Assay method for screening for inhibitors of proTNF conversion
AssigneeChiron Corporation
InventorsRobert F. Halenbeck, Michael Kriegler, Carl Perez, David A. Jewell, Kirston E. Koths
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Abstract
Methods and materials are disclosed for the production of purified, active recombinant human neutrophil protease, PR-3, via activation of a pro-form herein referred to as proPR-3. Human PR-3 is useful for discovering inhibitors of excessive release of mature, active TNF.alpha.. Also disclosed are methods for the identification of inhibitors of the conversion of the pro-form of TNF.alpha. to its mature active form.
Claims2
A method of screening for a prophylactic or therapeutic medicament for a disease caused by, exacerbated by, or associated with the production of a mature tumor necrosis factor (TNF.alpha.), from a 26 kD proTNF.alpha., the method comprising the steps of:
- (a) contacting a 26 kD proTNF.alpha. or a proteolytically cleavable fragment thereof with a TNF.alpha. convertase from HL60 cells in an amount effective for cleaving the pro-TNF.alpha. or the proteolytically cleavable fragment, said proTNF.alpha. or said proteolytically cleavable fragment having the sequence:
- Gln-Ala-Val-Arg-Ser-Ser (SEQ ID NO:
- 24), within which proteolytic cleavage by TNF.alpha. convertase occurs;
- (b) measuring either conversion of the proTNF.alpha. to the mature TNF.alpha. or cleavage of the cleavable fragment in step (a);
- (c) repeating steps (a) and (b) further including a molecule sought to be identified as a prophylactive or a therapeutic medicament for diseases caused by the mature TNF.alpha.;
- (d) measuring conversion of the proTNF.alpha. to mature TNF.alpha. or cleavage of the cleavable fragment in step (c);
- and
- (e) comparing the conversion or the cleavage measured in step (b) with the conversion or the cleavage measured in step (d), wherein lesser cleavage in step (d) than in step (b) suggests that the molecule is potentially a prophylactic or therapeutic medicament for diseases caused by mature TNF.alpha..
Dependencies are inferred from the claim text.