Screening And Evaluation Of Selective Small Molecule Inhibitors Of TRPC6

Background:TRPC6 is a member of the transient receptor potential canonical family,which is widely distributed in the brain,heart,kidney,lung and other tissues of mammals and plays a pivotal role in many important physiological processes,such as vascular smooth muscle contraction,podocyte signal transduction,central nervous development and pain regulation.TRPC6 protein serves as the molecular basis for the calcium ion channel of cell membrane,its mutation and abnormal expression usually cause changes in intracellular calcium signaling pathways and induce many pathological changes,such as cardiac hypertrophy,pulmonary hypertension,abnormal neuronal development,and tumor cell proliferation.TRPC6,as a potential therapeutic target for many related diseases,has gradually become one of the hot spots of the new drug research.However,due to the high homology of amino acid sequences among different members,reports on TRPC6 specific small molecule regulator are rare.Objective:This research intends to screen small molecule compounds that canbind to TRPC6 protein from 150,000 natural small molecule compounds by means of computer aided drug design?CADD?,and on this basis,detect the interaction between TRPC6 protein and small molecule compounds using microscale thermophoresis?MST?technology.The tool cells that over-expressing TRPC6 protein were established,and the specific biological activities of the compounds were further determined by measuring the changes of intracellular calcium transients([Ca²⁺]_i).The FSGS disease model was established to verify the effect of hit compound in the disease from the holistic,tissue,cellular and molecular levels.The aim of the current study was to screen out a small molecule compound that can specifically bind to TRPC6 protein and inhibit its function,providing more experimental references for the discoveries of TRPC6 modulators and the researches of innovative drugs.Methods:?1?The protein structure simulation software MODELLER 8.0 was used to construct the TRPC6 protein model.150,000 natural compounds in“Natural Products”database were docked and scored by virtual screening software AutoDock Vina and PyRx,and the compounds with ideal scores were selected.?2?Virtual screening-identified compounds were classified based on their structures and those with a higher score and representative structure were chose for MST experiments to detect their interaction with TRPC6 protein.?3?TRPC6 over-expressing tool cells were constructed,and the biological activities of the compounds on TRPC6 protein were determined by monitoring the changes of[Ca²⁺]_i as well as the concentration-response relationships and the median inhibition concentrations(IC₅₀).Calcium ion imaging and MST assay were used to analyze the specificity of the hit compound to TRPC6 protein.The cytotoxicity of the hit compound was determined by a CCK-8 assay.?4?A reasonable synthetic route of the hit compound was designed according to literatures.The acylation and hydrolysis reaction were performed with the original materials of 1-methyl-indole-2-formic acid and?-ethyl alanine ester hydrochloride to get the hit compound 3-[?1-Methyl-1H-indole-2-carbonyl?amino]propionic acid.The maximum tolerable dose of oral administration in mice was determined by an acute toxicity test.?5?The 8-week-old male Balb/c mice were intravenously injected with 10 mg/kg adriamycin to establish the FSGS disease model.The mice were treated the hit compound with different doses by gavage every day for6 weeks,and the urine was collected in the metabolic cage.Coomasie brilliant blue staining was used to detect the urine protein level.Picric acid colorimetry was used to determine the creatinine level.Blood urea nitrogen levels were measured by diacetyl monoxime colorimetry.H&E staining was used to evaluate the renal morphological changes.Masson staining was used to evaluate the renal fibrosis level.PAS staining was used to evaluate the glomerular mesangial area and basement membrane lesions.The expression levels of podocyte injury biomarkers Nephrin and Desmin in kidney were detected by immunofluorescence staining.TUNEL method was used to detect the apoptotic podocytes in kidney tissue.Results:?1?Using 5IRX?PDB ID?as the template,the virtual spatial structure model of TRPC6 protein was obtained by adjusting the position of each atom of the main chain in the amino acid sequence of the TRPC6 protein.Converted TRPC6 into a PDBQT file and set as a receptor in AutoDock Vina,and then we got 8 binding sites that may interact with the compounds.A total of 263 compounds with a score of less than or equal to-32 were selected by docking the protein receptor 5IRX to 150,000small molecule compounds.We classified the 263 compounds based on their structures and purchased 17 compounds with novel structure and top rated.?2?The interaction between TRPC6 protein and the 17 small molecule compounds were detected by MST experiments and the results showed that 8 out of the 17 compounds can actually bind to TRPC6 protein.They are zinc7042?Kd=30.49?M?,zinc0071?Kd=283.62?M?,zinc3571?Kd=29.78?M?,zinc4330?Kd=94.39?M?,zinc7978?Kd=178.87?M?,zinc0737?Kd=11.03?M?,zinc2840?Kd=26.24?M?,zinc6085?Kd=32.11?M?.?3?The HEK293_hTRPC6TRPC6 tool cells were constructed successfully to monitor the changes of intracellular calcium concentration([Ca²⁺]_i)after different compounds treatment.We found that compounds zinc0737,zinc3571 and zinc7042 in the above 8 compounds can inhibit the opening of TRPC6 channels and reduce the extracellular Ca²⁺influx.The calcium entered from the extracellular via TRPC6channel were reduce by 34.84%?0.14±0.02 vs.0.22±0.01,P<0.001?,52.94%?0.10±0.01 vs.0.22±0.01,P<0.001?and 38.46%?0.14±0.01 vs.0.22±0.01,P<0.001?,respectively,the IC₅₀0 values of this 3 compounds were further determined by measuring the dose-effect relationships,and they were 6.6?M,24.1 nM and 208.3nM,respectively.Compounds zinc7978,zinc6085 and zinc0071 in the above 8compounds can activate the TRPC6 channels as well as increase the extracellular Ca²⁺influx.The calcium entered from the extracellular via TRPC6 channel were increase by 71.04%?0.38±0.01 vs.0.22±0.01,P<0.001?,25.34%?0.28±0.01 vs.0.22±0.01,P<0.001?and 14.93%?0.25±0.01 vs.0.22±0.01,P<0.05?,respectively.Compounds zinc2840 and zinc4330 showed no obvious biological activities against TRPC6channels.By comparing the structure and IC₅₀0 values of the above 3 inhibitory compounds,the compound zinc3571 with the smallest IC₅₀0 value and the simple structure was identified as the hit compound.We used the sarco/endoplasmic reticulum Ca²⁺-ATPase Thapsigargin?Tg?as the tool drug to tested the effect of hit compound on the TRPC1/4/5 channels function.The results showed that there was no significant difference in the extracellular Ca²⁺influx through the TRPC1/4/5 channels after Tg activation between the compound zinc3571 pretreatment group and the control group.We further used MST technology to detect the interaction between hit compound and TRPC3/6/7 proteins.The results showed that the binding of compound zinc3571 to TRPC6 protein was highly specific.Its affinity to TRPC6 was 27 times higher than that of TRPC3 and 35 times higher than that of TRPC7.This part of the experiments indicated that the hit compound zinc3571 had no effect on the TRPC1/4/5 channels function and its affinity to TRPC6 was much stronger than that of TRPC3 and TRPC7.Therefore,we can conclude that the hit compound is specific for TRPC6 protein.When the concentration of the hit compound was less than 100?M in CCK-8 assay,the cell viability was not significantly different from that of the blank group?P>0.05?,indicating that the hit compound had no obvious cytotoxicity.?4?4.152 g of the hit compound was synthesized,and the structure of the compound was characterized by ¹H NMR,¹³C NMR,MS and IR to further determine its configuration.In the acute toxicity test,2.0 g/kg of the hit compound was infused to stomach and the mice were observed for 15 days,we found that the spirit,appetite and excretion of the mice were normal,the body weight was increasing for15 days,and no severe poisoning as well as death were observed.?5?The FSGS disease model was successfully established,and the hit compound was given by gavage at different doses for 6 weeks.We found that after 6 weeks,the body weights and the kidney weights in the model group were reduced by 29.31%?18.72±0.48 g vs.26.48±0.72 g,P<0.001?and 31.22%?0.15±0.01 g vs.0.22±0.01 g,P<0.001?.The low,medium and high dose groups of the hit compound dose-dependently inhibited the reductions of body and kidney weights.The body weights and kidney weights of the 10 mg/kg group were 24.52%?19.99±0.68 g vs.26.48±0.72 g,P<0.01?and 19.31%?0.17±0.01 g vs.0.22±0.01 g,P<0.01?lower than that of the normal group.The 25 mg/kg group were 22.91%?20.40±1.47 g vs.26.48±0.72 g,P<0.05?and 17.90%?0.18±0.01 g vs.0.22±0.01 g,P<0.01?lower than that of the normal group,and there was no significant difference in body and kidney weights between the 50 mg/kg group?P>0.05?and the normal group.The ratio of urinary protein to urinary creatinine in the model group increased about 1.89-fold?P<0.001?compared with the standard value before modeling.The ratio of urinary protein to urinary creatinine in the 10 mg/kg group,25 mg/kg group and 50 mg/kg group at sixth week were decreased by 23.20%?1.54±0.07 vs.2.00±0.07,P<0.001?,40.17%?1.20±0.13 vs.2.00±0.07,P<0.001?and 59.05%?0.82±0.05 vs.2.00±0.07,P<0.001?,respectively,compared with that in the model group.In the 50 mg/kg group,there was no significant difference in the ratio of urinary protein to urinary creatinine at sixth week compared with the standard value before modeling.The serum creatinine and the plasma urea-nitrogen in the model group were increased by 43.34%?175.60± 8.10?mol/L vs.132.30±8.01?mol/L,P<0.05?and 25.64%?11.82±0.46 mmol/L vs.9.41±0.16 mmol/L,P<0.01?compared with normal group.There was no significant difference in serum creatinine between the 10 mg/kg group and the model group?P>0.05?,but the urea-nitrogen content was decreased by 14.21%?10.14±0.33mmol/L vs.11.82±0.46 mmol/L,P<0.05?.These two indexes were also decreased by21.36%?138.11±4.40?mol/L vs.175.60±8.10?mol/L,P<0.05?and 19.66%?9.50±0.25 mmol/L vs.11.82±0.46 mmol/L,P<0.05?in 25 mg/kg group and 27.51%?127.32±4.64?mol/L vs.175.60±8.10?mol/L,P<0.01?as well as 19.89%?9.46±0.12 mmol/L vs.11.82±0.46 mmol/L,P<0.01?in 50 mg/kg group,respectively.H&E,Masson and PAS staining of renal tissue showed that the glomerular mesangial area in the model group was severely hyperplasia,and the basement membrane was thickened.The percent of the glomerulus with epithelial hyperplasia lesions?EPHL?was as high as 65.33%,and it was reduced by 61.73%?25.00±3.22 vs.65.33±9.74,P<0.05?in the 50 mg/kg group compared with the model group.Obviously glomerular fibrosis also occurred in the model group,and the glomerular fibrosis area in the model group was 5.75-fold?38.53±5.71 vs.5.71±1.42,P<0.01?higher than that in the normal group.Compared with the model group,the fibrosis area were reduced by 67.69%?12.45±3.68 vs.38.53±5.71,P<0.05?and 87.39%?4.86±1.49vs.38.53±5.71,P<0.01?in 25 mg/kg and 50 mg/kg,respectively.The immunofluorescence staining results of the expressions of Nephrin and Desmin proteins showed that the expression of Nephrin in the model group was down-regulated by 68.94%?0.31±0.09 vs.1.00±0.16,P<0.05?compared with the normal group,and Nephrin expression in the 50 mg/kg group was 1.59-fold?0.80±0.10 vs.0.31±0.09,P<0.05?higher than that in the model group.The Desmin expressions between different groups were in an opposite tendency of Nephrin.The Desmin expression in the model group was 1.20-fold?2.20±0.30 vs.1.00±0.16,P<0.05?higher than that in the normal group,and it in the 50 mg/kg group was49.32%?1.11±0.17 vs.2.20±0.30,P<0.05?lower than that in the model group.TUNEL staining results showed that there were a large number of apoptotic podocytes in the model group.The apoptotic podocytes in 25 mg/kg group and 50 mg/kg group were reduced by 45.60%?1.53±0.30 vs.2.81±0.27,P<0.05?and 83.26%?0.47±0.14 vs.2.81±0.27,P<0.01?,respectively,compared with the model group.Conclusion:The compound zinc3571 can specifically bind to TRPC6 protein with a high affinity and drastically inhibit the calcium influx via TRPC6.Besides,it also can significantly alleviate the TRPC6-mediated podocyte apoptosis and reduce the production of proteinuria as well as slow down the development of FSGS.The compound zinc3571,with the characteristics of simple structure,easy synthesis and low toxicity,can serve as an ideal small molecule inhibitor of TRPC6.