Complement Proteins Factor B and C2 Complement Inhibitor That Targets

Identification of Complin, a Novel Complement Inhibitor that Targets Complement Proteins Factor B and C2

This information is current as Archana P. Kadam and Arvind Sahu of September 24, 2021. J Immunol published online 12 May 2010 http://www.jimmunol.org/content/early/2010/05/12/jimmun ol.1000200 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 12, 2010, doi:10.4049/jimmunol.1000200 The Journal of Immunology

Identiﬁcation of Complin, a Novel Complement Inhibitor that Targets Complement Proteins Factor B and C2

Archana P. Kadam and Arvind Sahu

Complement factor B (fB) is a key constituent of the alternative pathway (AP). Its central role in causing inflammation and tissue injury through activation of the AP urges the need for its therapeutic targeting. In the current study, we have screened phage-displayed random peptide libraries against fB and identified a novel cyclic hendecapeptide that inhibits activation of fB and the AP. Structure- activity studies revealed that: 1) the cysteine-constrained structure of the peptide is essential for its activity; 2) Ile5, Arg6, Leu7, and Tyr8 contribute significantly to its inhibitory activity; and 3) retro-inverso modification of the peptide results in loss of its activity. Binding studies performed using surface plasmon resonance suggested that the peptide has two binding sites on fB, which are located on the Ba and Bb fragments. Studies on the mechanism of inhibition revealed that the peptide does not block the interaction of fB

with the activated form of C3, thereby suggesting that the peptide inhibits fB activation primarily by inhibiting its cleavage by factor Downloaded from D. The peptide showed a weak effect on preformed C3 and C5 convertases. Like inhibition of fB cleavage, the peptide also inhibited C2 cleavage by activated C1s and activation of the classical as well as lectin pathways. Based on its inhibitory activities, we named the peptide Complin. The Journal of Immunology, 2010, 184: 000–000.

he complement system is one of the strong bulwarks of the of the amplification loop of the AP (11, 12, 14). It is therefore not http://www.jimmunol.org/ host immune system against a wide repertoire of patho- surprising that in many of the above-mentioned diseases, com- T gens (1, 2). During the complement system surveillance, plement-mediated pathologies are galvanized by the amplification pathogens are marked as foreign by covalent tethering of activated loop of the AP (11). Recently, it has been reported that under C3 on their surface and then killed by various mechanisms (3–5). in vitro conditions, as much as 80% of the total complement ac- Although the system is designed to target pathogens, its excessive tivation initiated by the CPs and LPs is contributed by the AP activation as well as inappropriate intrinsic regulation leads to de- alone (12). Together, these data clearly point out a need to target struction of the host tissues (6–8). Over the years, a wealth of the AP for therapeutic benefit. knowledge has been accumulated revealing the hazardous effects of The complement component factor B (fB), which circulates in complement during various diseases and pathological conditions, blood as a proenzyme, is the key protease of the AP. It forms by guest on September 24, 2021 which include diseases linked to deficiencies, mutations, and poly- a three-lobed structure: one corresponds to three complement morphisms in complement regulatory proteins and/or its compo- control protein domains, the second represents a von Willebrand nents, such as paroxysmal nocturnal hemoglobinuria, hereditary factor type A domain, and the third corresponds to a serine protease angioedema, age-related macular degeneration, atypical hemolytic domain (15, 16). Association of fB with fluid phase C3(H2O) uremic syndrome, and membranoproliferative glomerulonephritis formed by the process of tick over, or C3b attached to the acti- type II (9–12). Thus, recent times have seen a wide research focus vating surface, and its cleavage by factor D (fD) leads to activation on therapeutic targeting of the complement system (7, 9, 13). of fB and formation of the initial [C3(H2O),Bb] or amplification The complement system is activated by three major pathways: C3 convertase (C3b,Bb) of the AP, respectively (17–19). The C3b, classical (CP), alternative (AP), and lectin (LP). Consequently, the Bb formed on the activating surface triggers the amplification loop damage mediated by complement can occur as a result of activation of the AP, leading to the attachment of millions of C3b molecules of any of the three pathways. It is, however, important to point out on the target surface within minutes and activation of the terminal in this paper that even if tethering of activated C3 (C3b) onto the complement cascade (20, 21). Thus, blocking fB activation would host tissues is initiated by the CPs or LPs, this results in activation lead to inhibition of generation of C3a, C5a, and the membrane attack complex, which are responsible for causing tissue injuries (6, 11, 22). Previous efforts to identify small molecule inhibitors National Centre for Cell Science, Pune University Campus, Ganeshkhind, Pune, India against fB led to the identification of a substrate-based peptide in- Received for publication January 26, 2010. Accepted for publication April 4, 2010. hibitor (Ac-SHLGLAR-H) that showed inhibition of fB-mediated This work was supported by a project grant from the Department of Biotechnology, cleavage of C3 with an IC50 of 19 mM at nonphysiological alka- New Delhi, India (to A.S.). This work was done in partial fulfillment of the Ph.D. thesis of A.P.K. to be submitted to the University of Pune, Pune, India. line pH (23). In addition, complement C2 receptor inhibitor tris- Address correspondence and reprint requests to Dr. Arvind Sahu, National Centre for panning-17 peptide (HEVKIKHFSPY) that inhibits C2 activation Cell Science, Pune University Campus, Ganeshkhind, Pune 411007, India. E-mail also showed inhibition of fB activation, but only at a millimolar address: [email protected] concentration (24). Abbreviations used in this paper: AP, alternative pathway; C3b, activated form of C3; In the current study, we have screened phage-displayed random CP, classical pathway; CVF, cobra venom factor; EA, Ab-coated sheep erythrocyte; ESI-MS, electrospray ionization-MS; fB, factor B; fD, factor D; GVB, gelatin Vero- peptide libraries to identify a small peptide inhibitor of fB. Our efforts nal buffer; LP, lectin pathway; MS, mass spectrometry; NA, not applicable; RU, led to the identification of a novel cyclic hendecapeptide that inhibits response unit; SA, streptavidin; SPR, surface plasmon resonance; VBS, Veronal- activation of fB (IC50 =9.5mM). The peptide also showed inhibition buffered saline. of C2 activation (IC50 = 11.3 mM), suggesting that it has the potential Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 to inhibit pathologies mediated by all the three pathways.

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000200 2 COMPLIN INHIBITS FACTOR B AND C2 ACTIVATION

Materials and Methods (Bio-Rad, Segrate, Italy). The data obtained were normalized as percent Reagents and buffers inhibition. In a variant of this assay, CVF was used instead of C3b. In this study, Polyclonal Ab against human complement C2 was purchased from Cal- preincubated reaction mixtures containing fB (2.5 mg) and various con- biochem (La Jolla, CA). Ab-coated sheep erythrocytes (EAs) were made by centrations of peptides were further incubated at 37˚C for 1 h with 3 mg incubating sheep erythrocytes with anti-sheep erythrocyte Ab procured CVF and 80 ng fD in a total volume of 20 ml PBS containing 5 mM from ICN Biomedical (Irvine, CA). Veronal-buffered saline (VBS) contained MgEGTA. The reaction mixtures werethensubjectedtoSDS-PAGE 5 mM barbital, 145 mM NaCl, and 0.02% sodium azide (pH 7.4). VBS++ was analysis, and the percentage of fB cleaved was quantitated densito- made by adding 0.5 mM MgCl2 and 0.15 mM CaCl2 to VBS. Gelatin metrically as described above. Veronal buffer (GVB) was VBS containing 0.1% gelatin, GVB++ was GVB To measure the effect of Complin on C2 cleavage, indicated concentra- m ++ containing 0.5 mM MgCl2 and 0.15 mM CaCl2, and GVB-EDTA was GVB tions of the peptide were preincubated with C2 (150 ng) in 3.5 l VBS with 10 mM EDTA. MgEGTA contained 0.1 M MgCl2 and 0.1 M EGTA, for 15 min at 37˚C and 100 ng activated C1s was then added to each and PBS contained 10 mM sodium phosphate and 145 mM NaCl (pH 7.4). reaction mixture, volume was adjusted to 10 ml by adding VBS++,and TBS was 50 mM Tris-HCl and 150 mM NaCl (pH 7.5). reactions were further incubated for 1 h at 37˚C. Inhibition of C2 cleavage by the peptide was then determined by running the reaction mixtures on an Complement proteins and their proteolytically cleaved fragments 8.5% SDS-PAGE gel under reducing conditions followed by Western blot analysis using anti-C2 Ab and anti-goat HRP conjugate (Bio-Rad). The The human complement proteins C3 (25) and fB (26) were purified from blot was developed using SuperSignal West Pico chemiluminescent sub- plasma as described earlier. Human complement factor H and fD were strate (Pierce, Rockford, IL) and subjected to densitometric analysis to kindly provided by Prof. Michael K. Pangburn (University of Texas Health determine the percentage of C2 cleaved. The data obtained were nor- Center, Tyler, TX), and C2 and C1s were purchased from Calbiochem. The malized as percent inhibition. proteolytically cleaved fragments of fB (Ba and Bb) were generated by incubating fB with C3b, fD, and factor H and purified on a Mono Q Hemolytic assays Downloaded from column (Amersham Biosciences, Uppsala, Sweden) (27). C3b, the proteolytically activated form of C3, was generated by limited tryptic di- Effect of peptides on activation of the AP and CP of complement was gestion of C3 and purified on a Mono Q column (28). Native C3 and C3 measured by using hemolytic assays as described (32). (H2O) were separated by running the sample on a Mono S column (Amersham Biosciences) (29). Cobra venom factor (CVF) was purified ELISA for measurement of effect on CPs, APs, and LPs from Naja naja kaouthia venom as previously described (30). The effect of Complin on the various complement pathways was also mea-

Synthetic peptides sured using commercially available Wieslab complement system screen http://www.jimmunol.org/ ELISA kit (Euro-Diagnostica, Malmo, Sweden), wherein individual path- All of the peptides used in this study were synthesized by Genomechanix ways are activated by pathway-specific activators. In brief, suggested dilu- (Gainesville, FL). The peptides were purified by the supplier on a reversed tions of normal human serum were mixed with graded concentrations of phase C-18 column, and purity was verified by analytical HPLC and mass Complin and incubated at 22˚C for 15 min. These reaction mixtures were then . spectral analysis. The purity of all of the peptides was 95%. For cyclic added to microtiter wells precoated with IgM (for CP), mannan (for LP), or peptides, cyclization was confirmed by Ellman’s reagent by the supplier. LPS (for AP), incubated at 37˚C for 1 h, and washed three times before addition of the conjugate (alkaline phosphatase-labeled anti–C5b-9). The Biopanning of the phage libraries plate was then incubated at 22˚C for 30 min and washed, and the color was Affinity selection for isolation of fB-binding phage clones was performed as developed by adding the substrate. The absorbance was read at 405 nm, and data were normalized as percent inhibition.

described earlier (31) with minor modifications. In brief, microtiter wells by guest on September 24, 2021 were coated with fB (20 mg/well) in PBS by incubating the plate overnight at 4˚C. The wells were then blocked with 1% BSA for 30 min at 22˚C. After Surface plasmon resonance measurements washing the wells once with PBS containing 0.1% Tween 20 (v/v), 2 3 1011 The kinetics of binding of Complin to fB and its fragments (Ba and Bb) was PFUs of each of the M13 libraries (AC-X -CGGGS or X GGGS, where 7 12 measured using the surface plasmon resonance (SPR)-based biosensor X = any amino acid; New England Biolabs, Ipswich, MA) was added to the Biacore 2000 (Biacore, Uppsala, Sweden). All of the binding measure- wells and incubated overnight at 4˚C. The next day, the wells were washed ments were performed in PBS containing 0.05% Tween 20 and 5 mg/ml five times with washing buffer (PBS with 0.1% BSA and 0.1% Tween CM Dextran (Fluka, Buchs, Switzerland) at 25˚C. These additions to PBS 20 [v/v]). Bound phages were then eluted with 100 mM glycine-HCl (pH blocked the nonspecific adsorption of the analytes to the sensor chip. To 2.3) for 5 min and neutralized immediately with an equal volume of Tris- measure binding, ∼2000 response units (RUs) of fB or its fragments (Ba HCl (pH 8.5). The recovered phage particles were amplified in ER2738 or Bb) was immobilized on a test flow cell of a CM5 chip using amine- Escherichia coli, and the biopanning procedure was repeated twice as de- coupling chemistry; nonimmobilized (blank) flow cell served as a control scribed above. After the third round of biopanning, the phage mixture was flow cell. Various concentrations of the analyte were then injected for plated, and the fB-binding phages were identified by ELISA. 120 s at a flow rate of 50 ml/min, and dissociation was followed for 180 s. ELISA for measurement of binding of phages to fB The chip was regenerated by injecting brief alternate pulses of 4.0 M MgCl2 and 0.2 M sodium carbonate (pH 9.5). The biosensor data obtained Binding of phage clones to fB was evaluated by ELISA. Microtiter plates for the control flow cell were subtracted from those obtained for test flow were coated overnight at 4˚C with 100 ml fB (20 mg/ml). The wells were cell and evaluated using BIAevaluation software version 4.1 using global then blocked with 0.5% BSA for 1 h at 22˚C and washed once with TBS fitting (Biacore). containing 0.1% Tween 20 (v/v). Thereafter, the phage supernatant from the individually amplified plaques from the third round of biopanning was Measurement of effect on the AP C3 convertase added to the wells and incubated at 22˚C for 1 h. The wells were washed five times with washing buffer, and the bound phages were detected by The effect of Complin on the preformed AP C3 convertase was measured adding 1:5000 diluted HRP-labeled anti-M13 Ab (Amersham Biosciences) as below. The AP C3-cleaving enzyme [C3(H2O),Bb] was formed by in- m m m followed by ABTS HRP-substrate (Roche Applied Science, Indianapolis, cubating 1 g C3(H2O) with 2 g fB and 40 ng fD in 7 l PBS containing IN). The OD was read at 414 nm. 5 mM MgEGTA for 5 min at 22˚C. The reaction was stopped by adding 10 mM EDTA. Immediately thereafter, 3 mg C3 was added along with Measurement of inhibition of fB and C2 cleavage by peptides indicated concentrations of the peptide in a total volume of 20 ml PBS and incubated at 37˚C for 45 min. The amount of C3 cleaved (indicated by the The effect of the peptides on fB activity was studied by utilizing a fluid- generation of a9-chain) in the presence/absence of peptide was determined phase assay (31). Briefly, various concentrations of the peptides were by running the reaction mixtures on a 6% SDS-PAGE under reducing preincubated with fB (2 mg) for 15 min at 37˚C. A mixture of 2.5 mg C3b conditions and staining with Coomassie blue. and 50 ng fD in a total volume of 20 ml PBS containing 5 mM MgEGTA The effect of Complin on C3 convertase was also evaluated by was then added to each reaction mixture and further incubated at 37˚C for forming CVF,Bb. In order to form CVF,Bb, 1.5 mgCVFwasincubated 45 min. The inhibition of fB cleavage was visualized by running the re- with 2 mg fB and 80 ng fD in 7 ml PBS containing 5 mM MgEGTA for action mixtures on an 8.5% SDS-PAGE gel under reducing conditions and 15 min at 22˚C. After stopping the reaction by adding 10 mM EDTA, staining the gel with Coomassie blue. The percentage of fB cleaved was C3 (3 mg) along with the indicated concentrations of the peptide in quantitated by densitometric analysis using the VersaDoc XRS system 20 ml PBS was added to the convertase and incubated for additional 1 h The Journal of Immunology 3

1.4 amount of C5 cleaved. The C5 cleavage was indicated by the generation of Without fB a9 1.2 With fB -chain of C5.

1.0 414nm 0.8 Results Isolation and characterization of fB-binding phages 0.6

0.4 By virtue of its rich structural diversity, the phage-displayed peptide

Absorbance, 0.2 librarieshaveemergedasapowerfultoolforisolatingligandsagainst

0.0 variousproteins.Inviewofthis,wescreened12-merlinearand7-mer Clone L14 L41 L52 L61 C5 L15 cysteine-constrained phage-displayed peptide libraries for isolating ligands against human fB. The affinity selection of clones was performedbybiopanningagainstfBcoatedonmicrotiterplates.Both Clone Peptide length Phage displayed amino of the libraries were subjected to three rounds of biopanning against acid sequence fB,andat theendofthe thirdround,isolatedphageswerescreenedby L14 13 aqS---FYHI RLYS-----G g ELISA. The screening of ∼500 clones from the cysteine-constrained L41 13 --S---LLHV RIFSYEp--G - L52 13 eap-----Hw --YPYDWLLG - library yielded 25 positive clones. Further DNA sequencing re- L61 13 sndmrlLTHI RL------G - vealed that all of the clones had identical sequences. Concurrent C5 11 aca-----Hr g----DLSCG - screening of ∼1000 clones from the linear library yielded 51 positive *** *** ********* * **** **** **** **** clones. Sequencing of these clones confirmed the presence of four ** ** unique clones. Binding of these five unique clones displaying con- Downloaded from strained and linear peptides to fB is depicted in Fig. 1. FIGURE 1. Binding of phages isolated from phage-displayed random peptide libraries to fB and sequence alignment of the phage-displayed peptides. Effect of phage-displayed peptides on activation of fB Top panel, ELISA depicting binding of the phage clones isolated from phage- We next determined whether binding of these phage-displayed displayed peptide libraries to fB. Microtiter wells were coated with fB and peptides to fB inhibits its activation. During AP activation, fB blocked with BSA, and phage clones were allowed to bind fB. Binding of phages was detected by anti-M13 HRP Ab. Phage clone numbers starting with binds to C3b and is then cleaved by fD into Bb and Ba fragments. http://www.jimmunol.org/ L and C denote clones isolated from linear and constrained libraries, re- The cleavage of fB results in its activation. Thus, to examine the spectively. Clone L15 served as a negative control. Bottom panel, alignment of effect of peptides on fB activation, we synthesized five peptides peptide sequences displayed on the fB-binding phages using DIALIGN 2.2.1. corresponding to the peptide sequences displayed on the fB- binding clones and tested them in a fluid-phase assay wherein purified C3b, fB, and fD were incubated with or without peptides at 37˚C. The effect on C3 cleavage was determined by SDS-PAGE in the presence of MgEGTA, and then fB cleavage was monitored analysis as above. by SDS-PAGE analysis. In this assay, inhibition of fB cleavage Measurement of effect on the AP C5 convertase into Ba and Bb indicated the inhibition of fB activation. Of the five synthetic peptides examined, only peptide 1 corre- by guest on September 24, 2021 The effect of Complin on the preformed C5 convertase was assessed by sponding to the clone L14 displayed inhibitory activity. The IC50 of measuring CVF,Bb-mediated cleavage of C5. Briefly, CVF,Bb was formed m as described above, and then it was incubated in a total volume of 20 ml with this linear peptide for inhibition of fB activation was 56 M(Fig.2, indicated amount of peptide and C5 (2 mg) for 1 h at 37˚C. The reaction Table I). Thus, 50% inhibition of fB activation required ∼52-fold mixtures were then subjected to SDS-PAGE analysis to determine the molar excess of this peptide.

Peptide 1 Peptide 19 (Complin) Peptide 19 (Complin) Pep. conc. Pep. conc. Pep. conc. μ - - μ - - μ - - ( M) 625 313 156 78 39 19.5 9.8 ( M) 125 62.5 31.3 15.6 7.8 3.9 ( M) 100 50 25 12.5 6.3 3.1 fB +++++++++ fB ++++++++ fB + + + + ++ + + C3b -++++++++ C3b -+++++++ CVF -+++++++ fD - +++ +++ + + fD - +++ +++ + fD - +++ +++ + α α ’ ’ fB fB fB β α β CVF Bb Bb Bb CVFβ Ba Ba Ba CVFγ

100 100 35 80 30 80 60 25 60 40 20 40 15 20 20

50 for fB cleavage 10

0 IC 0 on 5 inhibiti lysis AP % 1 10 100 1000 0 51015 20 25 30 10 100

% fB cleavage inhibition cleavage fB % 1 Peptide conc. (μM) Days post solution preparation Complin conc. (μM) FIGURE 2. Inhibition of fB and AP activation by peptides. Top panels, effect of peptides on fB activation was studied by incubating C3b/CVF, fB, fD and various concentrations of the indicated peptides in the presence of MgEGTA for 45 min at 37˚C. The reaction mixtures were analyzed for fB cleavage (indicated by generation of Ba and Bb) on an 8.5% SDS-PAGE gel under reducing conditions, and the cleavage products were visualized by Coomassie blue staining. Bottom left panel, graphical representation of percentage inhibition of fB cleavage. The intensities of fB in gels were quantitated densitometrically, and percentage of fB cleavage inhibition is represented graphically against concentration. Bottom middle panel, graph showing increase in the inhibitory activity of Complin over a period of time on storage on ice. Bottom right panel, inhibition of AP-mediated lysis of rabbit erythrocytes by Complin. In- hibition of C3b associated fB cleavage by peptide 1 (N); inhibition of C3b-associated fB cleavage by Complin (d); inhibition of CVF-associated fB cleavage by Complin (s); inhibition of AP-mediated lysis of erythrocytes by Complin (n). 4

Table I. Amino acid sequences, mass analyses, and inhibitory activities of phage-displayed peptides, Complin, and its analogs

Inhibition of Human Complement IC50 Mass Spectral Analyses (mM)

Peptide Number Amino Acid Sequencea Peptide Length Expected Observed B Cleavageb AP Activityc Phage-displayed peptides 1 AQSFYHIRLYSGG 13 1499 1498 56.2 2700 2 SLLHVRIFSYEPG 13 1518 1514 .1000 ND 3 EAPHWYPYDWLLG 13 1645 1643 .1000 ND 4 SNDMRLLTHIRLG 13 1526 1523 .1000 ND 5 a*caHRgDLSC*G 11 1086 .500d ND Motif-based cyclic peptides 6_A*CYHIRLYSC*G 11 1282 1283 622 1170 7 A*CFYHIRLYSC*G 12 1429 1431 .500d 1060 8_A*CTHIRLYSC*R 11 1320 1324 553 640 9 A*CLTHIRLYSC*R 12 1434 1434 49.8 140 10 _A*CAHWRLYSC*R 11 1364 1366 .500d 680 11 _A*CAHIRLYSYC*R 12 1453 1453 .500d 570 12 _A*CAHIRLYSYDC*R 13 1568 1568 .500d .500d 13 A*CLLHIRLYSC*R 12 1445 1445 775 240 14 A*CLAHIRLYSC*R 12 1403 1403 .500d 730 15 A*CAHIRLYSC*G 11 1191 1193 44.2 145 16 E*CAHIRLYSC*G 11 1247 1248 .500d .500d R*CAHIRLYSC*G . d

17 11 1275 1275 500 790 ACTIVATION C2 AND B FACTOR INHIBITS COMPLIN 18 E*CAHIRLYSC*R 11 1347 1348 55 118 19 (Complin) A*CAHIRLYSC*R 11 1289 1290 9.5 33 20 L*CAHIRLYSC*R 11 1332 1332 .500d .500d 21 _AAAHIRLYSAR 11 1227 1228 .500d .500d Ala scan analogs of Complin 22 A*CAAIRLYSC*R 11 1224 1224 16.6 90 23 A*CAHARLYSC*R 11 1248 1248 361 327 24 A*CAHIALYSC*R 11 1205 1205 .500d .174d 25 A*CAHIRAYSC*R 11 1248 1248 455 351 26 A*CAHIRLASC*R 11 1198 1198 .500d 355 27 A*CAHIRLYAC*R 11 1274 1274 13.3 35 Retro-inverso analog of Complin 28 dR*dCdSdYdLdRdIdHdAdC*dA 11 1291 1291 .500d .500d Biotinylated analog of Complin 29 A*CAHIRLYSC*RGGGSAK-Biotin 17 1973 1974 .500d .500d aInhibition assays were performed after keeping the peptide solutions for seven days on ice. Asterisk indicate oxidized cysteines. bInhibition of fB cleavage was measured using a ﬂuid phase assay (see Materials and Methods). cInhibition of AP activity was measured by hemolytic assay (see Materials and Methods).

dMaximum concentration of the peptide that could be tested in the assay due to solubility limitations.

Downloaded from from Downloaded http://www.jimmunol.org/ by guest on September 24, 2021 24, September on guest by The Journal of Immunology 5

Design and characterization of motif-based cyclic peptides and Ile5,Arg6,Leu7, and Tyr8 play a critical role in maintaining the identification of Complin functional activity of the peptide. In an effort to design a peptide with enhanced inhibitory activity, we Retro-inverso analogs are considered important tools in dis- examined the phage-displayed peptide sequences for the presence of secting the relative role of the main-chain and side-chain inter- a motif. Alignment of five phage-displayed peptide sequences using actions in ligand-protein recognition because in such analogs, the DIALIGN (33), which constructs multiple alignments by compar- conformation of the side-chains are same as that in the L-isoform, but conformation of the main-chain atoms is reversed (37). Syn- ing whole segments of the sequences but does not use gap penalty, thesis and functional analysis of a retro-inverso analog of Complin showed the presence of eight conserved residues with a highly (peptide 28) showed that it was completely inactive (Table I). conserved four-residue core (Fig. 1). Next, based on the motif, we From these results, it can be inferred that in addition to the side synthesized 15 different disulfide-constrained peptides. Although chains, the main-chain atoms of Complin also contribute in its all the phage-displayed peptides except peptide 5 (corresponding to interactions with fB. the phage clone C5) were linear, we designed motif-based peptides as cyclic peptides, as they are likely to be structured and have Kinetic analysis of interaction of Complin with fB and its increased t1/2 in serum as compared with the linear peptides (34, fragments Ba and Bb 35). To optimize the structure of the peptide, we primarily varied To examine the binding kinetics of Complin with fB, we undertook the ring size and residues outside the four-residue core. Functional Biacore studies. We first synthesized biotinylated peptides for ori- characterization of these peptides for inhibition of fB activation entation onto the streptavidin (SA) chip. Addition of biotin at the C demonstrated that only 4 of the 15 peptides (peptides 9, 15, 18, and terminus of Complin resulted in loss of activity of Complin (peptide Downloaded from 19) showed increased inhibitory activity compared with peptide 1 29; Table I). We therefore reversed the setup and coupled fB or its (Fig. 2, Table I). The most active peptide was peptide 19 with an fragments onto CM5 chips by amine coupling chemistry and flowed m ∼ IC50 of 9.5 M, indicating that 9-fold molar excess concentration Complin; the control flow cell was either left blank or immobilized of this peptide was sufficient to inhibit 50% of fB activation. As with C3b. Complin bound to fB in a dose-dependent manner. Its expected, peptide 19 also inhibited AP-mediated lysis of rabbit linear analog (peptide 21), however, showed negligible binding to erythrocytes (IC =33mM; Fig. 2, Table I). Based on its activities 50 fB, suggesting that the cyclic nature of the peptide is important for http://www.jimmunol.org/ (described here and below), we have named this peptide Complin binding to fB (Fig. 3). To analyze the binding affinity and the nature (complement inhibitor). of interaction, the association and dissociation data were fitted to Apart from its association with C3b, fB activation also occurs various models. Data showed a good fit to a bivalent analyte model 2 when it is associated with C3b-like molecule CVF followed by its (x = 1.58); the apparent equilibrium rate constants (KD1 and KD2) cleavage by fD. Moreover, recently it has been demonstrated that were 3.05 mM and 4.16 mM, respectively (Fig. 3, Table II). CVF-bound fB show a difference in conformation compared with Because binding data fit well to a bivalent analyte model, we C3b-bound fB (36). Thus, we also sought to determine if Complin asked whether binding sites are localized only in one of the has the ability to inhibit the activation of CVF-associated fB. Data subfragments of fB or on both Ba and Bb. We generated Ba and Bb presented in Fig. 2 (top right panel) clearly show that Complin is by cleaving C3b-associated fB by fD, purified them on a Mono Q by guest on September 24, 2021 equally efficient in inhibiting the activation of CVF-associated fB

(IC50 =12mM). An intriguing observation we made during the course of this study was that Complin showed a gain in activity when stored on ice over 7 d (Fig. 2). We suspected that the peptide might be forming multimers upon storage. To verify this, we performed MALDI-mass spectrometry (MALDI-MS) as well as electrospray ionization-MS (ESI-MS) analyses of a 1-mo-old sample stored on ice. The stored sample did not show the presence of multimers by MALDI-MS, but did confirm their presence (2–6M species) when analyzed by ESI-MS (data not shown). Identification of structural features important for inhibitory activity of Complin During designing the motif-based constrained peptides, we made an assumption that cyclization would result in stabilization of peptide structure. Thus, to validate our premise, we synthesized a linear analog of Complin wherein cysteines were replaced with Ala (peptide 21). This analog did not show any inhibitory activity even at 500 mM concentration (Table I), suggesting that, indeed, the di- FIGURE 3. SPR analysis of binding of Complin to fB and its fragments Ba sulfide bond plays an important role in the maintenance of a pre- and Bb. fB and its fragments were coupled to CM5 chip and Complin, or its ferred structure of the peptide required for its inhibitory activity. linear analog was injected to measure binding. Top left panel, binding of To determine the importance of amino acid residues of Complin Complin (10 mM) and the linear analog of Complin (peptide 21; 10 mM) to fB. between Cys 2 and Cys 10 in its inhibitory activity, we performed an Top right panel, sensogram overlay for the interaction between fB and Complin. The solid lines represent the global fitting of data to a bivalent an- Ala scan analysis. We synthesized a set of six Ala analogs (peptides alyte model. Bottom left panel, sensogram overlay for the interaction between 22–27; Table I) and examined their effect on fB activation using the Complin and Bb fragment. The solid lines represent the global fitting of data to fluid-phase assay described above. Substitutions of each of the a 1:1 Langmuir binding model. Bottom right panel, sensogram overlay for the residues between Cys 2 and Cys 10 with Ala showed that sub- interaction between Complin and Ba fragment. Data were fitted (solid lines) stitutions at all the positions except at positions 4 and 9 drastically using a 1:1 Langmuir binding model. In the overlay plots, the concentrations hampered its activity (Table I). These data therefore suggested that of Complin injected are indicated at the right of the sensograms. 6 COMPLIN INHIBITS FACTOR B AND C2 ACTIVATION

Table II. Kinetic and afﬁnity data for the interactions of Complin with fB and its fragments

a 2 Ligand Analyte kd1 (1/s)/ka1 (1/Ms) SE (kd1/ka1) KD1 (mM) kd2 (1/s)/ka2 (1/Ms) SE (kd2/ka2) KD2 (mM) x fB Complin 1.58 3 1023/518 5.08 3 1025/9.25 3.05 0.0529/1.27 3 104 2.77 3 1023/817 4.16 1.58b Ba Complin 2.42 3 1023/2.3 3 103 5.39 3 1025/50.8 1.05 NA NA NA 0.299c Bb Complin 2.04 3 1023/1.3 3 103 3.09 3 1025/21.5 1.57 NA NA NA 0.889c

a ka2 (1/Ms) = ka2 (1/RU) 3 100 3 (m.w. of the ligand). bData were calculated by global fitting to a bivalent analyte model (BIAevaluation 4.1). cData were calculated by global fitting to a 1:1 Langmuir binding model (BIAevaluation 4.1). NA, not applicable. column (Amersham Biosciences), and coupled them onto different premise, we studied the effect of Complin on preformed C3 and C5 flow cells of a CM5 chip. When Complin was flown over Ba and convertases. The fluid-phase C3 convertases C3(H2O),Bb and CVF, Bb, binding was observed to both the fragments, suggesting that Bb were formed by incubating C3(H2O) or CVF with fB and fD in binding sites are located in Ba as well as Bb fragments (Fig. 3). As the presence of Mg2+. These were then incubated with native C3 in expected, binding data for both Ba and Bb fit well to the 1:1 the presence of increasing concentrations of Complin. The peptide Langmuir binding model (Fig. 3, Table II). had very weak effect on C3(H2O),Bb-mediated cleavage of C3 even at 100 mM concentration, which was ∼10 times higher than that

Complin does not inhibit binding of fB to C3b required for 50% inhibition of fB activation (Fig. 5). It, how- Downloaded from Inhibition of fB activation by Complin could occur as a result of ever, showed ∼30% inhibition of CVF,Bb-mediated C3 cleavage at blocking of fB binding to C3b. To delineate this, we designed the following experiment. We coupled C3b onto an SA chip in its physiological orientation and injected either fB or fB preincubated 120 100 with Complin or its linear analog (peptide 21). A positive control C3α 80 α was formed by injecting fB preincubated with anti-fB mAb NCCS C3 ’ 60 http://www.jimmunol.org/ fB 40 34.78, which binds to Ba and inhibits fB activation. fB showed good C3β Bb chain % C3 α ’ 20 binding response, but preincubation of fB with either 10- or 30-fold 0 fB ++++++ 100 10 1 100 molar excess of Complin compared with fB did not result in a de- C3(H2O) ++++++ crease in fB binding response (Fig. 4). No effect on the binding fD - + + + + + Complin Peptide 21 C3 + + + + + - (μM) (μM) response was also observed when fB preincubated with 30-fold Complin (μM) - -1-100 10 μ molar excess of the linear analog (peptide 21) was injected onto the Peptide 21 ( M) ---- - 100 120 chip. As expected (19), preincubation of mAb resulted in a signif- 100 icant decrease in fB binding response (Fig. 4). It is therefore ap- C3α 80

C3α’ 60 by guest on September 24, 2021 parent that Complin does not inhibit interaction of fB with C3b. fB C3β CVF α 40 Effect of Complin on the AP C3 and C5 convertase activities Bb CVFβ chain % C3 α ’ 20 0 fB ++++++ - 100 10 1 100 Our SPR data indicated that Complin binds to Bb as well as Ba CVF ++++++ + fragments of fB (Fig. 3). The Bb fragment provides the catalytic fD - ++ ++ + - Complin Peptide 21 C3 ++++++ - (μM) (μM) subunit to the AP C3 and C5 convertases. It was therefore likely that Complin (μM) - -1100 10 - - in addition to inhibition of fB activation, the observed inhibitory Peptide 21 (μM) --- -- 100 - effect of Complin on activation of the AP (Fig. 2) could also be in 120 100 part due to its effect on the C3 and C5 convertases. To verify this C5α 80 C5α’ 60 fB 40 300 C5β CVF α ’ chain % C5 α ’ Bb CVFβ 20 250 0 fB ++ + ++ + 100 10 1 100 CVF -++ ++ + 200 fD -++ ++ + fB+Complin Complin Peptide 21 C5 ++ + ++ + (μM) (μM) Complin (μM) - -1100 10 - 150 fB Peptide 21 (μM) ---- - 100

100 FIGURE 5. Effect of Complin on C3 and C5 convertases. Top and

Response (RU) fB+mAb middle panels, effect of Complin on C3 convertases. The C3 convertases 50 fB+Peptide 21 C3(H2O),Bb (top left panel) and CVF,Bb (middle left panel) were formed 0 by incubating fB, fD, and C3(H2O) or CVF in the presence of MgEGTA at 25˚C for 5 min or 15 min, respectively. The formation of the convertases -50 was stopped by adding 10 mM EDTA. Native C3 and Complin or the linear -100 0 100 200 300 400 analog of Complin (peptide 21) were then added to the preformed C3 Time (s) convertases [C3(H2O),Bb or CVF,Bb]. The reaction mixtures were ana- FIGURE 4. Effect of Complin on binding of fB to C3b. fB (100 nM) lyzed for C3 cleavage (revealed by the generation of a9-chain) on a 6% preincubated with Complin (1 mMor3mM), linear analog of Complin SDS-PAGE gel under reducing conditions. Bottom left panel, effect of (peptide 21; 3 mM), or a control anti-fB mAb (400 nM; it binds to Ba Complin on C5 convertase. CVF,Bb was formed as described above and fragment and inhibits fB activation) at 25˚C for 15 min was injected on incubated with C5 and Complin or the linear analog (peptide 21). The an SA chip coated with C3b, and binding and dissociation were mea- reaction mixtures were then analyzed for C5 cleavage (revealed by the sured. The C3b-coated ﬂow cell was generated by immobilizing bio- generation of a9-chain) on a 6% SDS-PAGE gel under reducing conditions. tinylated C3b (189 RUs) on an SA chip followed by deposition of All of the gels were stained by Coomassie blue to visualize the cleavage additional C3b molecules (∼4000 RUs) by forming AP C3-convertase products and scanned for densitometric analysis. Results of the respective onto the ﬂow cell (26, 41). densitometry are presented in the right panels. The Journal of Immunology 7

100 mM concentration (Fig. 5). The CVF-derived convertase CVF, on the LP and compared it with that on the CP and AP using Bb is also known to cleave C5. Therefore, to study the effect of a commercially available ELISA-based functional assay kit Complin on C5-convertase activity, we measured inhibition of C5 (Wieslab kit, Euro-Diagnostica) (Fig. 7). Complin showed in- cleavage by CVF,Bb. The peptide displayed a weak effect up to hibition of all the pathways with IC50 of 3.1 mM for the LP and 6.1 10 mM concentration (14% inhibition), but showed ∼70% inhibition mM and 31.5 mM for the CP and AP, respectively. of C5 cleavage at 100 mM concentration (Fig. 5). Discussion Effect of Complin on activation of C2 and CPs and LPs Therapeutic targeting of the complement system has garnered Human fB is homologous in structure and function to the com- much attention in the past decade due to its involvement in plement protein C2. We therefore also sought to examine whether a plethora of pathologies (7, 10–13). Because the AP has been Complin inhibits C2 activation. To measure the effect of Complin implicated in mediating tissue damage in many human diseases on C2 activation, purified C2 was incubated with activated C1s in (12, 38), we sought to identify a small molecule inhibitor against the presence of various concentrations of Complin and C2 cleavage the AP component fB. Our efforts led to the identification of an was quantitated. The results showed that like fB, Complin also 11-mer cyclic peptide that showed inhibition of fB and C2 activation. The peptide has been named Complin. inhibited C2 activation in a dose-dependent manner. The IC50 for inhibition of C2 activation was 11.3 mM (Fig. 6). In the current study, we screened random peptide phage-display Because Complin inhibited C2 activation, we next examined its libraries against fB as a means to identify small molecule inhibitors effect on the CP using hemolytic assay. To selectively activate against fB because this technique has been successful in identifying the CP, EAs were incubated with fB-depleted sera, and the degree peptide ligands against various protein targets (39, 40), including Downloaded from of lysis was measured. As expected, Complin inhibited the CP- complement protein C3 (31). It is believed that this method is mediated lysis of erythrocytes in a concentration-dependent more successful in identifying ligands against various proteins because the peptide ligands favor binding to recesses or cavities in manner. Its IC50 for inhibition of CP-mediated lysis of erythrocytes was 2 mM (Fig. 6). the target proteins by displacing water molecules, and typically, Inhibition of fB and C2 activation by Complin (Figs. 2, 6) clearly such sites are biologically important (39). Our screening led to the suggested that Complin has an ability to inhibit all the three identification of five peptide ligands against fB, but only one http://www.jimmunol.org/ pathways of complement activation. We thus measured its effect showed inhibition of fB activation at a high micromolar concentration (IC50 =56mM) (Fig. 1, Table I). The same peptide (peptide 1) also inhibited AP-mediated lysis of erythrocytes, but Complin only at a millimolar concentration (IC50 = 2.7 mM) (Table I), Pep. conc. (mM) --20 10 5 2.5 1.3 0.6 0.3 presumably because of proteolysis of the peptide by serum en- C1s -+++++++ + zymes. Thus, to design analogs with increased efficacy and t1/2 in C2 + + + + + + + + + serum, we first searched for the presence of a motif in these fB- C2 C2a binding peptides. Interestingly, sequence alignment showed the presence of eight conserved residues with a highly conserved by guest on September 24, 2021 four-residue core (Fig. 1). Consequently, we synthesized a series C2b of constrained peptides (peptides 6–20; Table I) based on this

100 100 80 80 60 60 40 40 20 20 0 % CP activity inhbition 1 10 100 0 % C2 cleavage inhibition % C2 cleavage 1 10 100 100 100 80 80 60 60 40 40 20 20 0 % LP activity inhbition 1 10 100

% CP lysis inhibition 0 0.1 1 10 100 Complin conc.(mM) FIGURE 6. Inhibition of C2 and CP activation by Complin. Top panel, 80 inhibition of C2 cleavage by Complin. C2 was preincubated with various 60 concentrations of Complin at 37˚C for 15 min, and then activated C1s was 40 mixed and incubated for additional 1 h at 37˚C. The reaction mixtures were 20 analyzed for C2 cleavage on an 8.5% SDS-PAGE gel under reducing con- 0 % AP activity inhibtion ditions followed by Western blotting using anti-C2 Ab. Middle panel, 1 10 100 graphical representation of percentage inhibition of C2 cleavage. The in- Complin conc.(mM) tensities of the C2 were quantitated densitometrically and represented FIGURE 7. Inhibition of CP, LP, and AP activation by Complin. Effect graphically against concentration. Bottom panel, inhibition of CP-mediated of Complin on the CP, LP, and AP was examined using Wieslab total lysis of EAs by Complin. EAs were incubated with fB-depleted serum in the complement screen kit (see Materials and Methods). Top panel, inhibition presence of various concentrations of the Complin, and lysis was measured of CP by Complin. Middle panel, inhibition of LP by Complin. Bottom colorimetrically. panel, inhibition of AP by Complin. 8 COMPLIN INHIBITS FACTOR B AND C2 ACTIVATION motif with variations in ring size and residues flanking the four- could also serve as an important tool to study fB and C2 activation residue core. This exercise resulted in the identification of Com- process, the molecular basis of which is still poorly understood. plin, which was ∼6-fold more potent in inhibiting fB activation and 82-fold more potent in inhibiting the AP (Fig. 2, Table I) than Acknowledgments the parent peptide. We thank Profs. John D. Lambris (Department of Pathology and Laboratory Human fB is known to interact with C3b, the proteolytically Medicine, University of Pennsylvania, Philadelphia, PA) and Michael K. Pan- activated form of C3, and CVF, the C3 homolog present in Indian gburn (Department of Biochemistry, University of Texas Health Center, Tyler, cobra venom. We therefore examined whether there is a sequence TX) for continuous support. We also thank Dr. Jayati Mullick (National In- similarity between Complin and any part of C3b/CVF. We, how- stitute of Virology, Pune, India) for comments and critical reading of the man- ever, did not observe any obvious sequence similarity between uscript, Dr. Nicholas E. Sherman (Biomolecular Research Facility, University Complin and these proteins. Further, there was also no sequence of Virginia, Charlottesville, VA) for MALDI-MS and ESI-MS analyses, Dr. similarity between Complin and any of the known complement John Bernet for CVF purification, and Yogesh Panse and Sarang Satoor for regulators, indicating Complin is a novel peptide inhibitor of fB. excellent technical assistance. Structure-activity relationship studies in Complin showed that oxidation of cysteine residues is important for its binding to fB as well Disclosures as its functional activity(Fig. 3, Table I). These data therefore indicate The authors have no financial conflicts of interest. that cyclization is a prerequisite for maintaining the preferred structure of Complin for its binding and inhibitory function. Ala scan

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