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  波谱学杂志   2019, Vol. 36 Issue (2): 148-154.  DOI: 10.11938/cjmr20182696
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引用本文 [复制中英文]

TANG Heng, Gilbert NSHOGOZA, LIU Ming-qing, et al. Identification of Novel Hits of the NSD1 SET Domain by NMR Fragment-Based Screening[J]. Chinese Journal of Magnetic Resonance, 2019, 36(2): 148-154. DOI: 10.11938/cjmr20182696.
[复制英文]
汤衡, Gilbert NSHOGOZA, 刘明清, 等. 基于片段的核磁共振筛选方法识别NSD1 SET结构域的全新苗头化合物[J]. 波谱学杂志, 2019, 36(2): 148-154. DOI: 10.11938/cjmr20182696.
[复制中文]

Foundation item

National Key R & D Program of China (2016YFA0500700); the National Natural Science Foundation of China (21874123, 21703254, U1632153, 21807095)

Corresponding author

GAO Jia, Tel:18256925385, E-mail:jiagao@ustc.edu.cn

Article History

Received date: 2018-11-30
Available online: 2018-12-14
Identification of Novel Hits of the NSD1 SET Domain by NMR Fragment-Based Screening
TANG Heng 2, Gilbert NSHOGOZA 1, LIU Ming-qing 1, LIU Ya-qian 1, RUAN Ke 1, MA Rong-sheng 1, GAO Jia 1     
1. Hefei National Laboratory for Physical Sciences at the Microscale, School of Life Sciences, University of Science and Technology of China, Hefei 230027, China;
2. General Hospital of Wanbei Coal-electric Group, Suzhou 234011, China
Abstract: Nuclear receptor binding SET domain protein 1 (NSD1), which is a family member of histone methyltransferases, functions to methylate histone H3 on lysine 36 (H3K36). NSD1-related abnormalities are the major cause of Sotos syndrome, and also known to be associated with other human diseases. Inhibitors targeting histone methyltransferases DOT1L and EZH2 have been reported recently. However, no chemical probes targeting NSD1 have been found so far. Here, we identified three hits targeting the NSD1 SET domain using ligand-observed nuclear magnetic resonance (NMR) fragment-based screening. The binding affinities of the hit compounds to the NSD1 SET domain were determined by dose-dependent chemical shift perturbation analysis. Furthermore, the potential binding modes of the hit compounds to NSD1 were obtained by molecular docking. The hit compound 1 was found to bind to the binding pocket of S-adenosylmethionine (SAM), an endogenous ligand of the protein, in the NSD1 SET domain. The study provided valuable information for further structure-guided hit-to-lead evolution towards the potent and specific inhibitors of the NSD1 SET domain.
Key words: NSD1 SET domain    NMR fragment-based screening    chemical shift perturbation    molecular docking    
基于片段的核磁共振筛选方法识别NSD1 SET结构域的全新苗头化合物
汤衡 2, Gilbert NSHOGOZA 1, 刘明清 1, 刘亚茜 1, 阮科 1, 马荣声 1, 高佳 1     
1. 合肥微尺度物质科学国家实验室, 中国科学技术大学 生命科学学院, 安徽 合肥 230027;
2. 皖北煤电集团总医院, 安徽 宿州 234011
摘要: 包含SET结构域的核受体结合蛋白1(NSD1)是一种组蛋白甲基转移酶,它能够特异性的甲基化组蛋白H3赖氨酸第36位(H3K36).异常表达的NSD1主要发现于Sotos综合症患者体内,但它同样也能导致其他多种人类疾病的发生.目前已有靶向组蛋白甲基转移酶DOT1L和EZH2的小分子抑制剂报道,然而,靶向NSD1的化学探针分子尚未被发现.本文使用基于片段的核磁共振(NMR)筛选方法寻找到3个以NSD1蛋白作为靶点的苗头化合物,利用化学位移扰动分析技术测定了这些化合物与NSD1的结合亲和力.另外,利用分子对接方法选择获得苗头化合物与NSD1蛋白的最可能的结合模型.结果显示苗头化合物1结合于NSD1天然底物S-腺苷酸甲硫氨酸(SAM)的结合口袋中.我们的研究成果为进一步以结构为指导的从苗头化合物到先导化合物的衍化奠定了基础.
关键词: NSD1 SET结构域    基于片段的核磁共振筛选    化学位移扰动    分子对接    
Introduction

In eukaryotes, the post-translational modifications (PTMs) of histones play an essential role in the regulation of chromatin structural dynamics and gene expression. Histone methylation, one of the key epigenetic modifications, usually occurs on the lysine (K) or arginine (R) residues at the N-terminal flexible tails of histones, and regulates transcriptional activation or inactivation of specific genes[1, 2]. The nuclear receptor binding SET domain (NSD) family has three SET domain-containing methyltransferases, i.e., NSD1, NSD2, NSD3[3]. NSD proteins specifically methylate K36 of histone H3 on lysine 36 (H3K36). Importantly, such PTM is associated with multiple human diseases including cancers[4-7]. Each NSD protein contains a SET domain, a PWWP domain and PHD fingers[8]. The catalytic SET domain is an evolutionarily conserved motif with 130~150 amino acids, while the PWWP and PHD domains are readers of other proteins or nucleic acids.

NSD1 mutations are closely relative to multiple human diseases. For instance, NSD1 mutations cause Sotos syndrome, which has the symptom of childhood overgrowth, learning disability, and mental retardation[4-6, 9, 10]. NSD1-NUP98 fusion protein, resulting from chromosomal translocations, is associated with childhood acute myeloid leukemia[7, 11]. The inactivating mutation of NSD1 is linked to multiple cancers as well[9, 12]. Therefore, it is of significance to discover small molecule inhibitors to probe the biologic functions of NSD1 and to exploit the potential therapeutic treatment. The SET domains are drug gable targets as have been validated by the inhibitors of DOT1L/KMT4, EZH2/KMT6, and G9a[13-15]. However, to our best knowledge, no small molecule inhibitors of the NSD1 has yet been identified.

Fragment-based drug discovery (FBDD) has been proven effective and fruitful for the past 20 years, even for challenging protein-protein interaction (PPI) targets with shallow pockets[16]. FBDD starts from low-molecular-weight compounds (fragments), and usually directly detects the weak interactions between targets and fragments by using biophysical methods, e.g., surface plasmon resonance (SPR), fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray crystallography[17, 18]. The combination of protein-observed and ligand-observed NMR spectroscopy enabled robust detection of ambiguous weak interactions, due to NMR nature of multi-valued structural output[19, 20].

Here, we applied an automated NMR fragment-based screening (FBS) and identified three weak binders of the NSD1 SET domain. To enhance the screening throughput, we first identified feasible hits from the fragment cocktails by using ligand-observed spectra, e.g., saturation transfer difference (STD) and WaterLOGSY. These experiments were then reproduced for each individual hit. The three specific binders were further validated by the dose-dependent chemical shift perturbations (CSPs) of 15N-labeled NSD1 SET domain. We finally analyzed the binding mode of the hit using molecular docking.

1 Materials and methods 1.1 Cloning, protein expression and purification

DNA encoding NSD1 SET domain (residues 1 852~2 082) was cloned into the pGEX4T-1 vector (GE Healthcare, Shanghai, China), which was modified with tobacco etch virus (TEV) cleavage sites. The construct was then transformed into Escherichia coli BL21(DE3)-RIL. Cells were grown in minimal medium supplemented with 15NH4Cl for NMR samples. The protein expression was generally induced at OD600 of 0.8~1.2 using 0.5 mmol/L isopropyl β-D-thiogalactos (IPTG) at 16 ℃ for 24 h. The glutathione S-transferase (GST)-tagged proteins were purified from pretreated bacterial lysates using GSTrap FF (GE Healthcare, Shanghai, China), then treated with TEV to cleave the N-terminal GST tag. Protein was purified further by size exclusion chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare, Shanghai, China). The purified protein was stored in Tris buffer [20 mmol/L Tris, 200 mmol/L NaCl, 2 mmol/L tris(2-carboxyethyl)phosphine (TCEP) at pH 6.8].

1.2 NMR fragment-based screening

The whole process of NMR fragment-based screening was performed at 25 ℃ on a 700 MHz spectrometer (Agilent Technologies Santa Clara, CA, USA) equipped with a 96-well autosampler and a cryoprobe[19]. In brief, the screening samples were prepared containing 0.4 mmol/L compounds in DMSO-d6 solution (cocktails for primary screening), 50% D2O and 10 μmol/L unlabeled protein. The binding hits were identified from the primary screening in cocktail and secondary screening for each individual compound based on the signals of STD and WaterLOGSY spectra.

The STD experiment was acquired using the acquisition time of 1 s, 32 dummy scans, and relaxation delay of 0.1 s, followed by a 2 s Gauss pulse train with the irradiation frequency at 20.7 ppm or 250 ppm alternatively. The total acquisition time was 15 min with 256 scans. WaterLOGSY was acquired for 15.1 min with 1 s acquisition time, 1 s relaxation and 1.3 s NOE mixing time and 256 scans.

1.3 NMR CSP calculation

The 15N-labeled NSD1 proteins were concentrated to 0.1 mmol/L in Tris buffer. The compounds were then titrated into 15N-labeled NSD1. The HSQC spectra were collected for each titration points on Agilent 700 MHz spectrometer at the ligand/protein molar ratios of 0, 0.25, 0.5, 1, 2, 5, respectively. The binding constant was best-fitted using the following equation assuming a 1:1 binding mode[24]

$ \begin{array}{l} \mathit{\Delta }\delta = \mathit{\Delta }{\delta _{\max }}\\ \left\{ {{K_D} + {{[P]}_t} + {{[L]}_t} - {{\left[ {{{\left( {{K_D} + {{[P]}_t} + {{[L]}_t}} \right)}^2} - \left( {4*{{[P]}_t}{{[L]}_t}} \right)} \right]}^{1/2}}} \right\}/\left( {2{{[P]}_t}} \right) \end{array} $ (1)

where [P]t and [L]t are total concentrations of ligand and protein, respectively, Δδ is the chemical shift change with respect to the free state; maximum chemical shift change (Δδmax) and binding affinity (KD) were best-fits from the dose-dependent chemical shift changes.

2 Results and discussion

NSD1 is a 2 969 amino-acid protein containing four PHD fingers, two PWWP domains, and one catalytic SET domain [Fig. 1(a)]. Although the crystal structure of NSD1 containing AWS, SET and post-SET sequence motifs (amino acids 1 852~2 082) has been resolved (PDB code: 3OOI), little is known about its solution structure and dynamics. The 1H-15N heteronuclear single quantum coherence (HSQC) spectrum is powerful to assess the protein folding and aggregation status. We hence expressed and purified the 15N-labeled protein fused to a GST tag with TEV protease cleavage site [Fig. 1(b) and 1(c)]. The transverse relaxation optimized HSQC (TROSY-HSQC) spectrum suggests that the NSD1 SET domain is mono-dispersed [Fig. 1(d)], suitable for following FBS.

Fig. 1 (a) The domain structure of NSD1 containing 2 969 amino acids. The black box represents each domain with the domain name annotated; (b) SDS-PAGE of the NSD1 SET domain. Lanes 1~6 represent marker, elution of NSD1, washing, flow through, supernatant, and precipitation, respectively; (c) The UV spectrum at wave length of 280 nm of NSD1 SET (domain 1 852~2 082 amino acid) purified by 16/60 Superdex 200 size exclusion columns; (d) The 1H-15N TROSY-HSQC spectrum of NSD1 SET domain

Our automated NMR FBS[19] was utilized to uncover hits of the NSD1 SET domain. NMR FBS integrates the ligand-observed and protein-observed experiments[17]. The protein-observed NMR FBS method provides valuable information of binding sites and affinity. However, protein-observed NMR FBS requires a significant amount of 15N-labeled proteins and a high quality of the 2D 1H-15N HSQC spectrum. Conversely, the ligand-observed NMR FBS experiments record the changes of ligand signals in the presence of a small portion of the unlabeled protein. STD and WaterLOGSY are the most widely used spectra in NMR FBS. To enhance the throughput of primary screening, we pooled the fragment library of 890 compounds into 89 cocktails with 10 compounds each. The compound and protein concentrations were set to 0.4 and 0.01 mmol/L, respectively. A total of 16 hits were identified as they presented signals in STD and inverted sign of signals in WaterLOGSY [Fig. 2(a)]. The 16 hits were further filtered by a secondary screening using the same set of aforementioned experiments for each individual compound [Fig. 2(b)]. Such a cross validation procedure produced 7 hits.

Fig. 2 Ligand-observed NMR fragment-based screening against the NSD1 SET domain. (a) The primary screening over a typical cocktail. The top 10 spectra are the reference 1H NMR spectra of each individual component of the cocktail, and the bottom 3 are WaterLOGSY, STD and the 1H NMR spectra of the cocktail; (b) The secondary screening for hit 1 alone, using the same set of screening spectra

The hits identified from the above ligand-observed FBS were then validated by CSPs of the 15N labeled NSD1 SET domain. The hits were titrated to 0.12 mmol/L 15N-labeled NSD1 SET domain at a final ligand/protein molar ratio varying from 0 to 5 [Fig. 3(a)]. Three hits perturbed a common set of residues, indicating that these hits bind specifically to the NSD1 SET domain. The binding affinities determined from the dose-dependent CSPs [Fig. 3(b)] were 0.12, 0.54 and 1.22 mmol/L for the three hits, respectively (Table 1). The hit rate and the reasonable ligand efficiencies (binding energy per non-proton atom) indicate the NSD1 SET domain is a drug gable target.

Fig. 3 Chemical shift perturbations induced by the fragment screening hit 1. (a) Chemical shift perturbations of 15N-labeled NSD1 SET domain upon titration of hit 1 at the indicated ligand/protein molar ratios; (b) The affinity of hit 1 to NSD1 SET domain is best-fitted from the dose-dependent chemical shift changes (Δδ). Δδ was calculated as [(ΔδH)2+(ΔδN/5)2]1/2, where ΔδH and ΔδN denote the chemical shift changes of 1H and 15N, respectively
Table 1 Structure and affinities of fragment screening hits of NSD1 SET domain

The target-ligand interaction modes are essential for rational drug discovery. However, we failed to crystallize the NSD1 SET domain complexes, probably due to the low binding affinities of the hits. We hence simulated the complex structural models using the molecular docking software AutoDock, in a way as has been published previously[21-23]. Notably, the lowest-energy docking pose demonstrated compound 1 bound to the AdoMet binding pocket [Fig. 4(a) and 4(b)] and formed hydrogen bonds with residues H2021 and K2071, respectively [Fig. 4(c)]. This interaction pattern is consistent with that of AdoMet as revealed in the complex crystal structure[8] [Fig. 4(d)]. We recently demonstrated that paramagnetic NMR spectroscopy facilitated the filtration of the best-fit docking pose[21-23]. We are now working on the backbone chemical shift assignment of NSD1 SET domain and subsequent acquisition of NMR structural restraints.

Fig. 4 Docking modes of hit 1 in comparison with the natural substrate AdoMet of the NSD1 SET domain. (a) Electrostatic surface representation of NSD1 SET domain (PDB code: 3OOI) with hit 1 (carbon atoms in yellow sticks), superimposed with the crystal structure in complex with AdoMet (carbon atoms in green sticks); (b) Binding site topology of the NSD1 SET domain in complex with AdoMet (green) and hit 1 (yellow), respectively; (c) The lowest-energy docking pose suggests the interaction details of hit 1, which may form hydrogen bonds (red dotted lines) with H2021 and K2071, respectively; (d) The binding pattern revealed from the crystal structure of NSD1 in complex with AdoMet
3 Conclusion

NSD1 is a potential therapeutic target for multiple human cancers and Sotos syndrome. We discovered new hits by using NMR FBS against the NSD1 SET domain. Three specific binders were confirmed by NMR CSPs. The molecular docking poses exhibited a similar pattern to the natural substrate AdoMet. Our study shall enable the following structure-guided hit-to-lead evolution targeting the NSD1 SET domain.


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