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• W2933578412 abstract "The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it also contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions. Mannan-binding lectin–associated serine proteinase 2 (MASP-2) is essential for LP activation, and therefore, it is a potential drug target. We have previously developed the first two generations of MASP-2 inhibitors by in vitro evolution of two unrelated canonical serine proteinase inhibitors. These inhibitors were selective LP inhibitors, but their nonhuman origin rendered them suboptimal lead molecules for drug development. Here, we present our third-generation MASP-2 inhibitors that were developed based on a human inhibitor scaffold. We subjected the second Kunitz domain of human tissue factor pathway inhibitor 1 (TFPI1 D2) to directed evolution using phage display to yield inhibitors against human and rat MASP-2. These novel TFPI1-based MASP-2 inhibitor (TFMI-2) variants are potent and selective LP inhibitors in both human and rat serum. Directed evolution of the first Kunitz domain of TFPI1 had already yielded the potent kallikrein inhibitor, Kalbitor® (ecallantide), which is an FDA-approved drug to treat acute attacks of hereditary angioedema. Like hereditary angioedema, acute IRI is also related to the uncontrolled activation of a specific plasma serine proteinase. Therefore, TFMI-2 variants are promising lead molecules for drug development against IRI. The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it also contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions. Mannan-binding lectin–associated serine proteinase 2 (MASP-2) is essential for LP activation, and therefore, it is a potential drug target. We have previously developed the first two generations of MASP-2 inhibitors by in vitro evolution of two unrelated canonical serine proteinase inhibitors. These inhibitors were selective LP inhibitors, but their nonhuman origin rendered them suboptimal lead molecules for drug development. Here, we present our third-generation MASP-2 inhibitors that were developed based on a human inhibitor scaffold. We subjected the second Kunitz domain of human tissue factor pathway inhibitor 1 (TFPI1 D2) to directed evolution using phage display to yield inhibitors against human and rat MASP-2. These novel TFPI1-based MASP-2 inhibitor (TFMI-2) variants are potent and selective LP inhibitors in both human and rat serum. Directed evolution of the first Kunitz domain of TFPI1 had already yielded the potent kallikrein inhibitor, Kalbitor® (ecallantide), which is an FDA-approved drug to treat acute attacks of hereditary angioedema. Like hereditary angioedema, acute IRI is also related to the uncontrolled activation of a specific plasma serine proteinase. Therefore, TFMI-2 variants are promising lead molecules for drug development against IRI. The complement system (CS) 2The abbreviations used are: CScomplement systemAPalternative pathwayCPclassical pathwayIRIischemia-reperfusion injuryLPlectin pathwayMBLmannan-binding lectinMASPMBL-associated serine proteinaseTFPI1tissue factor pathway inhibitorTFPI1 D2the second Kunitz domain of TFPI1TFMITFPI1-based MASP inhibitorPRMpattern recognition moleculeSFTIsunflower trypsin inhibitorNHSnormal human serumAcBSAacetylated BSASPSsodium polyanethole sulfonateTTthrombin timePTprothrombin timeAPTTactivated partial thromboplastin timeTBSTris-buffered saline. is an essential part of innate immunity. It is a network of more than 30 plasma and cell surface proteins that recognizes, labels, and eliminates microbial pathogens and dangerously altered (e.g. apoptotic) self-cells, triggers inflammation, and recruits immune cells (1Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis.Nat. Immunol. 2010; 11 (20720586): 785-79710.1038/ni.1923Crossref PubMed Scopus (2477) Google Scholar, 2Merle N.S. Church S.E. Fremeaux-Bacchi V. Roumenina L.T. Complement system part I—molecular mechanisms of activation and regulation.Front. Immunol. 2015; 6 (26082779): 26210.3389/fimmu.2015.00262Crossref PubMed Scopus (845) Google Scholar3Merle N.S. Noe R. Halbwachs-Mecarelli L. Fremeaux-Bacchi V. Roumenina L.T. Complement system part II—role in immunity.Front. Immunol. 2015; 6 (26074922): 25710.3389/fimmu.2015.00257Crossref PubMed Scopus (583) Google Scholar). complement system alternative pathway classical pathway ischemia-reperfusion injury lectin pathway mannan-binding lectin MBL-associated serine proteinase tissue factor pathway inhibitor the second Kunitz domain of TFPI1 TFPI1-based MASP inhibitor pattern recognition molecule sunflower trypsin inhibitor normal human serum acetylated BSA sodium polyanethole sulfonate thrombin time prothrombin time activated partial thromboplastin time Tris-buffered saline. The CS can be activated through three pathways. The classical pathway (CP) is activated primarily by immune complexes, but it can also recognize microbial surfaces and apoptotic and necrotic cells; it contributes to the elimination of unnecessary synapses during ontogenesis; and it is important for the clearance of immune complexes and cell debris (4Nayak A. Ferluga J. Tsolaki A.G. Kishore U. The non-classical functions of the classical complement pathway recognition subcomponent C1q.Immunol. Lett. 2010; 131 (20381531): 139-15010.1016/j.imlet.2010.03.012Crossref PubMed Scopus (85) Google Scholar, 5Presumey J. Bialas A.R. Carroll M.C. Complement system in neural synapse elimination in development and disease.Adv. Immunol. 2017; 135 (28826529): 53-7910.1016/bs.ai.2017.06.004Crossref PubMed Scopus (151) Google Scholar). The lectin pathway (LP) recognizes ancient surface-exposed molecular determinants on microbes via a diverse set of pattern recognition molecules (PRMs) and provides immediate defense against microbial pathogens, which does not depend on specific antibodies (6Garred P. Genster N. Pilely K. Bayarri-Olmos R. Rosbjerg A. Ma Y.J. Skjoedt M.-O. A journey through the lectin pathway of complement-MBL and beyond.Immunol. Rev. 2016; 274 (27782323): 74-9710.1111/imr.12468Crossref PubMed Scopus (232) Google Scholar). The alternative pathway (AP) continuously challenges all surfaces by spontaneous low-level activation, but it activates productively only on those that lack protecting complement regulator molecules. Additionally, the AP provides an important amplification loop for complement activation (7Harrison R.A. The properdin pathway: an “alternative activation pathway” or a “critical amplification loop” for C3 and C5 activation?.Semin. Immunopathol. 2018; 40 (29167939): 15-3510.1007/s00281-017-0661-xCrossref PubMed Scopus (43) Google Scholar, 8Lachmann P.J. Looking back on the alternative complement pathway.Immunobiology. 2018; 223 (29525356): 519-52310.1016/j.imbio.2018.02.001Crossref PubMed Scopus (27) Google Scholar). Danger signal recognition triggers the activation of pathway-specific serine proteinase zymogens. The activated proteinases cleave downstream complement components that form surface-bound C3 convertases: C4b2a for the CP and the LP and C3bBb for the AP. At this point, the three activation pathways converge to a common effector route leading to the labeling and lysis of the pathogens, recruitment of immune cells, and triggering of inflammation. Normally, complement activation is tightly regulated (9Zipfel P.F. Skerka C. Complement regulators and inhibitory proteins.Nat. Rev. Immunol. 2009; 9 (19730437): 729-74010.1038/nri2620Crossref PubMed Scopus (896) Google Scholar). Lack of complement inhibition is responsible for the pathomechanism of paroxysmal nocturnal hemoglobinuria, and inappropriate complement activation contributes to the development of diseases such as ischemia-reperfusion injury (IRI), rheumatoid arthritis, age-related macular degeneration, and neurodegenerative diseases including Alzheimer’s disease. Therefore, there is a great need for potent and specific anti-complement drugs that could provide targeted therapies for these complement-related diseases (10Dobó J. Kocsis A. Gál P. Be on target: strategies of targeting alternative and lectin pathway components in complement-mediated diseases.Front. Immunol. 2018; 9 (30135690)185110.3389/fimmu.2018.01851Crossref PubMed Scopus (46) Google Scholar). Whereas many anti-complement compounds are under development (11Ricklin D. Mastellos D.C. Reis E.S. Lambris J.D. The renaissance of complement therapeutics.Nat. Rev. Nephrol. 2018; 14 (29199277): 26-4710.1038/nrneph.2017.156Crossref PubMed Scopus (248) Google Scholar), there are only two molecules that have been approved for clinical use: the anti-C5 antibody Soliris® (eculizumab) and C1 inhibitor. Even of these two, only eculizumab is a dedicated complement-targeted drug. One of the most promising candidates among complement-targeted lead molecules is compstatin and its derivatives (12Berger N. Alayi T.D. Resuello R.R.G. Tuplano J.V. Reis E.S. Lambris J.D. New analogs of the complement C3 inhibitor compstatin with increased solubility and improved pharmacokinetic profile.J. Med. Chem. 2018; 61 (29920096): 6153-616210.1021/acs.jmedchem.8b00560Crossref PubMed Scopus (21) Google Scholar). These compounds effectively block the interaction of the C3 convertases with C3 to inhibit erroneous complement activation. In most complement-related diseases, the contribution of only one of the three pathways is dominant. The CP participates in Alzheimer’s disease (13Hong S. Beja-Glasser V.F. Nfonoyim B.M. Frouin A. Li S. Ramakrishnan S. Merry K.M. Shi Q. Rosenthal A. Barres B.A. Lemere C.A. Selkoe D.J. Stevens B. Complement and microglia mediate early synapse loss in Alzheimer mouse models.Science. 2016; 352 (27033548): 712-71610.1126/science.aad8373Crossref PubMed Scopus (1547) Google Scholar) and myasthenia gravis (14Tüzün E. Christadoss P. Complement associated pathogenic mechanisms in myasthenia gravis.Autoimmun. Rev. 2013; 12 (23537510): 904-91110.1016/j.autrev.2013.03.003Crossref PubMed Scopus (82) Google Scholar), the LP contributes to IRI of various tissues (15Hart M.L. Ceonzo K.A. Shaffer L.A. Takahashi K. Rother R.P. Reenstra W.R. Buras J.A. Stahl G.L. Gastrointestinal ischemia-reperfusion injury is lectin complement pathway dependent without involving C1q.J. Immunol. 2005; 174 (15879138): 6373-638010.4049/jimmunol.174.10.6373Crossref PubMed Scopus (176) Google Scholar16Schwaeble W.J. Lynch N.J. Clark J.E. Marber M. Samani N.J. Ali Y.M. Dudler T. Parent B. Lhotta K. Wallis R. Farrar C.A. Sacks S. Lee H. Zhang M. Iwaki D. Takahashi M. Fujita T. Tedford C.E. Stover C.M. Targeting of mannan-binding lectin-associated serine protease-2 confers protection from myocardial and gastrointestinal ischemia/reperfusion injury.Proc. Natl. Acad. Sci. U.S.A. 2011; 108 (21502512): 7523-752810.1073/pnas.1101748108Crossref PubMed Scopus (145) Google Scholar, 17Asgari E. Farrar C.A. Lynch N. Ali Y.M. Roscher S. Stover C. Zhou W. Schwaeble W.J. Sacks S.H. Mannan-binding lectin-associated serine protease 2 is critical for the development of renal ischemia reperfusion injury and mediates tissue injury in the absence of complement C4.FASEB J. 2014; 28 (24868011): 3996-400310.1096/fj.13-246306Crossref PubMed Scopus (67) Google Scholar18Orsini F. Chrysanthou E. Dudler T. Cummings W.J. Takahashi M. Fujita T. Demopulos G. De Simoni M.-G. Schwaeble W. Mannan binding lectin-associated serine protease-2 (MASP-2) critically contributes to post-ischemic brain injury independent of MASP-1.J. Neuroinflammation. 2016; 13 (27577570): 21310.1186/s12974-016-0684-6Crossref PubMed Scopus (42) Google Scholar), and the AP plays a significant role in age-related macular degeneration (19Tan P.L. Bowes Rickman C. Katsanis N. AMD and the alternative complement pathway: genetics and functional implications.Hum. Genomics. 2016; 10 (27329102): 2310.1186/s40246-016-0079-xCrossref PubMed Scopus (45) Google Scholar) and atypical hemolytic uremic syndrome (20Le Quinterec M. Roumenina L. Noris M. Frémeaux-Bacchi V. Atypical hemolytic uremic syndrome associated with mutations in complement regulator genes.Semin. Thromb. Hemost. 2010; 36 (20865641): 641-65210.1055/s-0030-1262886Crossref PubMed Scopus (39) Google Scholar). Pathway-specific inhibitors should be useful tools for academic research to identify individual roles of the three pathways in physiologic and pathologic processes and ideal therapeutics that selectively block the derailed pathologic pathway while leaving the protecting functions of the other two pathways undisturbed. Pathway-specific proteinases are obvious targets of pathway-selective drug development. However, as most plasma serine proteinases have trypsin-like substrate specificity, selectively targeting a single complement proteinase is a formidable challenge. Small molecules that target only the active site are rarely monospecific. Fragment-based drug discovery (21Murray C.W. Rees D.C. The rise of fragment-based drug discovery.Nat. Chem. 2009; 1 (21378847): 187-19210.1038/nchem.217Crossref PubMed Scopus (518) Google Scholar) has been successfully applied to develop highly selective small-molecule inhibitors against factor D, a key enzyme of the AP (22Maibaum J. Liao S.-M. Vulpetti A. Ostermann N. Randl S. Rüdisser S. Lorthiois E. Erbel P. Kinzel B. Kolb F.A. Barbieri S. Wagner J. Durand C. Fettis K. Dussauge S. et al.Small-molecule factor D inhibitors targeting the alternative complement pathway.Nat. Chem. Biol. 2016; 12 (27775713): 1105-111010.1038/nchembio.2208Crossref PubMed Scopus (57) Google Scholar). These compounds target factor D in its unique, self-inhibited conformation that is characteristically different from other proteinases. Proteins such as mAbs and canonical serine proteinase inhibitors have much larger interacting surfaces and have therefore greater potential to provide monospecificity. In the last decade we have developed the first selective LP inhibitors by directed evolution of canonical serine proteinase inhibitors (23Kocsis A. Kékesi K.A. Szász R. Végh B.M. Balczer J. Dobó J. Závodszky P. Gál P. Pál G. Selective inhibition of the lectin pathway of complement with phage display selected peptides against mannose-binding lectin-associated serine protease (MASP)-1 and -2: significant contribution of MASP-1 to lectin pathway activation.J. Immunol. 2010; 185 (20817870): 4169-417810.4049/jimmunol.1001819Crossref PubMed Scopus (66) Google Scholar, 24Héja D. Harmat V. Fodor K. Wilmanns M. Dobó J. Kékesi K.A. Závodszky P. Gál P. Pál G. Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 are essential for lectin pathway activation and reveal structural plasticity of MASP-2.J. Biol. Chem. 2012; 287 (22511776): 20290-2030010.1074/jbc.M112.354332Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar25Héja D. Kocsis A. Dobó J. Szilágyi K. Szász R. Závodszky P. Pál G. Gál P. Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22691502): 10498-1050310.1073/pnas.1202588109Crossref PubMed Scopus (156) Google Scholar). The first-generation LP inhibitors were based on the 14-amino acid sunflower trypsin inhibitor (SFTI) scaffold, resulting in SFMI-1 and SFMI-2 (23Kocsis A. Kékesi K.A. Szász R. Végh B.M. Balczer J. Dobó J. Závodszky P. Gál P. Pál G. Selective inhibition of the lectin pathway of complement with phage display selected peptides against mannose-binding lectin-associated serine protease (MASP)-1 and -2: significant contribution of MASP-1 to lectin pathway activation.J. Immunol. 2010; 185 (20817870): 4169-417810.4049/jimmunol.1001819Crossref PubMed Scopus (66) Google Scholar). The second-generation compounds were developed on the scaffold of the 35-amino acid Schistocerca gregaria proteinase inhibitor 2 (SGPI-2), yielding SGMI-1 and SGMI-2 (24Héja D. Harmat V. Fodor K. Wilmanns M. Dobó J. Kékesi K.A. Závodszky P. Gál P. Pál G. Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 are essential for lectin pathway activation and reveal structural plasticity of MASP-2.J. Biol. Chem. 2012; 287 (22511776): 20290-2030010.1074/jbc.M112.354332Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 25Héja D. Kocsis A. Dobó J. Szilágyi K. Szász R. Závodszky P. Pál G. Gál P. Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22691502): 10498-1050310.1073/pnas.1202588109Crossref PubMed Scopus (156) Google Scholar). With these inhibitors, we revealed that both MASP-1 and MASP-2 are essential for LP activity in human serum (23Kocsis A. Kékesi K.A. Szász R. Végh B.M. Balczer J. Dobó J. Závodszky P. Gál P. Pál G. Selective inhibition of the lectin pathway of complement with phage display selected peptides against mannose-binding lectin-associated serine protease (MASP)-1 and -2: significant contribution of MASP-1 to lectin pathway activation.J. Immunol. 2010; 185 (20817870): 4169-417810.4049/jimmunol.1001819Crossref PubMed Scopus (66) Google Scholar, 24Héja D. Harmat V. Fodor K. Wilmanns M. Dobó J. Kékesi K.A. Závodszky P. Gál P. Pál G. Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 are essential for lectin pathway activation and reveal structural plasticity of MASP-2.J. Biol. Chem. 2012; 287 (22511776): 20290-2030010.1074/jbc.M112.354332Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar25Héja D. Kocsis A. Dobó J. Szilágyi K. Szász R. Závodszky P. Pál G. Gál P. Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22691502): 10498-1050310.1073/pnas.1202588109Crossref PubMed Scopus (156) Google Scholar), and therefore, both enzymes are promising targets for drug development. However, MASP-1 has several functions outside the LP (26Megyeri M. Makó V. Beinrohr L. Doleschall Z. Prohászka Z. Cervenak L. Závodszky P. Gál P. Complement protease MASP-1 activates human endothelial cells: PAR4 activation is a link between complement and endothelial function.J. Immunol. 2009; 183 (19667088): 3409-341610.4049/jimmunol.0900879Crossref PubMed Scopus (102) Google Scholar27Dobó J. Major B. Kékesi K.A. Szabó I. Megyeri M. Hajela K. Juhász G. Závodszky P. Gál P. Cleavage of kininogen and subsequent bradykinin release by the complement component: mannose-binding lectin-associated serine protease (MASP)-1.PLoS One. 2011; 6 (21625439)e2003610.1371/journal.pone.0020036Crossref PubMed Scopus (88) Google Scholar, 28Jenny L. Dobó J. Gál P. Pál G. Lam W.A. Schroeder V. MASP-1 of the complement system enhances clot formation in a microvascular whole blood flow model.PLoS One. 2018; 13 (29324883)e019129210.1371/journal.pone.0191292Crossref PubMed Scopus (24) Google Scholar29Paréj K. Kocsis A. Enyingi C. Dani R. Oroszlán G. Beinrohr L. Dobó J. Závodszky P. Pál G. Gál P. Cutting edge: a new player in the alternative complement pathway, MASP-1 is essential for LPS-induced, but not for zymosan-induced, alternative pathway activation.J. Immunol. 2018; 200 (29475986): 2247-225210.4049/jimmunol.1701421Crossref PubMed Scopus (18) Google Scholar), and MASP-2 has a significantly lower plasma concentration than MASP-1 (30Thiel S. Jensen L. Degn S.E. Nielsen H.J. Gál P. Dobó J. Jensenius J.C. Mannan-binding lectin (MBL)-associated serine protease-1 (MASP-1), a serine protease associated with humoral pattern-recognition molecules: normal and acute-phase levels in serum and stoichiometry of lectin pathway components.Clin. Exp. Immunol. 2012; 169 (22670777): 38-4810.1111/j.1365-2249.2012.04584.xCrossref PubMed Scopus (60) Google Scholar). Therefore, MASP-2 might be a better target for the development of highly selective LP inhibitors. Nonhuman origin rendered the previously developed MASP inhibitors suboptimal for subsequent drug development. Therefore, we decided to develop a third generation of MASP-2 inhibitors based on a human scaffold to reduce the risk of immunogenicity in humans. We chose the factor Xa–inhibiting second Kunitz domain of tissue factor pathway inhibitor 1 (TFPI1 D2), as it is normally present in the plasma and because it has already been shown to be a low-affinity inhibitor of MASP-2 (31Keizer M.P. Pouw R.B. Kamp A.M. Patiwael S. Marsman G. Hart M.H. Zeerleder S. Kuijpers T.W. Wouters D. TFPI inhibits lectin pathway of complement activation by direct interaction with MASP-2.Eur. J. Immunol. 2015; 45 (25359215): 544-55010.1002/eji.201445070Crossref PubMed Scopus (24) Google Scholar). Here, we present how we developed TFPI1-based MASP-2 inhibitor (TFMI-2) variants that potently inhibit both human and rat MASP-2, enabling their use in proof-of-concept studies in rats. We followed the same strategy we already applied for developing TFPI1 D2–based MASP-3 inhibitors (32Dobó J. Szakács D. Oroszlán G. Kortvely E. Kiss B. Boros E. Szász R. Závodszky P. Gál P. Pál G. MASP-3 is the exclusive pro-factor D activator in resting blood: the lectin and the alternative complement pathways are fundamentally linked.Sci. Rep. 2016; 6 (27535802)3187710.1038/srep31877Crossref PubMed Scopus (82) Google Scholar). We randomized the P3–P4′ region (33Schechter I. Berger A. On the size of the active site in proteases. I. Papain.Biochem. Biophys. Res. Commun. 1967; 27 (6035483): 157-16210.1016/S0006-291X(67)80055-XCrossref PubMed Scopus (4759) Google Scholar) of TFPI1 D2 (UniProt ID P10646) except the P2 Cys that forms a structurally indispensable disulfide (Fig. S1). The inhibitor-phage library of 5 × 108 clones was selected for binding to the catalytic fragment of human MASP-2 (hMASP-2cf) or that of rat MASP-2 (rMASP-2cf) in separate experiments. Target-binding clones were identified and sequenced to determine sequence patterns that enable binding to hMASP-2cf or rMASP-2cf. Amino acid and DNA sequences of hMASP-2– and rMASP-2–binding clones are listed in Tables S2 and S3, respectively. The codon bias normalized sequence pattern of the hMASP-2–binding TFPI1 D2 clones is presented in Fig. 1A as a sequence logo. We compare this logo with those we obtained previously when generating the SFTI-based (23Kocsis A. Kékesi K.A. Szász R. Végh B.M. Balczer J. Dobó J. Závodszky P. Gál P. Pál G. Selective inhibition of the lectin pathway of complement with phage display selected peptides against mannose-binding lectin-associated serine protease (MASP)-1 and -2: significant contribution of MASP-1 to lectin pathway activation.J. Immunol. 2010; 185 (20817870): 4169-417810.4049/jimmunol.1001819Crossref PubMed Scopus (66) Google Scholar) and the SGPI-based MASP-2 inhibitors (24Héja D. Harmat V. Fodor K. Wilmanns M. Dobó J. Kékesi K.A. Závodszky P. Gál P. Pál G. Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 are essential for lectin pathway activation and reveal structural plasticity of MASP-2.J. Biol. Chem. 2012; 287 (22511776): 20290-2030010.1074/jbc.M112.354332Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The three unrelated scaffolds stabilize the canonical loop conformation through different intramolecular interactions, including disulfides that we kept intact. Therefore, only the three central loop positions, P1, P1′, and P2′, were randomized on all three scaffolds. Human MASP-2 selected only Lys and/or Arg at the P1 position on all scaffolds, but the relative frequencies of these residues are scaffold-dependent. The enzyme preferred Lys on the SGPI-2 and Arg on the SFTI, whereas it exclusively selected an Arg on the TFPI1 D2 scaffold. Similar scaffold-dependent differences are observed at the P1′ and P2′ positions. At P1′, hMASP-2 preferred Ala/Gly/Ser/Thr on TFPI1 D2, whereas it accepted only Ser/Gly on SFTI and Leu/Ala on SGPI-2. At P2′, on TFPI1 D2 hMASP-2 preferred aliphatic Val/Ala/Ile/Leu, whereas on SGPI-2, it selected only aromatic Trp/Tyr. On SFTI, the preference was a mixture of the previous two, as mostly Tyr/Phe and, to a lesser extent, Ile/Met/Leu were selected. The observed differences between the three selected sequence patterns demonstrate that each unrelated scaffold uniquely affects the side-chain preference of the same enzyme at analogous canonical loop positions. Logos derived from the sequences of unique hMASP-2– and rMASP-2–binding TFPI1 D2 clones are shown in Fig. 1, A and B, respectively. At the two energetically most important positions, the two enzymes selected the same amino acids: Arg at P1 and Ala/Gly/Ser at P1′. At P2′, both enzymes selected hydrophobic side chains; hMASP-2 preferred aliphatic, whereas rMASP-2 both aliphatic and aromatic residues. At P3′ and P4′, hMASP-2 shows a weak preference for positively charged residues, whereas rMASP-2 lacks any observable selectivity. At P3, there is a clear species-specific difference in amino acid preferences, but even here, there is an overlapping set of selected residues. Whereas rMASP-2 selected only small hydrophobic residues, Val/Pro/Ile/Gly, hMASP-2 preferred Phe/Tyr and selected smaller residues, such as Pro/Val, at lower frequencies. We designed three TFMI-2 variants along the notion that normalized amino acid frequencies generally correlate with binding energy contributions of individual amino acid residues (34Weiss G.A. Watanabe C.K. Zhong A. Goddard A. Sidhu S.S. Rapid mapping of protein functional epitopes by combinatorial alanine scanning.Proc. Natl. Acad. Sci. U.S.A. 2000; 97 (10908667): 8950-895410.1073/pnas.160252097Crossref PubMed Scopus (262) Google Scholar35Pál G. Kouadio J.-L. K. Artis D.R. Kossiakoff A.A. Sidhu S.S. Comprehensive and quantitative mapping of energy landscapes for protein-protein interactions by rapid combinatorial scanning.J. Biol. Chem. 2006; 281 (16762925): 22378-2238510.1074/jbc.M603826200Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 36Szabó A. Héja D. Szakács D. Zboray K. Kékesi K.A. Radisky E.S. Sahin-Tóth M. Pál G. High-affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4′ acidic residues.J. Biol. Chem. 2011; 286 (21515688): 22535-2254510.1074/jbc.M111.235754Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar37Boros E. Sebák F. Héja D. Szakács D. Zboray K. Schlosser G. Micsonai A. Kardos J. Bodor A. Pál G. Directed evolution of canonical loops and their swapping between unrelated serine proteinase inhibitors disprove the interscaffolding additivity model.J. Mol. Biol. 2019; 431 (30543823): 557-57510.1016/j.jmb.2018.12.003Crossref PubMed Scopus (10) Google Scholar). TFMI-2a carries the hMASP-2–selected consensus P3-P4′ sequence (FCRAVKR) and is expected to have the highest affinity toward hMASP-2. On the other hand, due to its bulky P3 Phe, it should be only a weak inhibitor of rMASP-2. Therefore, we designed two additional inhibitors to efficiently inhibit both hMASP-2 and rMASP-2. Such variants can serve as surrogate compounds in studies investigating the in vivo effects of MASP-2 inhibition in rats. As rMASP-2 mostly preferred a Pro or Val at P3, we substituted the P3 Phe with Pro in TFMI-2b (PCRAVKR) and Val in TFMI-2c (VCRAVKR). TFPI1 D2 and TFMI-2a-c were expressed in Escherichia coli and purified to homogeneity. Their equilibrium inhibitory constants (KI) were determined against the catalytic fragments of human MASP-1, human and rat MASP-2, and human MASP-3 (Table 1).Table 1KI values of TFPI1 D2, TFMI-2a–c variants, and SGMI-2 on the catalytic fragments of human MASP-1, -2, and -3 and rat MASP-2InhibitorSequence (P4–P4′)KIHuman MASP-1cfHuman MASP-2cfRat MASP-2cfHuman MASP-3cfnmTFMI-2aGFCRAVKRNEaNE, not effective; no inhibition could be detected.2.0 ± 0.1bAverage ± range (n = 2).640 ± 20bAverage ± range (n = 2).NETFMI-2bGPCRAVKRNE7.9 ± 0.3bAverage ± range (n = 2).7.2 ± 0.2cAverage ± S.D. (n = 3).30,000dApproximation based on a single measurement.TFMI-2cGVCRAVKRNE36.7 ± 0.7bAverage ± range (n = 2).7.5 ± 0.3bAverage ± range (n = 2).NETFPI1 D2GICRGYITNE1883 ± 48bAverage ± range (n = 2).185 ± 4bAverage ± range (n = 2).NESGMI-2VCTKLWCNNE6eData from Ref. 24.22.7 ± 1.6cAverage ± S.D. (n = 3).5200 ± 300fData from Ref. 25.a NE, not effective; no inhibition could be detected.b Average ± range (n = 2).c Average ± S.D. (n = 3).d Approximation based on a single measurement.e Data from Ref. 24Héja D. Harmat V. Fodor K. Wilmanns M. Dobó J. Kékesi K.A. Závodszky P. Gál P. Pál G. Monospecific inhibitors show that both mannan-binding lectin-associated serine protease-1 (MASP-1) and -2 are essential for lectin pathway activation and reveal structural plasticity of MASP-2.J. Biol. Chem. 2012; 287 (22511776): 20290-2030010.1074/jbc.M112.354332Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar.f Data from Ref. 25Héja D. Kocsis A. Dobó J. Szilágyi K. Szász R. Závodszky P. Pál G. Gál P. Revised mechanism of complement lectin-pathway activation revealing the role of serine protease MASP-1 as the exclusive activator of MASP-2.Proc. Natl. Acad. Sci. U.S.A. 2012; 109 (22691502): 10498-1050310.1073/pnas.1202588109Crossref PubMed Scopus (156) Google Scholar. Open table in a new tab TFPI1 D2 was previously shown to inhibit hMASP-2 with low affinity (31Keizer M.P. Pouw R.B. Kamp A.M. Patiwael S. Marsman G. Hart M.H. Zeerleder S. Kuijpers T.W. Wouters D. TFPI inhibits lectin pathway of complement activation b" @default.
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• W2933578412 title "Novel MASP-2 inhibitors developed via directed evolution of human TFPI1 are potent lectin pathway inhibitors" @default.
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