Method for screening inhibitors of interleukin-32 (IL-32)
The present invention relates to methods of screening for modulators of interleukin 32 (IL-32), to modulators of IL-32 and to their use.
1. A method of screening for an inhibitor of activation of interleukin 32 (IL-32) based on the proteolytic activity of proteinase 3 (PR-3) on IL-32, the method comprising the steps of
(a) contacting a composition comprising an IL-32 with protein PR3 in the presence of a test agent;
(b) detecting the presence of an IL-32 proteolytic fragment; and
(c) identifying the test agent as the inhibitor of activation of IL-32 when the presence of the proteolytic fragment is less and when presence of the intact IL-32 protein is more than that in a control without the test agent.
2. The method of claim 1 , wherein the detection is performed using SDS-PAGE.
3. The method according to claim 1 , wherein the intact IL-32 protein is about 20 kDa and wherein said IL-32 proteolytic fragment is about 16 kDa.
4. The method according to claim 1 , wherein the intact IL-32 protein is about 20 kDa and wherein said IL-32 proteolytic fragment is about 13 kDa.
5. The method of claim 1 , further comprising assaying the inhibiting effect of the identified inhibitor of activation of IL-32 in IL-32 induced secretion of a cytokine, which comprises contacting the IL-32 composition and PR-3 with IL-32 responsive T-cell or macrophage cell in a culture medium in the presence of the inhibitor, measuring a cytokine concentration in the culture medium, and comparing the cytokine concentration in the culture medium to the cytokine concentration in a control culture medium absent said inhibitor, wherein lower cytokine concentration is detected in the culture medium comprising the inhibitor as compared to the control.
6. The method of claim 5 wherein the cytokine is selected from tumor necrosis factor alpha, Interleukin-8, and macrophage inflammatory protein-2.