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Caspase-3 inhibitor and purpose

An inhibitor, aspartic acid technology, is applied in the direction of medical preparations containing active ingredients, tetrapeptide components, digestive system, etc., and can solve problems such as limited application, poor membrane permeability, and unsatisfactory cell effects

Active Publication Date: 2020-02-07
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Peptide inhibitors, represented by Z-DEVD-FMK, are designed from the specific amino acid sequence of the recognition site of PARP protein digested by caspase-3, and have certain effects on the treatment of burns, sepsis and spinal cord injuries (Discovery of novel aspartyl ketonedipeptides as potent and selective caspase-3 inhibitors. Bioorg Med Chem Lett, 2004.14, 805-808; J Clin Neurosci, 2008.15, 672-678), the disadvantage is that it has irreversible inhibition and non-specific inhibition of other cysteines Protease, and poor membrane permeability, the effect on cells is not ideal, thus limiting its clinical application

Method used

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  • Caspase-3 inhibitor and purpose
  • Caspase-3 inhibitor and purpose
  • Caspase-3 inhibitor and purpose

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Molecular structural formula of Ac-DMPD / DMLD-CMK (982478 / 982479, China Peptide Biochemical Co., Ltd.). The specific steps of the MST instrument (NanoTemper, Germany) detection are as follows: the concentration gradient of the inhibitor is set to: 0.8, 1.6, 3.2, 6.25, 12.5, 25, 50, 100, 200 μM, and diluted to the above concentration gradient with PBS before use. Dilute the active protein of caspase-3 (Nanjing Jingmai Biotechnology Co., Ltd.) fused with GFP to an appropriate concentration. The peak intensity detected by the microthermophoresis instrument is preferably 200-600 peak intensity. GFP recombinant protein (Nanjing Detai Biotechnology Co., Ltd.) was used as a control to observe whether the interaction between the inhibitor and caspase-3 was specific. Each concentration of inhibitor and protein was mixed at a ratio of 1:1 (4+4μl) for 10 minutes and then tested on the machine. Select blue light as the excitation condition, set the excitation parameters (20%-50%), a...

Embodiment 2

[0073] The inhibitor concentration gradient was set to 1.5, 3.125, 6.25, 12.5, 25, 50, 100 μM, the dosage of active caspase-3 was 1 unit, and the composition of warm compress system was: 50mM HEPES (pH7.5), 150mM NaCl, 3mM EDTA and 0.005% (v / v) Tween-20 and 10mM DTT. Add 1 unit caspase-3 / 4 protein to 100μl reaction system, shake well, then add concentration gradient inhibitors and incubate at room temperature for 2h, then add 200μM corresponding substrate, among which caspase- 3 protein uses Ac-DMPD / DMLD-AMC as the substrate, and caspase-4 (1084, Biovision) uses Ac-LEVD-pNA (1110, Biovision) as the chromogenic substrate. The instrument detects the fluorescence value of caspase-3 activity at Ex 365nm and Em 450nm, and detects the activity of caspase-4 at the absorption wavelength of 405nm. Using Graphpad prism6.0 software in the nonlinear fitting mode log (inhibitor concentration) vs enzyme activity ratio (%) to obtain the IC50 value and the corresponding inhibition curve. Fi...

Embodiment 3

[0076] HepG2 (ATCC, HB-8065) ​​cell level verification of inhibitor efficacy

[0077] First determine the dose and action time of anti-tumor drugs, and found that in the case of 70-80% cell confluency, pre-administered 10 μg / ml cycloheximide CHX or 400nM actinomycin D ActD for half an hour, and then co-administered 20ng After 16 hours of TNFα / ml TNFα treatment, the cells appeared massive necrosis, and the caspase-3, PARP and GSDME proteins were obviously activated after 8 hours. Based on this, the inhibitor concentration gradient was set to 3.125, 6.25, 12.5, 25, 50, 100, and 200 μM. After pre-administrating the HepG2 cell concentration gradient inhibitor for 2 hours, 10 μg / ml CHX or 400 nM ActD was synergistically pre-administered for 0.5 h. In the end, 20ng / ml TNFα was administered synergistically for 16 hours, and the lactate dehydrogenase detection kit (C0017, Biyuntian Biotechnology Co., Ltd.) was used to detect the release of lactate dehydrogenase in the cell culture sup...

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Abstract

The invention discloses a caspase-3 inhibitor and a purpose. The caspase-3 inhibitor comprises two molecule forms, core function regions are respectively DMPD (aspartic acid-methionine-proline-aspartic acid) and DMLD (aspartic acid-methionine-leucine-aspartic acid) polypeptide fragments, and two ends are respectively subjected to chemical modification to be used for improving biological characteristics, activity and druggability of polypeptide. The inhibitor can directly be combined with active caspase-3 to restrain enzymic activity, and effectively restrain caspase-3 / PARP mediated apoptosis and apoptosis necrosis, and caspase-3 / GSDME mediated cell apoptosis. The caspase-3 inhibitor can be used for preparing cell death resistant relevant tool molecules or reagents, and powerful medicines for resisting cell death or histoorgan apoptosis relevant diseases.

Description

technical field [0001] The invention relates to a caspase-3 inhibitor and its use. Background technique [0002] Cell death, as the core event in liver tissue organ failure, runs through the whole process of liver tissue failure, and it is also the cause of myocardial infarction (Acute myocardial infarction and heart failure: Role of apoptosis. Int J Biochem Cell B, 2006.38, 1834-1840) , the common cause of many diseases such as chronic renal failure (Apoptosis inglomerular sclerosis.Kidney Int, 1993.49, 103-111). For a long time, the high mortality rate caused by complications such as coagulation mechanism disorder, jaundice, hepatic encephalopathy and ascites caused by liver function injury has remained high. At present, clinical treatment for liver failure includes antiviral therapy (Hepatitis B virus associated hepatic failure) (Antiviraltherapy for hepatitis B virus associated hepatic failure. Hepatob Pancreat Dis8, 2009.17-24), hormone therapy (glucocorticoid) (Import...

Claims

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Application Information

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IPC IPC(8): C07K5/113A61K38/07A61P1/16
CPCA61K38/00A61P1/16C07K5/1021
Inventor 郝海平曹丽娟许万峰张权
Owner CHINA PHARM UNIV
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