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Novel ligands and methods for preparing same

a technology of ligands and receptors, applied in the field of modified ligands, can solve the problem of lack of rational design methods currently available for rationally designing ligands to irreversibly bind a target receptor

Inactive Publication Date: 2001-12-13
NAT UNIV OF SINGAPORE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

0049] In this respect, the cell is preferably an animal cell, more preferably a mammalian cell, and more preferably a human cell. Alternatively, th

Problems solved by technology

Several other enzymes are inhibited by covalent attachment of the inhibitor, giving rise to irreversibility.
Despite the availability of several agents that bind their targets irreversibly, there is a dearth of methods currently available for rationally designing ligands to irreversibly bind a target receptor.

Method used

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  • Novel ligands and methods for preparing same
  • Novel ligands and methods for preparing same
  • Novel ligands and methods for preparing same

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0189] Effects of N-ethylmaleimide on the Equilibrative Nucleoside Transporter in Murine Myeloma SP2 / 0-Ag14 Cells.

[0190] Exemplary N-maleimide derivatives were used as a sulfhydryl reagent that is advantageously specific to the sulfhydryl group, reacting only with certain accessible sulfhydryl groups on the proteins, making it possible for specific inhibitions, and good penetration into cells due to the uncharged nature of the compound. N-Ethylmaleimide (NEM) is the smallest maleimide reagent capable of forming stable thio esters with the reactive sulfhydryl groups of proteins. The sensitivity of es and ei nucleoside transport systems towards NEM was demonstrated. Various different cell lines were used to show that the sensitivity of nucleoside transport system(s) toward NEM is a general phenomenon and not restricted to certain cell or tissues types.

[0191] Previous studies of the es nucleoside transporter system (Paterson et al. (1977), Mol. Pharmacol., vol. 13, pp. 114; Gati et al....

example 2

[0195] Synthesis and Characterization of CrMCC

[0196] The strategy to selectively irreversibly inhibit the es transporter protein is to attach a reactive group specific covalent binding agent (maleimide) to a driver so that it can deliver the covalent binding agent to the desired target. The most suitable driver will be the physiological ligand itself (nucleoside). Cytidine, a pyrimidine nucleoside, is selected among other physiological nucleosides due to its "function inertness", thus minimizes "non-specific" drug binding. As for the spacer arm that links the maleimide to the cytidine, a cyclohexane carboxylic acid is chosen for pilot studies. This configuration is to mimic the chemical structure of NBMPR (FIG. 5b). Furthermore, other advantages like hydrophobicity provided by cyclohexane) and stability (provided by carboxylic acid) are also taking into consideration.

[0197] A direct, rapid synthetic route used to synthesize CrMCC or 1-[[4-[(4-amino-1-.beta.-D-ribofuranosyl-2(1H)-pyr...

example 3

[0201] Effects of CrMCC on the Binding of .sup.3H-NBMPR in Human HL-60 Promyelocytic Leukemia Plasma Membranes

[0202] To synthesize .sup.3H-CrMCC, radioactive cytidine (.sup.3H-cytidine) was used. Since high concentration of substrate increases the yield of CrMCC, nonradioactive cytidine was pre-mixed with radioactive .sup.3H-cytidine at a concentration ratio of 100:1 prior to reaction with SMCC (see Example 2).

[0203] Purified HL-60 plasma membranes suspended in reaction buffer (0.13 M NaCl, 0.02 M NaHCO.sub.3, pH 7.0) were pretreated with graded concentrations of cytidine, SMCC, and CrMCC for 5 min prior exposed to .sup.3H-NBMPR (5 .mu.M) for additional 30 min. The reaction was terminated by membrane filtration method (Lee & Jarvis (1988), Biochem. J., vol 249, pp. 557-564). The data shown in FIG. 6 were corrected for non-specific binding determined in the presence of 20 .mu.M of NBTGR. FIG. 6 shows the specific of .sup.3H-NBMPR to HL-60 unsealed plasma membranes was inhibited by Cr...

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Abstract

A process is disclosed for modifying a parent ligand by attaching to the parent ligand a conjugation agent that is reactive with a moiety of a target receptor to which the parent ligand binds such that a covalent bond is formable between the conjugation agent and the receptor moiety. Also disclosed are compositions, probes and methods of detecting and / or quantifying receptors using the modified ligands of the invention.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 60 / 178,756, filed Jan. 28, 2000, the disclosure of which is incorporated herein by reference in its entirety.TECHNICAL FIELD[0002] This invention relates generally to ligand-receptor interactions. In particular, the present invention relates to modified ligands that bind irreversibly to their cognate receptors, and to methods for preparing such ligands. The novel ligands of the invention have utility inter alia for investigating protein function and as drugs (in healthcare, agricultural and environmental applications) for more effectively inhibiting or stimulating cognate receptor function.BACKGROUND ART[0003] Pharmacological receptors are intracellular or membrane-bound proteins which produce a pharmacological effect after binding with a specific ligand. In this regard, a pharmacological receptor has a dual function to (a) detect a ligand signal by forming ...

Claims

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Application Information

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IPC IPC(8): A61K47/48C07D403/06C07D403/12C07D405/14C07D473/34G01N33/531G01N33/566G01N33/68
CPCA61K47/48023A61K47/48092C07D403/04C07D403/06C07D403/12C07D405/14G01N33/6842C07H19/06C07H19/16G01N33/531G01N33/566G01N33/6803C07D473/34A61K47/54A61K47/549
Inventor LEE, CHEE WEE
Owner NAT UNIV OF SINGAPORE