Methods for identifying inhibitors of mannan-binding lectin associated serine protease (MASP) proteins and uses thereof

A method of screening for one or more small molecules capable of inhibiting MBL-associated serine protease (MASP) protein activity, the method comprising:

• (a) contacting a MASP protein, or a portion thereof comprising a CUB1 or a CUB2 domain, with at least one or more small molecule(s) in the presence of a polypeptide comprising at least one collagen-like binding domain of a mannose-binding lectin (MBL) or ficolin, and Ca2+, wherein the collagen-like binding domain comprises one or more of the amino acid sequence OGKXGP (SEQ ID NO:3), wherein O is hydroxyproline and X is an aliphatic amino acid residue or methionine residue;
• (b) performing an assay to determine the affinity of Ca2+-dependent binding between the MASP protein, or portion thereof comprising a CUB1 or a CUB2 domain, and the polypeptide comprising the at least one collagen-like binding domain comprising one or more of the amino acid sequence OGKXGP (SEQ ID NO:3), wherein O is hydroxyproline and X is an aliphatic amino acid residue or methionine residue, identifying small molecule(s) that reduce the affinity of Ca2+-dependent binding between the MASP protein or the portion thereof comprising a CUB1 or a CUB2 domain and the polypeptide comprising the collagen-like binding domain of MBL or ficolin;
• and
• (c) determining the level of MASP protein activity in the presence and absence of the small molecule(s) identified in accordance with step (b);
• wherein reduced MASP protein activity determined in the presence of the small molecule in step (c) compared to the level of MASP protein activity determined in the absence of the small molecule indicates the capacity of the small molecule to inhibit MASP protein activity.