荧光偏振法筛选组蛋白去甲基化酶抑制剂及HPS1缺失对眼黑色素体影响
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摘要
研究背景及目的
组蛋白甲基化是组蛋白共价修饰中的一种重要形式。一直以来被认为是一个稳定且不可逆的过程,直到2004年发现第一个组蛋白去甲基化酶LSD1。经过近年的研究,已发现了超过20种的组蛋白去甲基化酶(histone demethylases,HDMs)。根据去甲基化酶的结构和不同的催化机理,可以将去甲基化酶分为两类：一类是黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)依赖性去甲基化酶,LSD1和LSD2；另一类是依赖于Fe2+和共同作用因子α-酮戊二酸(αKG),并含有Jumonji结构域的去甲基化酶(JmjC domain-containing histone demethylases,JHDMs).它们通过调控与特定基因的转录激活或抑制相关。大量的研究发现它们参与包括癌症在内的多种疾病的发生和进行发展,如前列腺癌、乳腺癌、膀胱癌、食管鳞状细胞癌以及原发性纵隔B细胞淋巴瘤等。
组蛋白去甲基化酶在生理和病理中的重要作用使其成为新药开发的靶点,对其抑制剂的研发和筛选也已成为了现阶段组蛋白去甲基化酶研究的热点。基于去甲基化酶作用机制以及蛋白质晶体结构的特点,已经有大量的小分子抑制剂被设计并合成出来。因此,迫切需要一个方便、快速、高效、低廉以及高通量的方法对其进行大规模筛选,发现有效的小分子去甲基化酶抑制剂。
研究方法
我们通过建立荧光偏振方法应用于组蛋白去甲基化酶小分子抑制剂的筛选。基于实验室已发现的JHDMs小分子抑制剂methyl stat的结构,我们设计合成了荧光探针SY-Ⅱ-082、WQX-Ⅶ-291和WQX-Ⅶ-417等,并在荧光偏振实验过程中进行结构优化。在建立荧光偏振结合体系时,我们首先优化了缓冲液条件和荧光探针浓度。由于探针本身含有αKG结构,我们又对金属离子Fe(?)和外层电子排布与Fe(?)相似的Ni(?)对结合体系的影响进行了检测,并进一步优化了Ni'浓度；还对信号的稳定性进行检测。
通过优化的结合体系,我们对荧光探针分子与去甲基化酶的亲和能力进行测定,并利用改良的解离常数计算公式在KaleidaGraph中拟合曲线得到两者的Kd值。接下来,通过对所需蛋白浓度的优化以及小分子抑制剂与荧光探针分子背景结合信号的检测,我们优化了荧光偏振竞争体系。我们通过对JHDMs常用抑制剂的滴定(IC50)来确定荧光偏振竞争实验的可行性后,还检测了Z’因子(0 研究结论
利用对实验体系的一系列优化,我们最终建立了一个稳定的可用于去甲基化酶抑制剂筛选的荧光偏振方法,可在同一背景下检测所有小分子抑制剂与去甲基化酶的结合能力Ki,避免了不同方法检测到的半数抑制浓度IC50值无可比性的缺点；同时筛选出一个特异性结合JHDM1A的荧光探针WQX-Ⅶ-417,可用于针对JHDM1A的小分子抑制剂的筛选。
荧光偏振结合实验的优化结果显示,1nM的荧光探针WQX-VII-417本身具有较强的稳定的荧光信号；Tris缓冲液较HEPES缓冲液能拟合出更优曲线；Fe(?)无论在还原剂是否存在的条件下,都会干扰结合信号,不能产生正常的结合曲线；Ni(?)能够在体外反应中保持结合信号的稳定,替代体内反应中所需的Fe(?)。相比JMJD2A,探针WQX-Ⅶ-417能够特异性地较强地结合JHDM1A (ΔmP约为280,Kd值仅为10.8nM)。这一结果为小分子抑制剂的筛选提供了较大筛选窗,利用此探针可以筛选出K大于10nM的JHDMs小分子抑制剂。
荧光偏振竞争实验的优化结果显示,60nM的JHDM1A即可用于竞争结合实验。常用小分子抑制剂滴定结果显示methylstat能够将相似结构的探针分子WQX-Ⅶ-417竞争下来,亲和能力较高(Ki约为12.8nM),证明了其作用机制及设计理念的合理性,并第一次测出了共同作用因子aKG与去甲基化酶的亲和能力；滴定结果还说明此方法可用于抑制剂的筛选。此外,测定此实验方法的Z’因子为0.75显示其较适用于高通量筛选(>0.7)。
研究意义
荧光偏振方法通过检测小分子抑制剂与不同去甲基化酶的亲和能力,实现对小分子抑制剂文库的初步筛选,得到具有较高亲和力的抑制剂,为体内实验提供候选抑制剂小分子,并为优化设计新的小分子抑制剂以及研究小分子抑制剂的结构活性关系(Structure activity relationship, SAR)提供重要的线索。此外,此方法还可用于筛选合成修饰的肽链库,通过体外检测不同结构修饰的肽链与组蛋白去甲基化酶结合的特异性,为体内发现新的去甲基化酶提供线索。
综上所述,此种荧光偏振方法不仅为去甲基化酶小分子抑制剂筛选提供高效便利的方法,也为生物过程的机理研究提供重要的工具和线索,对建立成熟高效的去甲基化酶小分子抑制剂筛选平台以及研究相关疾病的机理具有重要的应用价值,应用前景较好。
研究的背景及目的
HPS综合征是一种常染色体隐性遗传病,是一种特殊的白化病,可由多个基因突变引起。目前已发现并克隆了导致小鼠HPS的15个基因以及所对应人的8个基因。临床上,HPS综合征可导致白化、出血倾向、肺部纤维化引起的呼吸衰竭、肠道感染和视力降低。
目前,对HPS1突变小鼠模型pale ear (ep)背部皮肤的色素沉着已有深入研究,发现HPS1突变并不影响毛囊中的黑色素体,但是会降低表皮和真皮毛囊间黑色素体的数目,引起尾部黑色素体的不成熟,从而形成了ep小鼠的特殊毛色模式—背部色素沉着正常呈黑色,而耳朵、尾巴和爪子着色较浅呈近白色。对于同样含有黑色素体的眼则未见系统的报道。在人类中已发现HPS1患者视力下降并伴随着眼球水平摆动震颤。为对HPS1突变所导致视力下降的原因提供研究线索,我们利用HPS1突变小鼠ep为模型,对ep小鼠眼黑色素的起源、分布以及形态进行系统研究。
研究方法及结果
采集不同发育阶段ep小鼠的眼球,通过黑色素含量测定、冷冻切片、HE染色、免疫荧光和电镜等方法,我们对眼不同发育阶段含有黑色素细胞的组织进行了生化、光镜和电镜的检测。结果发现,ep小鼠在出生时整体黑色素含量就低于正常小鼠,这种差异随着发育逐步增大；通过进一步的光学显微镜检测发现,虹膜、睫状体以及脉络膜/视网膜色素上皮的色素沉着都低于同时期的正常小鼠；电镜结果显示虹膜内起源于神经脊的虹膜基质中黑色素体异常,表现为巨大黑色素体,而在来源于视杯上皮的色素细胞,前虹膜色素上皮和后虹膜上皮中黑色素体数目大量减少,并没有产生巨大黑色素体。
结论
HPS1的缺失可导致起源于神经脊的黑色素细胞形成巨大黑色素体,而使来源于视杯色素上皮的黑色素细胞中黑色素体数目降低但并不产生巨大黑色素体。这个结果与之前对ep小鼠中起源于神经脊的脉络膜与起源于视杯的视网膜色素上皮中黑色素体的形态研究一致,证明了所得结论的正确性。
研究意义
此研究结果详细的阐述和证明了HPS1基因缺失对眼黑色素体的影响,为HPS1患者眼内黑色素体的分布及异常提供了详细的形态学资料及发育起源证据；但仍需进一步研究HPS1对不同发育来源的黑色素体形态造成影响的机制,探寻HPS1在眼黑色素体发生、成熟和运输过程中的作用,从而为研究HPS1患者视力下降以及开展相关治疗提供有力线索。
BACKGROUND AND OBJECTIVE
Histone methylation is an important modification of histone modifications, which was thought to be stable and irreversible until the first histone demethylase LSD1was identified in2004. So far, more than20histone demethylases (HDMs) have been found and studied. According to their structure and different catalytic machinism, they can be devided into two classes. One class is flavin adenine dinucleotide (FAD) dependent demethylase, LSD1and LSD2. And the other class compromises the recently discovered JmjC domain-containing histone demethylases (JHDMs), which are Fe2+and a-ketoglutarate (aKG)-dependent enzymes. These HDMs can regulate target gene expression by transcriptional activation or repression. Studies have also found HDMs to be associated with many diseases, specifically the initiation and progression of cancer. Examples of HDMs related cancers are prostate cancer, breast cancer, bladder cancer, and esophageal squamous cell carcinoma and primary mediastinal B cell lymphoma.
Due to HDMs play important roles in physiology and pathology, they have emerged as attractive targets for the development of new therapeutic drugs. In pursuit of developing HDM therapeutics, the design and screening of HDM inhibitors has become to focus of current HDM research. Based on the mechanism of histone demethylase function and their X-Ray crystal structure, the abundance of small molecules produced and necessitates the development of inexpensive, efficient high throughput assay to screen functional inhibitors of histone demethylases.
METHODS
We developed a fluorescence polarization (FP) assay for the screening of histone dementylase inhibitors. Based on the structure of methylstat, a demethylase inhibitor found recently in the lab, fluorophores, including SY-Ⅱ-082, WQX-Ⅶ-291and WQX-Ⅶ-417, were synthesized and optimized for the FP assay. During the foundation of FP assay, conditions of assay buffer and fluorophore concentration were optimized first. Due to the fluorophore structure already containing aKG mimic part, metal ions of FeⅡ and NiⅡ, which has a similar electron configuration as FeⅡ were tested to see if they could affect the binding between fluorophore and demethylases. After found NiⅡ could replace FeⅡ in vitro, we further optimized NiⅡ concentration for the binding assay. In addition, we also checked the signal stablility of FP assay.
By using the binding system that has been optimized, we examined the binding affinity between fluorophores and demethylases, and calculated their dissociation constant (Kd) values via plotting AmP values in KaleidaGraph software with an improved Kd equation. We then optimized the FP competition assay by optimization of protein concentration needed in competition system and examination of signal background between fluorophore and inhibitors. By titration of common HDM inhibitors, we confirmed the FP competition assay works well. Finally, we also checked Z'factor of this FP assay for the high throughput screening.
RESULTS AND DISCUSSION
After optimization of assay conditions, we finally developed a sensitive and stable FP assay to screen JHDM inhibitors, which can determine Ki(inhibit constant) of all the inhibitors to demethylases under the same condition, avoiding noncomparable IC50(half maximal inhibitory concentration) values by different assays. Meanwhile, we also obtained a fluorophore that specificly bound to JHDM1A, and can be used in JHDM1A specific inhibitor screening.
The optimization result of FP binding assay showed that1nM fluorophore has had a strong and stable signal. Tris buffer exhibited a better curve than HEPES buffer. For the metal ions, FeⅡ can interrupt the binding signal no matter where is ascorbic or not, and not produce a normal binding curve, while NiⅡ could stabilize binding signal in vitro by replacing FeⅡ that is needed in vivo. Compared to JMJD2A, fluorophore WQX-VII-417can specificly bind to JHDM1A with a higher binding affinity (Kd10.8nM) and a wider signal range (AmP280), which supply a wider window for inhibitor screening to screen all the JHDMs inhibitors that have a higher Ki value.
The optimization result of FP competition assay showed that only60nM concentration of JHDM1A could be used in the competition system. Titration result of common inhibitors showed methylstat can compete out fluorophore WQX-Ⅶ-417with a similar structure in a high binding affinity (Ki around12.8nM), which proved its inhibion machinism and resonablilty of design. The titration result also demonstrated FP competiton sassay works for inhibitor screening. Moreover, Z'factor of this assay proves it is suitable for high throughput screening applications.
SIGNIFICANTS
Fluorescence polarization could achive the preliminary screening of small molecule inhibitors by testing their binding affinity with different demethylases. Inihibitor candidates with higher binding affinity will be obtained for in vivo assay, and also supply important cues for the design of new inihibitors and the study of their structure activity relationship (SAR). Besides, this assay could also be used for screening of modified peptide library by checking the binding specificity between different peptides and different demethylases, to provide cue for finding new demethylases in vivo.
In summary, this fluorescence polarization method provides not only an efficient and convenient way for screening small molecule inhibitors of demethylase. but also an important tool and clues for the mechanism research of the biological process. The advantages of this method, which has promising application prospects, will also benefit the establishment of a mature and efficient platform for demethylase small molecule inhibitors screening, and could help us to study the mechanism of related diseases.
BACKGROUND AND OBJECTIVE
Hermansky-Pudlak syndrome (HPS) is a collection of autosomal resessive disorders, which is genetically heterogeneous. As a special albinism, several genes can cause it. So far, fifteen and eight HPS genes of mouse and human have been found and cloned, respectively. This syndrome is characterized by oculocutaneous albinism, prolonged bleeding, respiratory failure due to pulmonary fibrosis, granulomatous colitis and visual disturbances etc.
The mutant mouse model of HPS1pale ear (ep) shows a special pattern of coat color:dorsal coat color similar to parental C57BL/6J and significant pigment dilution of ears, tail and paws, which has been well studied. It suggest that HPS1mutation does not affect melanosomes in hair follicle, but causes decreased number of melanosomes in the interfolliar epidermis and dermis, and severe melanosome inmaturity in tail epidermis. However, few reports talked about melanosomes in the eye of ep mouse. In human, clinical research have found HPS1patient has visual defects combined with congenital nystagmus. To find its reason, we took advantages of HPS1muant mouse model ep to study the origin, distribution and morphology of ocular melanosome.
METHODS AND RESULTS
We collected eyeballs of ep mouse at different stages of eye development, and studied melanocytes in different tissues of eye by frozen section, HE staining, immunostaining and transmission electron microscope examination. Based on the results, we found the whole melanin level in ep mouse eye has already decreased after birth, compared to narmal mouse. This difference increased significantly along with eye development. We then further emamined this phenomenon under light microscope. It showed that all the pigmentation level of iris, clliary body and chorid/retina in ep mouse is lower than that of normal mouse. To confirm these finding, we then checked details of iris by electron microscope. Enlarged pigmented structures termed as macromelanosome were observed in iris stroma, which migrated from neural crest. No similar structures were found in anterior iris epithelium and posterior iris epithelium that originated from the epithelium of optic cup, only showing decreased number of melanosomes.
CONCLUSION
In summary, mutantions in HPS will casue macromalenosomes in ocular melanocytes that migrated from neural crest, while only results decreased numbers of melanosomes but no macromelanosomes in ocular melanocytes that originated from epithelium of optic cup. This conclusion agrees with the same phenomenon in both choroidal melanocytes that migrate from neural crest and retina pigment epithelial cells that originate from optic cup.
SIGNIFICANCE
Here we have elaborated and proved HPS1function in ocular melanosome, providing detailed morphological information and original proof for HPS1patient. However, we still need futher study the mechanism of effect of HPS1mutation on ocular melanosome morphology, explore HPS1function in ocular melanosome genesis, maturation and transportation, providing a useful cue for the study of visual defects in HPS1patients and its therapy development.
组蛋白甲基化是组蛋白共价修饰中的一种重要形式。一直以来被认为是一个稳定且不可逆的过程,直到2004年发现第一个组蛋白去甲基化酶LSD1。经过近年的研究,已发现了超过20种的组蛋白去甲基化酶(histone demethylases,HDMs)。根据去甲基化酶的结构和不同的催化机理,可以将去甲基化酶分为两类：一类是黄素腺嘌呤二核苷酸(flavin adenine dinucleotide,FAD)依赖性去甲基化酶,LSD1和LSD2；另一类是依赖于Fe2+和共同作用因子α-酮戊二酸(αKG),并含有Jumonji结构域的去甲基化酶(JmjC domain-containing histone demethylases,JHDMs).它们通过调控与特定基因的转录激活或抑制相关。大量的研究发现它们参与包括癌症在内的多种疾病的发生和进行发展,如前列腺癌、乳腺癌、膀胱癌、食管鳞状细胞癌以及原发性纵隔B细胞淋巴瘤等。
组蛋白去甲基化酶在生理和病理中的重要作用使其成为新药开发的靶点,对其抑制剂的研发和筛选也已成为了现阶段组蛋白去甲基化酶研究的热点。基于去甲基化酶作用机制以及蛋白质晶体结构的特点,已经有大量的小分子抑制剂被设计并合成出来。因此,迫切需要一个方便、快速、高效、低廉以及高通量的方法对其进行大规模筛选,发现有效的小分子去甲基化酶抑制剂。
研究方法
我们通过建立荧光偏振方法应用于组蛋白去甲基化酶小分子抑制剂的筛选。基于实验室已发现的JHDMs小分子抑制剂methyl stat的结构,我们设计合成了荧光探针SY-Ⅱ-082、WQX-Ⅶ-291和WQX-Ⅶ-417等,并在荧光偏振实验过程中进行结构优化。在建立荧光偏振结合体系时,我们首先优化了缓冲液条件和荧光探针浓度。由于探针本身含有αKG结构,我们又对金属离子Fe(?)和外层电子排布与Fe(?)相似的Ni(?)对结合体系的影响进行了检测,并进一步优化了Ni'浓度；还对信号的稳定性进行检测。
通过优化的结合体系,我们对荧光探针分子与去甲基化酶的亲和能力进行测定,并利用改良的解离常数计算公式在KaleidaGraph中拟合曲线得到两者的Kd值。接下来,通过对所需蛋白浓度的优化以及小分子抑制剂与荧光探针分子背景结合信号的检测,我们优化了荧光偏振竞争体系。我们通过对JHDMs常用抑制剂的滴定(IC50)来确定荧光偏振竞争实验的可行性后,还检测了Z’因子(0
利用对实验体系的一系列优化,我们最终建立了一个稳定的可用于去甲基化酶抑制剂筛选的荧光偏振方法,可在同一背景下检测所有小分子抑制剂与去甲基化酶的结合能力Ki,避免了不同方法检测到的半数抑制浓度IC50值无可比性的缺点；同时筛选出一个特异性结合JHDM1A的荧光探针WQX-Ⅶ-417,可用于针对JHDM1A的小分子抑制剂的筛选。
荧光偏振结合实验的优化结果显示,1nM的荧光探针WQX-VII-417本身具有较强的稳定的荧光信号；Tris缓冲液较HEPES缓冲液能拟合出更优曲线；Fe(?)无论在还原剂是否存在的条件下,都会干扰结合信号,不能产生正常的结合曲线；Ni(?)能够在体外反应中保持结合信号的稳定,替代体内反应中所需的Fe(?)。相比JMJD2A,探针WQX-Ⅶ-417能够特异性地较强地结合JHDM1A (ΔmP约为280,Kd值仅为10.8nM)。这一结果为小分子抑制剂的筛选提供了较大筛选窗,利用此探针可以筛选出K大于10nM的JHDMs小分子抑制剂。
荧光偏振竞争实验的优化结果显示,60nM的JHDM1A即可用于竞争结合实验。常用小分子抑制剂滴定结果显示methylstat能够将相似结构的探针分子WQX-Ⅶ-417竞争下来,亲和能力较高(Ki约为12.8nM),证明了其作用机制及设计理念的合理性,并第一次测出了共同作用因子aKG与去甲基化酶的亲和能力；滴定结果还说明此方法可用于抑制剂的筛选。此外,测定此实验方法的Z’因子为0.75显示其较适用于高通量筛选(>0.7)。
研究意义
荧光偏振方法通过检测小分子抑制剂与不同去甲基化酶的亲和能力,实现对小分子抑制剂文库的初步筛选,得到具有较高亲和力的抑制剂,为体内实验提供候选抑制剂小分子,并为优化设计新的小分子抑制剂以及研究小分子抑制剂的结构活性关系(Structure activity relationship, SAR)提供重要的线索。此外,此方法还可用于筛选合成修饰的肽链库,通过体外检测不同结构修饰的肽链与组蛋白去甲基化酶结合的特异性,为体内发现新的去甲基化酶提供线索。
综上所述,此种荧光偏振方法不仅为去甲基化酶小分子抑制剂筛选提供高效便利的方法,也为生物过程的机理研究提供重要的工具和线索,对建立成熟高效的去甲基化酶小分子抑制剂筛选平台以及研究相关疾病的机理具有重要的应用价值,应用前景较好。
研究的背景及目的
HPS综合征是一种常染色体隐性遗传病,是一种特殊的白化病,可由多个基因突变引起。目前已发现并克隆了导致小鼠HPS的15个基因以及所对应人的8个基因。临床上,HPS综合征可导致白化、出血倾向、肺部纤维化引起的呼吸衰竭、肠道感染和视力降低。
目前,对HPS1突变小鼠模型pale ear (ep)背部皮肤的色素沉着已有深入研究,发现HPS1突变并不影响毛囊中的黑色素体,但是会降低表皮和真皮毛囊间黑色素体的数目,引起尾部黑色素体的不成熟,从而形成了ep小鼠的特殊毛色模式—背部色素沉着正常呈黑色,而耳朵、尾巴和爪子着色较浅呈近白色。对于同样含有黑色素体的眼则未见系统的报道。在人类中已发现HPS1患者视力下降并伴随着眼球水平摆动震颤。为对HPS1突变所导致视力下降的原因提供研究线索,我们利用HPS1突变小鼠ep为模型,对ep小鼠眼黑色素的起源、分布以及形态进行系统研究。
研究方法及结果
采集不同发育阶段ep小鼠的眼球,通过黑色素含量测定、冷冻切片、HE染色、免疫荧光和电镜等方法,我们对眼不同发育阶段含有黑色素细胞的组织进行了生化、光镜和电镜的检测。结果发现,ep小鼠在出生时整体黑色素含量就低于正常小鼠,这种差异随着发育逐步增大；通过进一步的光学显微镜检测发现,虹膜、睫状体以及脉络膜/视网膜色素上皮的色素沉着都低于同时期的正常小鼠；电镜结果显示虹膜内起源于神经脊的虹膜基质中黑色素体异常,表现为巨大黑色素体,而在来源于视杯上皮的色素细胞,前虹膜色素上皮和后虹膜上皮中黑色素体数目大量减少,并没有产生巨大黑色素体。
结论
HPS1的缺失可导致起源于神经脊的黑色素细胞形成巨大黑色素体,而使来源于视杯色素上皮的黑色素细胞中黑色素体数目降低但并不产生巨大黑色素体。这个结果与之前对ep小鼠中起源于神经脊的脉络膜与起源于视杯的视网膜色素上皮中黑色素体的形态研究一致,证明了所得结论的正确性。
研究意义
此研究结果详细的阐述和证明了HPS1基因缺失对眼黑色素体的影响,为HPS1患者眼内黑色素体的分布及异常提供了详细的形态学资料及发育起源证据；但仍需进一步研究HPS1对不同发育来源的黑色素体形态造成影响的机制,探寻HPS1在眼黑色素体发生、成熟和运输过程中的作用,从而为研究HPS1患者视力下降以及开展相关治疗提供有力线索。
BACKGROUND AND OBJECTIVE
Histone methylation is an important modification of histone modifications, which was thought to be stable and irreversible until the first histone demethylase LSD1was identified in2004. So far, more than20histone demethylases (HDMs) have been found and studied. According to their structure and different catalytic machinism, they can be devided into two classes. One class is flavin adenine dinucleotide (FAD) dependent demethylase, LSD1and LSD2. And the other class compromises the recently discovered JmjC domain-containing histone demethylases (JHDMs), which are Fe2+and a-ketoglutarate (aKG)-dependent enzymes. These HDMs can regulate target gene expression by transcriptional activation or repression. Studies have also found HDMs to be associated with many diseases, specifically the initiation and progression of cancer. Examples of HDMs related cancers are prostate cancer, breast cancer, bladder cancer, and esophageal squamous cell carcinoma and primary mediastinal B cell lymphoma.
Due to HDMs play important roles in physiology and pathology, they have emerged as attractive targets for the development of new therapeutic drugs. In pursuit of developing HDM therapeutics, the design and screening of HDM inhibitors has become to focus of current HDM research. Based on the mechanism of histone demethylase function and their X-Ray crystal structure, the abundance of small molecules produced and necessitates the development of inexpensive, efficient high throughput assay to screen functional inhibitors of histone demethylases.
METHODS
We developed a fluorescence polarization (FP) assay for the screening of histone dementylase inhibitors. Based on the structure of methylstat, a demethylase inhibitor found recently in the lab, fluorophores, including SY-Ⅱ-082, WQX-Ⅶ-291and WQX-Ⅶ-417, were synthesized and optimized for the FP assay. During the foundation of FP assay, conditions of assay buffer and fluorophore concentration were optimized first. Due to the fluorophore structure already containing aKG mimic part, metal ions of FeⅡ and NiⅡ, which has a similar electron configuration as FeⅡ were tested to see if they could affect the binding between fluorophore and demethylases. After found NiⅡ could replace FeⅡ in vitro, we further optimized NiⅡ concentration for the binding assay. In addition, we also checked the signal stablility of FP assay.
By using the binding system that has been optimized, we examined the binding affinity between fluorophores and demethylases, and calculated their dissociation constant (Kd) values via plotting AmP values in KaleidaGraph software with an improved Kd equation. We then optimized the FP competition assay by optimization of protein concentration needed in competition system and examination of signal background between fluorophore and inhibitors. By titration of common HDM inhibitors, we confirmed the FP competition assay works well. Finally, we also checked Z'factor of this FP assay for the high throughput screening.
RESULTS AND DISCUSSION
After optimization of assay conditions, we finally developed a sensitive and stable FP assay to screen JHDM inhibitors, which can determine Ki(inhibit constant) of all the inhibitors to demethylases under the same condition, avoiding noncomparable IC50(half maximal inhibitory concentration) values by different assays. Meanwhile, we also obtained a fluorophore that specificly bound to JHDM1A, and can be used in JHDM1A specific inhibitor screening.
The optimization result of FP binding assay showed that1nM fluorophore has had a strong and stable signal. Tris buffer exhibited a better curve than HEPES buffer. For the metal ions, FeⅡ can interrupt the binding signal no matter where is ascorbic or not, and not produce a normal binding curve, while NiⅡ could stabilize binding signal in vitro by replacing FeⅡ that is needed in vivo. Compared to JMJD2A, fluorophore WQX-VII-417can specificly bind to JHDM1A with a higher binding affinity (Kd10.8nM) and a wider signal range (AmP280), which supply a wider window for inhibitor screening to screen all the JHDMs inhibitors that have a higher Ki value.
The optimization result of FP competition assay showed that only60nM concentration of JHDM1A could be used in the competition system. Titration result of common inhibitors showed methylstat can compete out fluorophore WQX-Ⅶ-417with a similar structure in a high binding affinity (Ki around12.8nM), which proved its inhibion machinism and resonablilty of design. The titration result also demonstrated FP competiton sassay works for inhibitor screening. Moreover, Z'factor of this assay proves it is suitable for high throughput screening applications.
SIGNIFICANTS
Fluorescence polarization could achive the preliminary screening of small molecule inhibitors by testing their binding affinity with different demethylases. Inihibitor candidates with higher binding affinity will be obtained for in vivo assay, and also supply important cues for the design of new inihibitors and the study of their structure activity relationship (SAR). Besides, this assay could also be used for screening of modified peptide library by checking the binding specificity between different peptides and different demethylases, to provide cue for finding new demethylases in vivo.
In summary, this fluorescence polarization method provides not only an efficient and convenient way for screening small molecule inhibitors of demethylase. but also an important tool and clues for the mechanism research of the biological process. The advantages of this method, which has promising application prospects, will also benefit the establishment of a mature and efficient platform for demethylase small molecule inhibitors screening, and could help us to study the mechanism of related diseases.
BACKGROUND AND OBJECTIVE
Hermansky-Pudlak syndrome (HPS) is a collection of autosomal resessive disorders, which is genetically heterogeneous. As a special albinism, several genes can cause it. So far, fifteen and eight HPS genes of mouse and human have been found and cloned, respectively. This syndrome is characterized by oculocutaneous albinism, prolonged bleeding, respiratory failure due to pulmonary fibrosis, granulomatous colitis and visual disturbances etc.
The mutant mouse model of HPS1pale ear (ep) shows a special pattern of coat color:dorsal coat color similar to parental C57BL/6J and significant pigment dilution of ears, tail and paws, which has been well studied. It suggest that HPS1mutation does not affect melanosomes in hair follicle, but causes decreased number of melanosomes in the interfolliar epidermis and dermis, and severe melanosome inmaturity in tail epidermis. However, few reports talked about melanosomes in the eye of ep mouse. In human, clinical research have found HPS1patient has visual defects combined with congenital nystagmus. To find its reason, we took advantages of HPS1muant mouse model ep to study the origin, distribution and morphology of ocular melanosome.
METHODS AND RESULTS
We collected eyeballs of ep mouse at different stages of eye development, and studied melanocytes in different tissues of eye by frozen section, HE staining, immunostaining and transmission electron microscope examination. Based on the results, we found the whole melanin level in ep mouse eye has already decreased after birth, compared to narmal mouse. This difference increased significantly along with eye development. We then further emamined this phenomenon under light microscope. It showed that all the pigmentation level of iris, clliary body and chorid/retina in ep mouse is lower than that of normal mouse. To confirm these finding, we then checked details of iris by electron microscope. Enlarged pigmented structures termed as macromelanosome were observed in iris stroma, which migrated from neural crest. No similar structures were found in anterior iris epithelium and posterior iris epithelium that originated from the epithelium of optic cup, only showing decreased number of melanosomes.
CONCLUSION
In summary, mutantions in HPS will casue macromalenosomes in ocular melanocytes that migrated from neural crest, while only results decreased numbers of melanosomes but no macromelanosomes in ocular melanocytes that originated from epithelium of optic cup. This conclusion agrees with the same phenomenon in both choroidal melanocytes that migrate from neural crest and retina pigment epithelial cells that originate from optic cup.
SIGNIFICANCE
Here we have elaborated and proved HPS1function in ocular melanosome, providing detailed morphological information and original proof for HPS1patient. However, we still need futher study the mechanism of effect of HPS1mutation on ocular melanosome morphology, explore HPS1function in ocular melanosome genesis, maturation and transportation, providing a useful cue for the study of visual defects in HPS1patients and its therapy development.
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