Protein inhibitor and application thereof
A technology of protein inhibitors and solvents, applied in the field of medicine, can solve the problems of complex molecular components, poor stability, and low bioavailability of natural medicines, and achieve inhibition of tumor cell metastasis and invasion, low threshold for industrial synthesis, and simple reaction conditions Effect
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Embodiment 1
[0037] The synthetic route of compound ① involved in this embodiment is as follows, and compounds ②~compound ⑧ can also be prepared in a similar manner:
[0038]
[0039] This embodiment specifically includes the following steps:
[0040] Step 1) will raw material S1, namely CH 2 CH(NH 2 )COOH was dissolved in a mixed solvent of ice water and methanol, and then NaBH 3 CN was added in portions and after a few minutes of stirring acetaldehyde was added dropwise (5 equiv first). After the dropwise addition, the reaction was carried out at room temperature for 6 hours, and the remaining 5 equivalents of acetaldehyde were added, and the reaction was carried out for two hours. Then, concentrated hydrochloric acid was slowly added to adjust the pH to 1.5, and the reaction was carried out at 40°C for 1.5 hours.
[0041] Post-processing procedure: TLC detection reaction (DCM:MeOH=7:1). After the reaction, spin off methanol first, then add dichloromethane to extract impurities, ...
Embodiment 2
[0051] This example relates to compound ① inhibiting the expression and activity of human Legumain protein, specifically including:
[0052] Step 1) AEP activity detection experiment: To determine the inhibitory effect of the inhibitor on AEP enzymatic activity, recombinant human legumain protein (rhLegumain, catalog #2199-CY) was diluted to 100 μg / mL in activation buffer containing 50 mM sodium acetate and 100 mM NaCl mL, pH 4.0 for 2h at 37°C. 30 ng / μl of activated rhLegumain diluted in assay buffer (50 mM MES, 250 mM NaCl), combined with 30 μl of various doses of inhibitors (0 nM, 10 nM, 100 nM and 1 μM, diluted in assay buffer) at pH 5.0, were then added into a black well plate for 30 minutes. Afterwards, 40 μl of 200 μM substrate Z-AAN-AMC diluted in assay buffer was added, including a control containing 60 μl of assay buffer (50 mM MES, 250 mM NaCl, pH 5.0) and 40 μl of 200 μM substrate. Enzyme activity was measured using a microplate reader (Bio-tek) at 380 and 460 nm...
Embodiment 3
[0057] This example relates to inhibitors that improve the ability of tumor cells to metastasize and invade by affecting the expression of key proteins in the process of epithelial-mesenchymal transition (EMT) such as Snail, E-cadherin, and MMP-9.
[0058] The EMT refers to the biological process by which epithelial cells are transformed into cells with a mesenchymal phenotype through a specific program. During the EMT process of tumor cells, the ability of tumor cells to migrate and invade, and to degrade extracellular matrix will be improved, which is an important biological process in which malignant tumor cells derived from epithelial cells acquire the ability to migrate and invade. EMT changes can be detected by detecting key proteins such as E-cadherin and Snail, and the ability of tumor cell migration and invasion can be judged.
[0059] In this example, the breast cancer 4T1.2 cells were stimulated with 100 nM, 1 μM and 0 nM inhibitors respectively, the culture medium ...
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