. b.
dATP_1	－26.7	0	0
dATP_2	－26.3	1.509	2.414
dATP_3	－26.3	2.614	4.567
cGAMP_1	－35.1	0	0
cGAMP_2	－34.7	3.053	10.525
cGAMP_3	－34.3	4.185	5.330
2 $′$ -脱氧鸟苷 3 $′$ -（磷酸二氢酯） 2 $′$ -deoxyguanosine 3 $′$ -(dihydrogen phosphate)	－23.4	0	0
2 $′$ -脱氧胞苷 2 $′$ -deoxycytidine	－22.1	0	0

Table 1 Simulated docking results of dATP and cGAMP small molecules

Fig. 2 Molecular structure of cGAMP

Fig. 3 Separation and purification of DNA polymerase ηA. Diagram of protein purification by Ni column affinity chromatography; B. Diagram of protein purification by desalt column; C. Diagram of protein purification by heparin column; D. Protein purity checked by SDS-PAGE (M: Protein marker). The black arrow indicates the target protein of DNA polymerase η.

Table 2 Kinetic parameters of DNA polymerase η (G-dCTP pairing)


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