SAE1与SAE2蛋白相互作用肽抑制剂的多种体外筛选体系的构建与评价

doi:10.3969/j.issn.1674-8115.2024.05.004

摘要/Abstract

摘要：

目的·构建用于发现小泛素样修饰蛋白（small ubiquitin-like modifier，SUMO）的活化酶亚基1（SUMO-activating enzyme subunit 1，SAE1）与亚基2（SUMO-activating enzyme subunit 2，SAE2）相互作用的肽抑制剂的多种体外筛选体系，并对不同筛选体系的优势与不足进行评价。方法·将编码SAE1和SAE2的目的基因分别插入pET-28a载体以构造原核蛋白表达质粒，在大肠埃希菌中表达并纯化人源SAE1和SAE2蛋白；利用纯化的蛋白先后构建等温滴定量热检测（isothermal titration calorimetry，ITC）实验、荧光偏振（fluorescence polarization，FP）实验、表面等离子共振（surface plasmon resonance，SPR）实验和基于SAE酶活的荧光实验等多种筛选体系。尝试利用不同的筛选体系检测候选多肽的抑制活性，基于检测结果，从灵敏度、稳定性、检测通量和检测成本等维度评价各筛选体系的优缺点与适用性。结果·经ITC测得SAE1和SAE2蛋白在体外相互作用的解离常数（K_d）为0.96 μmol/L，并将活性最好的多肽PEPT7改造为FP实验的示踪剂（tracer），但同SAE2的亲和力无法满足FP的要求；SPR测得SAE1和SAE2相互作用的K_d值为1.13 μmol/L，与ITC数据接近；基于SAE酶活的荧光实验筛选得到抑制活性最强的多肽HP1B［半数抑制浓度（half-maximal inhibitory concentration，IC₅₀）达15.72 μmol/L］，SPR进一步确定其同SAE1的亲和力为34.4 μmol/L。结论·尝试构建并比较了多种常见的蛋白-蛋白相互作用（protein-protein interaction，PPI）抑制剂的筛选体系。其中，ITC的检测通量低，且难以准确评估结合热不显著的低亲和力多肽；FP体系的可行性高度依赖于示踪剂同靶点蛋白之间的强亲和力，同样无法用于低亲和力多肽的筛选与优化；SPR检测的灵敏度高，但检测成本较高；酶活实验兼具高灵敏度、稳健性、高通量和可接受的检测成本，是最适宜的筛选方法。

关键词: SUMO活化酶亚基1, SUMO活化酶亚基2, 多肽抑制剂, 药物筛选, 等温滴定量热检测实验, 荧光偏振实验, 表面等离子共振实验, 基于酶活的荧光实验

Abstract:

Objective ·To establish various in vitro screening systems for the discovery of peptide inhibitors targeting the interaction between small ubiquitin-like modifier (SUMO)-activating enzyme subunit 1 (SAE1) and subunit 2 (SAE2), as well as to evaluate their advantages, disadvantages, and applicability to this research. Methods ·The DNA fragments encoding human SAE1 and SAE2 were cloned into vector pET-28a, respectively, to generate protein SAE1 and SAE2. Purified proteins were used to establish screening assays, including isothermal calorimetry (ITC), fluorescence polarization (FP), surface plasmon resonance (SPR) and a fluorescence assay based on the SAE enzyme activity. The inhibitory activity of peptide candidates in different screening systems was examined, and their performance in terms of sensitivity, robustness, throughput and cost was evaluated. Results ·The dissociation constant (K_d) of in vitro SAE1 and SAE2 interaction was determined to be 0.96 μmol/L by ITC, and PEPT7 was identified as the most potent peptide. However, the tracer of FP, which was derived from PEPT7, was not up to snuff due to its low affinity with SAE2. In the SPR assay, the K_d value (=1.13 μmol/L) of SAE1 and SAE2 interaction was in line with the results from ITC. The SAE enzyme activity-based screening assay revealed that HP1B, the most effective peptide, inhibited SAE with an half-maximal inhibitory concentration (IC₅₀) of 15.72 μmol/L. The affinity of HP1B for SAE1 was determined to be 34.4 μmol/L by SPR. Conclusion ·Several common screening systems for protein-protein interation (PPI) inhibitors are established and compared. Among them, ITC does not allow for high-throughput screening and it is difficult to accurately evaluate the low-affinity polypeptides with insignificant binding heat. The feasibility of FP relies heavily on the strong affinity between a tracer peptide and the protein target, making it unsuitable for the screening and optimization of low-affinity peptides. SPR is highly sensitive but the cost is high. The SAE enzyme activity-based assay stands out because it is a combination of high sensitivity, robustness, throughput and acceptable cost.

Key words: SUMO-activating enzyme subunit 1 (SAE1), SUMO-activating enzyme subunit 2 (SAE2), peptide inhibitor, drug screening, isothermal calorimetry (ITC), fluorescence polarization (FP), surface plasmon resonance (SPR), enzyme activity-based fluorescence assay

中图分类号:

R363.1

胡晨阳, 陆绍永, 杨秀岩. SAE1与SAE2蛋白相互作用肽抑制剂的多种体外筛选体系的构建与评价[J]. 上海交通大学学报（医学版）, 2024, 44(5): 567-575.

HU Chenyang, LU Shaoyong, YANG Xiuyan. Establishment and evaluation of various in vitro screening systems for peptide inhibitors targeting SAE1 and SAE2 interaction[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(5): 567-575.

图/表 7

表1 PCR引物序列

Tab 1 Primer sequences for PCR

Primer	Forward sequence (5′→3′)	Reverse sequence (5′→3′)
SAE1	GCAAATGGGTCGCGGATCCGAATTCATGGTGGAGAAGGAGGAG	GTGGTGCTCGAGTGCGGCCGCATCACTTGGGGCCAAGG
SAE2	GCAAATGGGTCGCGGATCCGAATTCATGGCACTGTCGCG	GTGGTGCTCGAGTGCGGCCGCATCAATCTAATGCTATGAC
SUMO1	GCGCGGCAGCCATATGGCTAGCATGTCTGACCAGGAGGC	GGTGGTGGTGGTGGTGCTCGAGTTAACCCCCCGTTTGTTCC

表2 不同蛋白的诱导表达条件

Tab 2 Conditions for protein expression

Protein	IPTG concentration/(mmol·L^-1)	Temperature/℃	Time/h
SAE1	0.3	10	36
SAE2	0.3	10	36
SUMO1	1.0	16	24

表3 不同蛋白使用的缓冲液

Tab 3 Buffers for different proteins

Buffer	Component
Buffer	Tris-HCl (pH 8.0)/(mmol·L^-1)	NaCl/(mmol·L^-1)	Imidazole/(mmol·L^-1)	DTT/(mmol·L^-1)
SAE1 lysis buffer	50	300	10	1
SAE1 Ni-NTA equilibration/wash buffer	50	50	20	1
SAE1 Ni-NTA elution buffer	50	50	40	1
SAE2 lysis buffer	50	300	/	1
SAE2 Ni-NTA equilibration/wash buffer	50	50	/	1
SAE2 Ni-NTA elution buffer	50	50	25	1
SAE2 anion exchange buffer A	50	50	/	1
SAE2 anion exchange buffer B	50	1	/	1
SUMO1 lysis buffer	50	500	20	/
SUMO1 Ni-NTA equilibration/wash buffer	50	500	20	/
SUMO1 Ni-NTA elution buffer	50	50	400	/

图1 ITC筛选体系的建立Note： A. ITC experiments for the binding of SAE1 to SAE2. B. ITC experiments for the binding of PEPT7 to SAE2. C. ITC experiments for the binding of PEPT2 to SAE2. 1 cal=4.184 J. N—the number of sites per SAE2; K—the binding constant; ΔH—heat change; ΔS—entropy change.

Fig 1 Establishment of ITC screening system

图2 FP筛选体系的建立Note： A. FP curve of tracer T1 and T2 at different concentrations. B. FP curve of tracer T1 binding with SAE2 at different concentrations. lg [Tracer (nmol·L-1)] means the logarithm of the concentration of Tracer, where the unit of concentration of Tracer is nmol·L-1. The concentration of T1 is 50 nmol/L.

Fig 2 Establishment of FP screening system

图3 SPR筛选体系的建立Note： A. SPR pH scouting results for SAE2 immobilization. B. SPR sensorgrams of SAE1 binding to immobilized SAE2. C. Affinities of YP3 peptides for SAE2 determined by SPR. D. Affinity of YP3D for SAE2 determined by SPR equilibrium analysis.

Fig 3 Establishment of SPR screening system

图4 基于SAE酶活的筛选体系的建立Note： A. Effect of the absence of SAE1 or SAE2 on the adenylation efficiency of SAE. B. The adenylation efficiencies of SAE under different concentrations of SUMO1. C. Optimal concentration of SAE; ΔRLU=RLUcontrol－RLUsample. D. Optimal concentration of SUMO1. E. Inhibition curve of COH000. F. Inhibition rates of peptides at 125 μmol·L-1. G. Inhibition curve of HP1B. H. Binding interaction between HP1B (blue) and SAE1 (gray). Key interacting residues of interaction are shown as sticks and labeled. Yellow dashed lines represent hydrogen bonds.

Fig 4 Establishment of a SAE enzyme activity-based screening system

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