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Methods and compositions for detecting receptor ligand mimetics

A technology for receptors and ligands, which is used in the field of detection of receptor ligand mimics and compositions, and can solve problems such as low activity

Inactive Publication Date: 2009-12-16
CELLPROTECT PATENT AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although no data were presented to support this claim, the reported dose would not reflect the low activity of AP-121 found in other in vitro assays

Method used

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  • Methods and compositions for detecting receptor ligand mimetics
  • Methods and compositions for detecting receptor ligand mimetics
  • Methods and compositions for detecting receptor ligand mimetics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Screening steps

[0086] In order to verify the practical applicability of the method, as an example, we chose the FAS receptor to find its small activating ligand, the agonistic antibody CH-11 as a suitable receptor ligand, and AP-121 as the putative CH-11 Simulant. The known drugs camptothecin, temozolomide, doxorubicin and Tarceva were selected as a group of other compounds for combined measurement.

[0087] As the biological test group, we selected the brain tumor cell lines U87MG, A172, LN-18, LN-229, U118MG and T98G, which have different levels of mutations in p53 and PTEN and FAS receptor expression:

[0088] #

Cell line

p53

pTEN

FasL

Fas

ATCC#

1

2

3

4

5

6

U87

A172

LN18

LN299

T98G

U-118MG

Wild type

Wild type

Mutant

Mutant

Mutant

Mutant

Mutant

(Missing)

Mutant

(Missing)

Wild type

Wild type

Mutant ...

Embodiment 2

[0095] Example 2: Cell lines and reagents

[0096] U-118MG (glioblastoma / astrocytoma, p53 mutation, PTEN mutation), T98G (glioblastoma multiforme, p53 mutation, PTEN mutation), A172 (glioblastoma, p53 wild Type, PTEN mutation) and U87MG (glioblastoma / astrocytoma, p53 wild type, PTEN mutation) were purchased from ATCC.

[0097] According to ATCC’s recommendation, U-118MG and A172 were incubated in DMEM medium supplemented with 10% FBS and P / S, and T98G and U87MG were supplemented with 0.1 mM non-essential amino acid solution (Gibco), 10% FBS and P / S. / S in MEM medium. AP-121 was prepared with sterile distilled water as a 5mM stock solution; Temozolomide (TMZ) (HaoruiPharma-Chem, Inc., Edison, NJ) was prepared with sterile filtered DMSO to make 100mM; Adriamycin (ADR) (Sigma) was used without Bacterial distilled water was prepared as 1 mM; camptothecin (CTP) (Sigma) was prepared as a 10 mM solution with 0.1N NaOH; Tarceva (protein kinase, Germany) was prepared as a 5 mM solution w...

Embodiment 3

[0098] Example 3: WST-1 proliferation analysis

[0099] 2000 cells were plated in each well of a 96-well flat bottom plate and incubated overnight at 37°C and 5% CO2. The growth of plated cells was determined by adding 7.5 μM WST-1 reagent (Roche Applied Sciences, Germany) to 3 control wells, and measuring the absorbance at 650 nm and 450 nm using a SpectraMax250 plate reader, respectively. If the OD650-OD450 value is higher than 0.5, the remaining wells of the plate can be used to incubate with AP-121, other agents or solvents for 96 hours.

[0100]After incubation, add WST-1 reagent to the well, and calculate the OD650-OD450 value as described above. Analyze 6 wells for each condition and determine the standard deviation: all experiments are performed independently at least three times. After elucidating the individual IC50 value of each compound, AP-121 and the chemotherapeutic agent were used for simultaneous combination treatment at their IC50 ratio; the combination index (...

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Abstract

A method to determine the utility of small molecules as functional replacements (mimetics) for protein receptor ligands is described. The method uses cellular biological assays on a systematic array of compounds, comprising known protein receptor ligands and other biologically active molecules to determine if a proposed small molecule is a functional equivalent of a receptor ligand, having therapeutic utility as a pharmaceutically relevant and useful agent either alone or in combination with other molecules.

Description

Invention field [0001] The present invention relates to methods for determining the utility of small molecules as receptor ligand mimics and their therapeutic applications. Background of the invention [0002] Difficulties in finding receptor ligand mimics. Regulating biological functions through agonistic or antagonistic receptor ligands is usually aimed at blocking or mitigating active biochemical pathways, or activating or restoring pathways that are believed to be related to a certain disease or its cause. [0003] Unlike endogenous or biologically heterologous proteins and antibodies, which tend to exhibit highly specific biological activities, small molecules have much lower specificity. Due to their limited size, small molecules bind to a variety of different targets, which can cause unwanted side effects, making it a challenge to develop them into therapeutically useful agents. For example, in order to establish a therapeutically useful dose window, to discover off-target...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50A61K31/66C12Q1/68
CPCG01N33/57423G01N2500/04A61K31/66G01N33/5011G01N33/5041G01N33/57407A61P31/12A61P35/00A61P35/04
Inventor 弗拉迪米尔·卡扎克卢茨·韦伯
Owner CELLPROTECT PATENT AG
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