Polypeptide epoxy ketone compound
A compound and solvate technology, applied in the direction of peptides, drug combinations, peptide/protein components, etc., can solve problems such as inability to inhibit T-L activity, PGPH activity, etc.
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Embodiment 1
[0210] Example 1: Determination of Inhibition of 20S Proteasome CT-L, T-L, and PGPH Activities in Vitro.
[0211]The purified human 20S proteasome was used for in vitro determination of CT-L, T-L and PGPH activities. The concentrations of 20S proteasome were 2, 4 and 8 nmol / L, respectively, and succinyl-Leu-Leu-Val-Tyr-AMC ( 10 μmol / L), Z-leu-Leu-Glu-AMC (10 μmol / L) and Boc-Leu-Arg-Arg-AMC (50 μmol / L) were used as substrates, and the reaction contained 0.5mM ethylenediaminetetraacetic acid, 0.001% sodium dodecyl sulfate (SDS) and 0.05% NP-40 in 20mM triisopropylethanesulfonyl buffer solution (pH8.0). Stock solutions of proteasome inhibitors were prepared in dimethylsulfoxide (DMSO), the final concentration of DMSO in the test mixture was 1%. The reaction was carried out at a temperature of 27°C. Proteasome activity was determined by detecting the cleaved 7-amino-4-methylcoumarin fluorophore with a fluorophotometer. IC 50 (Half inhibitory concentration) values were determ...
Embodiment 2
[0212] Example 2: Determination of the inhibitory effect on CT-L, T-L and PGPH activities of 20S proteasome in blood.
[0213] Take 800 microliters of fresh whole blood samples from CD-1 mice by cardiac puncture into test tubes containing sodium heparin, centrifuge at 150xg for 5 minutes at 4°C, and wash the centrifuged precipitate with ice-cold phosphate-buffered saline three times. Each time the pellet was redissolved in 1 mL of cold phosphate-buffered saline and centrifuged at 6000 x g for 10 min at 4 °C. After the last wash, 200 μL of dissociation solution (phosphate-buffered saline containing 5 mM EDTA, pH 8.0) was added, kept for 1 hour, and the cells were dissociated, and then centrifuged at 6000×g for 10 minutes at 4°C. The protein concentration in the above-mentioned blood lysate was determined by the bicincholic acid method (BCA). Add about 100 μ g protein to HEPES buffer solution (pH7.5); Add 0.2, 1 or 5 μ M of the compound of the present invention to the above mi...
Embodiment 3
[0214] Example 3: Determination of Inhibition of CT-L, T-L and PGPH Activities of 20S Proteasome in Vivo.
[0215] In vivo proteasome activity assays were performed in BALB / c nude mice. Take the polypeptide epoxy ketone compound described in the present invention as the test article and administer it to mice (every group of 5-6 mice) by intravenous injection in a tolerable dose, and the test article is prepared in a mixture containing 10-20 %(w / v) hydroxypropyl-B-cyclodextrin, 2.5% DMSO and 10mM sodium citrate in the vehicle (pH3.0-3.5). One hour after the injection, a whole blood sample was collected by cardiac puncture into a test tube containing sodium heparin and centrifuged at 150xg for 5 minutes at 4°C. The centrifuged pellet was washed three times with ice-cold phosphate-buffered saline. Each time the pellet was redissolved in 1 mL of cold phosphate-buffered saline and centrifuged at 6000 x g for 10 min at 4 °C. After the last wash, 200 μL of dissociation solution (p...
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