Modified proteases that inhibit complement activation

Abstract

Provided are methods for and compounds for modulating the complement system. In particular, compounds are provided that inhibit complement activation and compounds are provided that promote complement activation. The compounds are therapeutics by virtue of their effects on the complement system. Hence, the compounds that inhibit complement activation can be used for treatment of ischemic and reperfusion disorders, including myocardial infarction and stroke, sepsis, autoimmune diseases, inflammatory diseases and diseases with an inflammatory component, including Alzheimer's Disease and other neurodegenerative disorder.

1 . A method of modulating complement activation, comprising contacting a non-complement protease with a target substrate(s) of a complement pathway, wherein:
 the modified non-complement protease, or a catalytically active portion thereof, comprises modifications in a scaffold protease, or a catalytically active portion of a scaffold protease, at an amino acid residue that is a determinant of substrate specificity, whereby the modified amino acid residue(s) increases one or both of specificity of the modified non-complement protease for the target substrate(s) of a complement pathway compared to the scaffold protease;   the scaffold protease is an unmodified protease that is not part of a complement pathway; and   the modified non-complement protease, or a catalytically active portion thereof, cleaves the target substrate(s) of a complement pathway such that complement activation in a pathway comprising the target substrate is modulated.   
     
     
         2 . The method of  claim 1 , wherein complement activation is inhibited. 
     
     
         3 . The method of  claim 1 , wherein the target substrate is selected from among C1q, C2, C3, iC3, C4, iC4, C5, C6, C7, C8, C9, MBL, Factor B, Factor D, Factor P, MASP-1, MASP-2, C1r, C1s, C4b, C4a, C2b, C2a, C3b, C3a, Ba and Bb. 
     
     
         4 . The method of  claim 1 , wherein the scaffold protease is a serine protease, a cysteine protease, an aspartic protease, a threonine protease, or a metallo-protease. 
     
     
         5 . The method of  claim 4 , wherein the scaffold protease is selected from among a granzyme B, granzyme A, granzyme M, cathepsin G, MT-SP1, neutrophil elastase, chymase, alpha-tryptase, beta-tryptase I or II, chymotrypsin, collagenase, factor XII, factor XI, factor CII, factor X, thrombin, protein C, u-plasminogen activator (u-PA), t-plasminogen activator (t-PA), plasmin, plasma kallikrein, chymotrypsin, trypsin, a cathepsin, papain, cruzain, a metalloprotease and allelic variants, isoforms and catalytically active portions thereof. 
     
     
         6 . The method of  claim 1 , wherein the scaffold protease is an MT-SP1 protease or catalytically active portion thereof and the MT-SP1 protease or a catalytically active portion thereof comprises a sequence of amino acids set forth in SEQ ID NO: 2 or 10 or a sequence that exhibits at least 90% sequence identity to SEQ ID NO:2 or 10. 
     
     
         7 . The method of  claim 6 , wherein the scaffold protease is an MT-SP1 protease and the MT-SP1 protease consists of the sequence of amino acids set forth in SEQ ID NO:2 or a sequence that exhibits at least 90% sequence identity to SEQ ID NO:2. 
     
     
         8 . The method of  claim 6 , wherein the scaffold protease is a catalytically active portion of an MT-SP1 protease and the catalytically active portion consists of the sequence of amino acids set forth in SEQ ID NO:10 or a sequence that exhibits at least 90% sequence identity to SEQ ID NO:10. 
     
     
         9 . The method of  claim 6 , wherein target substrate is C2, C3 or C4. 
     
     
         10 . The method of  claim 6 , wherein the MT-SP1 protease or a catalytically active portion thereof comprises modification(s) at a position selected from among positions 41, 60c, 96, 97, 99, 143, 146, 147, 151, 172, 174, 175, 180, 192, 215, 217, 221a, and 224, based on chymotrypsin numbering. 
     
     
         11 . The method of  claim 10 , wherein the MT-SP1 protease or a catalytically active portion thereof comprises modification(s) selected from among I41T, I41A, I41L, I41F, I41D, I41E, R60 C D, R60 C W, D96A, D96V, D96F, D96S, D96T, F97N, F97D, F97E, F97A, F97W, F99A, F99S, F99G, F99Y, F99W, H143V, Y146N, Y146D, Y146E, Y146A, Y146W, Y146R, Y146F, G147E, G151L, L172N, Q174H, Q174A, Q174V, Q174F, Q174R, Q174K, Q174L, Q174Y, Q174V, Q175D, Q175E, Q175H, Q175L, Q175F, Q175W, Q175Y, Q175R, Q175K, M180E, Q192A, Q192R, Q192V, Q192F, Q192L, Q1921, Q192E, Q192K, Q192Y, W215F, W215Y, D217F, D217A, D217Q, D217N, D217H, Q221 a D, Q221 a L, Q221 a E, K224A, K224L, K224R, K224N, K224T, K224Y, K224S and K224F, based on chymotrypsin numbering. 
     
     
         12 . The method of  claim 6 , wherein the MT-SP1 protease or a catalytically active portion thereof comprises at least two modifications at a position selected from among 41, 60c, 96, 97, 99, 143, 146, 147, 151, 172, 174, 175, 192, 215, 217, 221a, and 224, based on chymotrypsin numbering. 
     
     
         13 . The method of  claim 12 , wherein the MT-SP1 protease or a catalytically active portion thereof comprises modifications selected from among Y146E/K224N, I41T/Y146E/Q175D/K224R, I41T/Y146D/K224F, I41T/Y146E/Q175D/K224N, I41T/Y146E/G151L/Q175D/K224L, Y146E/Q221aE/K224F, I41T/Y146E/G151L/Q175D/K224R, I41T/Y146E/G151L/Q175D/K224N, Y146E/K224R, Y146E/Q175D/K224N, Y146D/K224R, I41T/Y146E/G151L/Q175D/K224F, Y146E/Q175D/K224R, Y146E/K224L, Y146D/Q175D/K224R, Y146D/Q175L/K224L, Y146D/Q175W/K224L, Y146D/K224L, Y146E/Q221aE/K224R, Y146E/K224A, Y146D/Q175H/K224L, Y146D/Q175Y/K224L, Y146E/K224Y, Y146D/Q175F/K224L, Y146D/Q221aL/K224S, I41E/Y146D/K224L, Y146D/D217F/K224L, H143V/Y146D/K224F, Y146E/K224F, Y146A/K224F, Y146E/K224T, I41T/Y146E/K224L, I41F/Y146D/K224F, I41L/Y146D/K224F, I41T/Y146D/G151L/K224F, I41A/Y146D/K224F, I41E/Y146D/K224F, I41D/Y146D/K224L, I41D/Y146D/K224F, Y146N/K224F, I41T/Y146D/Q175D/K224F, Y146D/Q192A/K224F, I41T/Y146D/Q175D/K224L, I41T/Y146D/Q175D/K224R, I41T/Y146D/Q175D/K224N, I41T/Y146D/G151L/Q175D/K224F, I41T/Y146D/G151L/Q175D/K224L, I41T/Y146D/G151L/Q175D/K224R, I41T/Y146D/G151L/Q175D/K224N, I41T/Y146E/Q175D/K224F, I41T/Y146E/Q175D/K224L, I41T/Y146D/G151L/K224N, Y146D/Q175D/K224N, Y146D/G151L/K224N, Y146D/Q175R/K224N, Y146D/Q175K/K224N, Y146D/Q175H/K224N, I41T/Y146D/G151L/Q175K/K224F, I41T/Y146D/G151L/Q175R/K224F, I41T/Y146D/G151L/Q175H/K224F, I41T/Y146D/G151L/Q175Y/K224F, I41T/Y146D/G151L/Q175K/K224N, I41T/Y146D/G151L/Q175R/K224N, I41T/Y146D/G151L/Q175H/K224N Y146D/K224N, I41T/Y146D/K224L, I41T/Y146E/G151L/Q175D/K224N, I41T/Y146D/K224N and I41T/Y146D/G151L/Q175Y/K224N, based on chymotrypsin numbering. 
     
     
         14 . The method of  claim 1 , wherein:
 the modified-non complement protease is administered to a subject having a complement-mediated disease or disorder, whereby contacting the non-complement protease with a target substrate(s) of a complement pathway occurs in vivo upon administration; and   inhibition of complement activation leads to a reduction of inflammatory symptoms associated with the complement-mediated disorder.   
     
     
         15 . The method of  claim 14 , wherein the complement-mediated disorder is selected from among an inflammatory disorder, a neurodegenerative disorder and a cardiovascular disorder. 
     
     
         16 . The method of  claim 14 , wherein the wherein the complement-mediated disorder is selected from among sepsis, Rheumatoid arthritis (RA), membranoproliferative glomerulonephritis (MPGN), Multiple Sclerosis (MS), Myasthenia gravis (MG), asthma, inflammatory bowel disease, immune complex (IC)-mediated acute inflammatory tissue injury, Alzheimer's Disease (AD), and Ischemia-reperfusion injury. 
     
     
         17 . The method of  claim 14 , wherein the complement-mediated disorder is Guillan-Barre syndrome. 
     
     
         18 . The method of  claim 16 , wherein the complement-mediated disorder is ischemia-reperfusion injury and the ischemia-reperfusion injury is caused by an event or treatment selected from among myocardial infarct (MI), stroke, angioplasty, coronary artery bypass graft, cardiopulmonary bypass (CPB), and hemodialysis. 
     
     
         19 . The method of  claim 14 , wherein the modified non-complement protease or a catalytically active portion thereof is formulated for administration in combination with a second agent for treating the complement-mediated disorder. 
     
     
         20 . The method of  claim 19 , wherein the second agent is an anti-inflammatory agent or an anticoagulant. 
     
     
         21 . The method of  claim 19 , wherein the second agent is selected from among any one or more of a NSAID, antimetabolite, corticosteroid, analgesic, cytotoxic agent, pro-inflammatory cytokine inhibitor, anti-inflammatory cytokines, B cell targeting agents, compounds targeting T antigens, adhesion molecule blockers, chemokines receptor antagonists, kinase inhibitors, PPAR-γ ligands, complement inhibitors, heparin, warfarin, acenocoumarol, phenindione, EDTA, citrate, oxalate, argatroban, lepirudin, bivalirudin, and ximelagatran.