Development And Application Of Fluorescence Polarizaiton Assay In Histone Demethylase Inhibitor Screening And Study On Effect Of HPS1Mutation In Ocular Melanosome

BACKGROUND AND OBJECTIVEHistone 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.METHODSWe 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 DISCUSSIONAfter 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.SIGNIFICANTSFluorescence 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 OBJECTIVEHermansky-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 RESULTSWe 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.CONCLUSIONIn 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.SIGNIFICANCEHere 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.