© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. T B C 1 deposited is C3b C3b. and C3a into C3 cleaving (C3bBb), vertase B (FB) when it is complexed with C3b to generate an active C3 con quent amplification of central this activation step. FDcleaves factor and it plays a key role in both AP-initiated C3 activation and subse treatment may require inhibition systemically both and locally. effective their pathway,and the of also overactivation systemic diseases display these of many eye, the in or kidney the in example, (C3G) nephritis glomerulo C3 and (aHUS) syndrome uremic hemolytic atypical degeneration nocturnal age-related(AMD), hemoglobinuria(PNH), macular paroxysmal including disorders diverse predisposes to complexes individuals complement to antibodies stabilizing or proteins, regulatory complement to antibodies neutralizing tions, ruption and cell lysis. Dysregulation of AP activity by genetic muta dis membrane to leads which (MAC), complexmembrane-attack the of generation and elimination, phagocytic for important tion, complementopsonizaincludeC3-dependent are expressed. These ofmechanisms effector major the thatactivation C3 to subsequent (AP) pathway alternative the via of productionloop positive-feedback own the its of amplification in participates also C3b complement, C3, generating the C3a and C3b activation fragments. ways converge at proteolytic the path cleavage of third the component These of pathways. alternative the and lectin the classical, the initiated: be can activation which byroutes distinct three are there responses tory of complement-mediated diseases. the demonstrate feasibilitydata of inhibiting the AP with Thesesmall-molecule antagonists and support the mice.development of FD inhibitors FD-humanizedfor the treatment in activation AP lipopolysaccharide-induced inhibited administration oral inhibitors These FD. of efficiently block inhibitors alternative pathwaysmall-molecule (AP)selective activation and and prevent potent both C3 deposition of onto, and lysisidentification of, the PNH erythrocytes. Theirdescribe we Here (PNH). binuria hemoglo paroxysmalnocturnal and degeneration macular age-related including disorders diverse to individuals predisposes dysregulated, when which, process, amplification this for essential is (FD) D factor protease pathways.Thetwo other the distinct for three elimination. by initiated their be can promoting C3 and of Activation pathogenssystem. routes—the classical,the the recognizing lectin and of the alternative system,component pathways—with centralthe alternative immune pathwaythe also acting is as an innate (C3)amplification loop 3 the component Complement of component key a is Complement Bruce Jaffee Gould D E Jürgen Maibaum alternative complement pathway S nature CH D of Medicine, N asel, ambridge, Massachusetts, ivision ofHematology, dwige Lorthiois ovartis urand Complement factor D (FD) is the rate-limiting enzyme of the AP, mall-molecule factor pathogenic cell surfaces and trigger cell lysis and inflamma and lysis cell trigger and surfaces cell pathogenic of line defense against firstinvading pathogens. Its components effective recognize an constitutes system complement he B asel, Switzerland. 2 I nstitutes for , E 1 MIC A , KamalFettis C engus Mac ardiff A 1, L BIOLOGY 2 . Depending on the molecular recognition pattern,recognition molecular the on Depending . 4– 2 7 , U . While disease manifestation is often local, for local, often is manifestation disease While . R niversity, HenryWellcome B ichard Harrison ioMedical Research, N 1 5 1 , aples, D *,

| Adv P rug U S aul SA. ha-Mei Liao D S a I 1 taly. *e-mail: nce online public iscovery , weeney 3 E E S vonik Japan rbel olene D 1 , BerndKinzel epartment, Actelion epartment, N 5 [email protected] ovartis D , BerndGerhartz 6 C , B ussauge o., Shinjuku-ku, 2 uilding, Heath A , a A tion | ntonio Maria P nna Vulpetti harma AG, www.nature.com/naturechemicalbiology 1 , N P P 1 T , Fabrice ark, harmaceuticals okyo, Japan. N icola Hughes 2, ovartis 3 D . It is It . C ardiff, 1 , Frederic Cumin R 1 inhibitors targeting the ------, C isitano pu N ampus, U bli ciain oh ytmcly n i oua tsus Furthermore, tissues. ocular AP in and (LPS)-induced systemically both lipopolysaccharide FD-humanized activation to inhibited administration mice oral C57Bl/6 their and blood, human whole in activation AP blocked efficiently inhibitors These ing. structure- based design a approach in ofcombination with use fragment-based screen by FD of inhibitors small-molecule reversible to linked APdysregulation.eases FD inhibitorsspecific are currently available for oral therapy of dis degeneration (AMD), its use is limited to intravitreal injection macular age-related dry of treatment the for trials clinical in is ity activ FD neutralizing of capable (Fab) fragment antigen-binding multipleproteasesotherS1 prevents use clinical their toward specificity of lack however, reported; been have inhibitors thioesters lysine-derived synthetic against activity catalytic weak exerts still it but substrates, peptide pocket S1 the bottomof the at the non-prime recognition sites, and an Arg218–Asp189 salt bridge to access restricting 214–218) (aa loop self-inhibitory a formation, con inactive an in triad catalytic the comprisingarchitecture site C3bB substrate formationalchange uponendogenousbindingitssurface-bound to con a requires FD of activity proteolytic Full latent,form. self-inhibitedbut mature, a in plasma in circulates which kDa), 24 (aa), and lysis cell disruption membrane subsequent with surfaces targeted on convertase, leading to C5a release and MAC (C5b to C9) formation C5 a generatescomplex C3bBb membrane-bound the to molecule onto acceptor surfaces (opsonization). Recruitment of a further C3b ils Ostermann 4 A K. P harma Research and Kolb L s 7 In this report, we describe the discovery of potent, selective and potent,selective of discovery the describe we report, this In td., Allschwil, Switzerland. U h niversity of B or ed 7 asel, Switzerland. 1 , Jörg , Omar [email protected] 8 o . FD is a trypsin-like S1 serine protease (228 amino acids amino (228 protease serine S1 trypsin-like a is FD . 4 n , li S

ne: 24 O amuel Barbieri E N 9 Ltn F i caatrzd y n tpcl active atypical an by characterized is FD Latent . 1 aples, der , D S 1 elgado , tefanie Flohr E c 1 S arly &Karen 2 D t N tefan ob epartment of epartment ovartis D er 2016 | evelopment, Roche 6 I nstitute of 2 , 10, R I U nstitutes for 1 1 andl . The protease does not recognize not protease does The . 1 lrich Hommel , JuliaWagner A

d C 1 nderson o 2 linical Medicineand Surgery, 1 Irvril cvln FD covalent Irreversible . I , nfection and 3 i : ,

A 1 0 S nna . 1 imon B 0 ioMedical Research, article I 3 nnovation 8 / 2 S n * chubart c I R mmunity, School h 1 üdisser , e 13– 1 m T , Corinne C 1 enter y 5 b . Whilean. i o . 2 1 , 2 1 6 0 1 . No , 8 ------1

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. article to bind to cryptic or transient pockets of protein surfaces. Because Because surfaces. proteinof pockets transient or cryptic to bind to cally well below 300 Da, and high solubility, rendering them fragment-based suitable employed we screening selectivity, on-target and affinity improvedwith motifs To structural bindingalternative S1 discover G hydrophobic indentation of S2 the CF the and bond, disulfide Cys42–Cys58 the to proximity close in scaffold proline central the His57 side chain adopts an outward conformation to accommodate distance to the NH of Gly193 forming part of the oxyanion hole. The bottom of the S1 pocket. The urea spacer carbonyl is within binding atthe nitrogenArg218of the todistance 3.4-Å oxygenatestera lic carboxy the positioning bond, Cys191–Lys192 peptide the against packs and loop self-inhibitory the of Gly216–Arg218 by flanked is andS1 pocket occupying S1 the bentbindsconformation,a in molecule penetratingdeeply into the key binding features consistent with our library design concept. The optimization. 33 of value IC an with complex CVF–FB the against activity proteolytic FD data, biophysical and biochemical the with Consistent substrate endogenous surrogate a as complex (CVF)–FB factor ( cleav Ba B factor product age the of formation the of inhibition the determine 1c Fig. of binding direct 9 ( concentration(IC inhibitory half-maximum a showed compound identified substrate thioester-based a againstproteolytic activity FD compoundsinhibitionoffor these ( docking (GLIDE S2 and S1 the into attractive opportunity for aFD-tailored inhibitor design. of the topology the latent FD active site with compatible be might pose binding this that suggested tions, KLK7-bound of orientation The conformation. non-catalytic displaced a in chain (S1 site prime the into extends and pocket S1 buried the occupies effort, discovery drug separate ( protease serine compound of ( a approach in combination with deliver validated hits. toWe therefore failed applied a formation, structure-based library design MAC on or thioesterolysis on FD either based screens, high-throughput Two activity. proteolytic FD blocking of bioavailability,capable oral with molecules, small tive selec discover to out set we strategies, multiple-hit-findingUsing I RE currently either no or sub-optimal therapies. are there which for diseases complement-mediated treat to FD of of inhibiting the AP with highly specific small-molecule antagonists from erythrocytes with PNH patients. Taken together, confirmed these data demonstrate were the feasibility findings These rocytes. human of, lysis anassayin sensitivity thatlytic mimics oferyth PNH erythrocytes and on, deposition C3 prevented inhibition FD 2 bindinga of space chemical samplelarger-sized the tothanligands suited better much arecomplexity, lower they intrinsically their of dentification ofsmall-moleculefactor Dinhibitors Supplementary Table Supplementary 1 Results Supplementary

μ eneration ofpotent and selective inhibitors S The crystal structure ofstructure crystal The of analogs proline of set A M). S )-proline-based library was inspired by the co-crystal structure co-crystal the byinspired was library )-proline-based UL ). We further developed an developed Wefurther ). 1 TS H– 2 0 . Fragments are molecules of low molecular weight, typi 15 μ N HSQC NMR spectroscopy M, confirming M, 1 in complex with kallikrein-7 (KLK7), a related S1 related a (KLK7), kallikrein-7 with complex in Fig. 1a Fig. 1 7 1 upeetr Fg 2 Fig. Supplementary 2 ) of a virtual library to the crystal structure) of of library toa virtual FD the crystal frig eea ky yrgn od interac bond hydrogen key several forming , to uniformly to ′ okt, a slce fr ytei bsd on based synthesis for selected was pockets, , b ) and was equipotent against KLK7 (IC , ). Compound ). Supplementary Fig. 1a Fig. Supplementary 2 3 in complex with FD ( complexFD in with O group of group O 2 as a viable starting point for further further for point starting viable a as 1 in silico in ′ ′ , bearing moieties that could reach could that moieties bearing , –S2 and S2 15 ′ binding site. in vitro in N-labeled FD ( FD N-labeled ′ ) with the catalytic His57 side His57 catalytic the with ) docking of fragments. Use of 1 ′ 0 1 sites. The benzoate moiety , thus providing us with an 2 uig h cba venom cobra the using ) 1 , which originated from a from originated which , 8 is positioned in a small small a in positioned is was used to demonstrate FD proteolytic assay to assay proteolytic FD 2 ( , b Fig. 1 Fig. Supplementary Supplementary Fig. 1 Fig. ). Evaluation). of nature 50 2 ) of 14 of ) c inhibited ) revealed revealed ) a ), which which ), ch μ 50 e N mic M = 1 50 9 ------. at a l

to the 6-aza-indazole the to led cycles, design iterative of number a in optimization chemical in as scaffold, indole-like an with ( pose lapping itsbindwith toing computationalmodel by predicted was and collection compound Novartis the in search structure-similarity retrospective a by tified log prolineana bysupported was concept design inhibitors.The both the direct binding contacts to protein. the of moiety acid and the Thr214 carbonyl of the S1 andpocket, the tethered benzoic guanidine Arg218 the to bonded hydrogen is group carboxamide ( chain side Lys192 the and loop self-inhibitory the by flanked and S1 in positioned indole ide tureof ( 1 to water-solubleanalog, carboxylic acid more a synthesized affinity,we binding of quantification for allow identified FD of ence (WaterLOGSYNMR ligand-observation by tration) 100 at fragments top-scoring of set diverse PDB the on based mations 3a Fig. weight (molecular fragments 52,000 of set a selected first and approach structure-based focused, a chose we fragments, of libraries random screening than Rather affinity. 2 compound as such motifs, structural other with fragments bining com or growing necessitates This target. biological the to weakly only bind may they but campaigns, HTS in than screening based site is inaclosed,theHis57 sidechaininanon-catalytic‘outward’ complex with S2 site iscolored inyellow. ( bonds to Gly193 andHis41 are highlighted. ( compounds FD inhibitors. Figure 1|Structure-based evolution ofreversible small-moleculehuman moiety isexposed to solvent. hydrogen bondsofthe conformation. ( biology ure H– b K bc a in the case of FD, in order to generate compounds with higher higher with compounds generate to order in FD, of case the in ) d 3 MeO S2 The co-planar superposition of the S1 binding moiety of moiety binding S1 the of superposition co-planar The D102 ) = 1,600 = ) C 2 1 5 ortho , showed concentration-dependent chemical shift changes in the 15 1 KLK7IC o-crystal structure of . Consequently, hits are generally more frequent in fragment- in frequent more generally are Consequently,hits . (IC N HSQC NMR (averagespectrum NMR dissociation constant FD IC ), docking them them docking ), S1 4 ch

| Adv boundresolution) to(1.46-Å FD showed 2-carboxam the 50 H57 -benzoic ester portion of 50 50 = 5.8 1 S1 G193 0.7 >30 – emi ′ a 2 ( O 4 H N μ (orange) spanningS1–S2 nce online public d a determined by protein NH µ M; Fig. 2 Fig. ) Structure, bindingaffinitiesand ) Superpositionof O µ M H41 S2 4 N M μ ′ s xoe t te ovn sae ihu making without space solvent the to exposed is M, thioesterolysis assay; ca Supplementary Fig. 3b Fig. Supplementary N 4 b -methylindole portion to S1 in a similar oversimilar a in S1 to-methylindole portion carboxamide to Arg218/ ). Combining a carboxamide group, as in as group, carboxamide a Combining ). l biology 3 6 2 ( ( in silico in 1 KLK7IC (orange) incomplex withK Fig. 1 Fig. c HN FD IC S2 Fig. 2 Fig. CO ) Ribbondiagram oftheF 2 1DI N Me H57 i. 1 Fig. O a S1 50 50 a tion | O 9 14 C 2 into distinct FD active site confor site active FD distinct into a ) as a hit. To ascertain binding and bindingTo hit. ascertain a as ) N H S1 G193 (orange) and ) as a highly potent and selective selective and potent highly a as ) µ 1 µ ′ 4 M 2 crystal structure. Screening of a of Screening structure. crystal M 5 N d ( ′ , followed by further extensive further by followed , L41 www.nature.com/naturechemicalbiology ). The deeply buried terminal terminal buried deeply The ). MR andbiochemicalassays. OCF . S2 Fig. 1 4 T ′

T d ( 3 he self-inhibitory loop(pink) he surface oftheunoccupied o Fig.1 4 3 ≤ R=H R=CO 350 Da; Da; 350 i Fig. 2 , : d c

R 1 ). The co-crystal struc co-crystal The ). ) prompted us to merge 0 T μ in vitro d D189 4 . hr214. 1 a M (nominal concen (nominal M 2 (green) showing 0 H ), which, in contrastin which, ), a 3 FD NMR NMR solubility~30 D FD NMR NMR solubility2,000 ), which was iden 8 R218 T214 L active site in / potencies of K7. Hydrogen O Supplementary Supplementary n T 2 he c 2 S1 ) in the pres the in ) h K K 4 S1 e d d >30 benzoate ′ 1,600 m L41 N H b i µ o 4 M µ O NH . with with

µ M 2 2 M 2 µ 0

M 4 8 ------,

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. N in human versus mouse IC mousehumanversus in (IC Compound Compound strengthens interaction. this the Trp141; with bond halogen–carbonyl a carbonyl of TheLeu41. the and Gly193 of NH backbone the and S1 in Ile227 and Ser190 Arg218andforms additional water-mediated hydrogen bondswith to contact binding close in is ligand the of group carboxamide nal a adopting ( His57 conformation catalytic-triad ‘non-canonical’ catalytic the and site non-prime the shielding binding extendingpose from S1 to S2 ( 10 proteasesand kinases channels,receptors,ion of panel toassay broad (up effects significant no and Methods), Online (see activationcomplement-pathway lectin and classical of bition ( proteolyticactivity FB of tion Moreover, (IC blood whole human 50% anticoagulated (IC effec inhibitor serum human The 10% in hemolysisAP-mediated setting. both blocked tively physiological more a in activation was inhibited by alternative the assaybiochemical pathway. the in FD byProteolytic of FB cleavageof protease serine second the B, factor to binding protein–ligandcomplex ( ( plasmon surface resonance (SPR) spectroscopy, using assay binding direct a In FD. of inhibitor nature CH conformation distinct a adopts (Trp215–Arg218) self- loop the inhibitory that revealed which resolution, 1.25-Å at FD murine of closed conformation andHis57 in‘outward’ position. three technical replicates. Average using standard kineticsS P crystal structure of of Figure 2| Supplementary Table 1 2c Fig. d a roteins were exposed (arrows) to increasing concentrations of at 5 Response (RU) h cytl tutr of structure crystal The compound of ability the determined we Next 26 22 –2 10 50 50 14 18 (yellow andpinksticks) incomplex withF 6 2 0.86 = 0.006 = CV 0 20 ure , HN F–F N d 40 I E dentification andcharacterization ofpotent andselective humanFDinhibitor ;

MIC 60 6 B Supplementary Table 2 Table Supplementary is highly selective for human FD, showing no inhibi no showing FD, human for selective highly is 6 assubstrate. ( 80 N 6 ch μ demonstrated only modest inhibition of murine FD murine of inhibition modest only demonstrated O A 100 μ selectively blocks factor Dactivity ) I odr o nesad h srkn difference striking the understand to order In M). L BIOLOGY

M) and AP-induced MAC formation in lepirudin- in formation MAC AP-induced and M) 120 5 Time (s) P 6 4 140 O emi R. ( with an IC (green sticks) incomplex withF 160 N H

e 180

) 200 ca IC meta

| Adv 220 ). k 50 f

) 240 on determination ( 50 I 260 l biology = 1.64 × 10 × 1.64 = [nM] nhibition ofA 6 values, we solved the crystal structure crystal the solved we values, -Cl -Cl substituent positioned in S2 a 50 on t F cnimd h overall the confirmed FD to bound 280 nce online public 900 300 100 33.3 11.1 3.7 1.2 0.4 value of 0.03 6 IC Supplementary Fig. 2c Fig. Supplementary 300 showed an average 50 e value was 144nM.( H O ), with a fast association of the the of association fast a with ), % inhibition of Ba Formation 2 100 N ′ 40 80 60 20 , with the self-inhibitory loop 0 6 P 10 M D n -mediated MA –3 =4experiments) for inhibitionofF suggesting two distinctbindingconformations withintheS1pocket. oftheindoleportion N −1

N d μ 50 N s ortho M ( 10 o a −1 = 0.14 = N Fig. 2 Fig. tion | –2 i D ), and did not shownot did and ), : Compound

. ( 1 O Fig. 2 0 -F atom further further atom -F c 6 O . 10 1 ) H N K o nii AP inhibit to 0 www.nature.com/naturechemicalbiology g –1 g D d ). The termi The ). 3 μ ) of 0.006 6 C F irect bindingof , 8 in vivo e d M; C formation by 6 / ). ), no inhi no ), 6 rystal structure ofF 10 [ n μ IC (single-cycle kinetics). R µ 0 c ) n a in M) Fig. 2 Fig. M] 50 Cl ′ h =0.03 forms forms e 10 m μ 1 b f M ). µ i - - - - o M b

10 . 6 6 2 in50%wholebloodfrom four individualdonors. T214 2 to immobilized F 2 sions that can be drawn with respect to local retinal complement retinal local to respect with drawn be can that sions ration of retinal tissue from the eye vasculature, limiting the conclu compound LPS, after h 3.5 gavage oral by administered or C3d/iC3b were ~2- to 3-fold of those in the control group. When Fig. ( 4 tissue ocular and plasma both in iC3b and C3d productsbreakdown C3 the and Ba of levels rising by indicated as time-dependent increase in AP and activation after intraperitoneal dose- (i.p.) a injection of LPS, observed we mice KI human-FD In plement activation in mice by injection of lipopolysaccharide (LPS). com systemic We model tissue.to retinalsought in manifeststhat nents and evidence of systemic AP overactivity presencethe of compound alternativeinhibition complementthe of the that dence pathway in ( mice type expressingofmice human serum FD,ofwild- the butserum not in different the compoundarchitectures, site of active result a is which FD, of inhibition for seen tivity reported previously as FB, murine CFD for humans used mice KI 0.78 female of level blood enzyme orthologous human (human-FD knock-in (KI) mice) ( the only expressing mice C57Bl/6 inhibitor accommodate to order in required self- be thus FD would loop murineinhibitory the of movement conformational pocket. significant S1 A narrower the to binding inhibitor preventing Gly216 protease human the to compared 10 mg/kg ( at inhibition full with tissue, ocular and plasma both in post-dose h 4 determined as activation complement inhibited dependently D189 0 f

% inhibition of MAC formation 8 100 AMD patients commonly have genetic alterations in AP compo pursue to order In 110 40 80 60 90 20 50 70 30 D 10 D 0 −/− 10 activity by μ -bound ). At the 7.5-h time point after LPS administration, levels of Ba R218 –3 /L ( g/mL ie eosrtd ht ua F efciey activates effectively FD human that demonstrated mice 2 S1 4 Rcnttto eprmns ih eu otie from obtained serum with experiments Reconstitution . 6 10 U 6 in the S1binding inthe site ( Fig. 4a S1 Fig. 3 Fig. –2 , response units.( . ( 6 ′

a spanningtheS1–S2 i. 3 Fig. ) D 10 6 C (blue)andF asindicated by formation oftheF –1 d hemical structures of , b ). These resultsprovideevi strongThese experimental ). ). Technical limitations do not allow clean sepa µ 10 M c ad ec wti te ag rpre for reported range the within hence and ) 0 in vivo in 10 Donor 4 Donor 3 Donor 2 Donor 1 1 6 d B is indeed target is indeed specific. ) (red) determined by S pharmacology studies, we generated we studies, pharmacology B 10 inding of ′ c 2 pockets. Self-inhibitory loop(pink)isin Response (RU) Fig. 3 Fig. 3 2 26 22 –2 g 10 14 18 2 5 6 2 3 . In line with the species selec species the with line In . wt te abnl ru of group carbonyl the with , 6 5 0 Compound injections blocked C3 cleavage only in only cleavage C3 blocked and 100 6 R218 a b to immobilized F

). 200 ). The average human-FD H57 6

E 300 . ( rror bars denote s.d.of S1

b 400 n vivo in 24, ) B

C 500 cleavage product 26– Time (s) P article omputational model

R spectroscopy. 600 2 8 Supplementary Supplementary G193 S1 with pathology O 700 ′ verlaid isthe L41 tde was studies 800 Binding toFD Binding toFB D S2

by 900

′ 1,000 6

dose- 1,100 1,200 B a, 3 ------

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. article of glycosylphosphatidylinositolof(GPI)-anchoredcomplement regu in the and ment deposition onto, and hemolysis ( of, PNH erythrocytes Importantly, compound Factor Dinhibitionprevents lysis ofPNHerythrocytes concentrationmaximal efficacious (EC half- a with post-dose h 8 activationforatleast AP LPS-induced of ( tion in all animals to maintain a consistent interval of LPS exposure termina before h 7.5 given (i.p.)was LPS termination,while study separate groups of mice at time points ranging from 4 to 24 h before the duration of action, a single 30 mg/kg oral ofdose inhibition and blood-retina barrier penetration of mice (triangles; (circles; ( ± by western blottingwithanti-humanF male ( exons 3and4.( replacements ofmouseexons 2and5to complete deletion ofmouse indicated asdashedlines. top. encoding for thepropeptide andthemature F codon aswell asthestop codon are indicated. T ( pose incomplex withhumanF in yellow). of theself-inhibitory loopare highlighted assticks (pinkcolor, with Gly216 diagram ofapo-mouseF Figure 3| 4 vitro in (ref. CD59 and CD55 lators d b Supplementary Fig. 5 s.d.were 0.78 he five exons are displayed asblackboxes (

c b a ) Schematicrepresentation ofthemouse ) Human factor D (µg/ml) Mouse 0.0 C 2.0 0.5 1.0 1.5 I Supplementary Fig. 6 Fig. Supplementary ntegration sites ofthehuman ompound n PIGA locus (chr.10) =57) , normal human werered cells blood (RBCs) incubated with IC Cfd G 50 O genomic eneration ofhumanfactor Dknock-in mice. Female value of0.07 verlaid isthestructure of gene in hematopoietic stem cells, resulting in deficiency Human C 6 57 c n ± inhibits ) HumanF =2). 0.33 B W215 l/6 human-F CFD R218 ATG E1 cDNA S217 μ D g/m C ). Compound active site. I ± nsertion ofthehumanc nsertion 3 depositionin50%serumofhF Male 6 D 0.03 was active in an assay that mimics comple L levels inthebloodoffemale ( Propeptide G193 and0.63 ). PNH is caused by a somatic mutationsomatic a by caused is PNH ). D (protein structure omitted). D μ 5 D189 knock-in (hF M, . o iuae h pathophysiology the simulate To ). CFD S1 S1 ′ T E2 6 D n d rp215, Gly216,Ser217andArg218 c =4,mean (turquoise sticks) initsbinding antibody. Average plasmalevels ±

DN % inhibition of C3 deposition 0.24 6 –50 100 50 showed sustained inhibition Cfd 0 50 A inthemouse E E3 D ) of 0.034 1– 0.001 T gene locus(bottom). D protein are shown on μ he human E g/m K 5), andtheA Mature peptide DN ± I 0.01 ) mice were quantitated S2 s.d.),butnotwild-type L ′ A leads to partial A leadsto partial , respectively. E4 ( 6 0. μ a in mice. To test 11 M ( CFD µ ) Ribbon n D 6 M Cfd =101) and nature

was given to K T Fig. 4c c gene are I G start G start mice DN E5 10 Stop A Fig. 5 ch

, d 100 e ). N mic

a - - - at

a l

Methods and N oralafter dosingof inhibition ofplasma activation inC57 Figure 4| the the dependent examined wepatients, PNH from transfusion erythrocytes Furthermore,using remain patients eculizumab-treated of ~40% that finding the forexplanation plausible a provides coating liver.C3 and This spleen the in hemolysis extravascular to ceptible patientseculizumab-treatedPNH in monlyobserved 6 Fig. Supplementary rocytes that were extensively coated with C3 fragments ( standard of care for PNH contrast,anti-C5 the monoclonal antibodyeculizumab, current the 0.07 lysis and C3 deposition on the surface of with RBCs an IC acidification serum by vated acti was AP the and CD59, and CD55 againstantibodies blocking compound administration. confidence band. used to calculate duration-of-action studies( ( compared to positive control in D the level in T before tissue collection (see dose of indicated. ( lar hemolysis, but any also C3-driven pathology. tial advantage over anti-C5 therapy of blocking not only extravascu Targeting the AP amplification loop in PNH therefore( lysis has their the prevented poten and erythrocytes PNH negative compound F ability ( compoundfor P cells Caco-2 in efflux no and permeability higher showed but blood), whole human 50% using 1 Table which has similar biology ure d he level ofbreakdown products in cd ab 2%. n ie ih t atvt i te urgt PH assay, PNH surrogate the in activity its with line In 28%). = app ata shown asmean egative ( ) Relative plasma Ba or T (B-A) × 10 ex vivo erminal concentration of

μ ocular C3d/iC3b

0.0 Relative plasma Ba 2.0 0.5 2.5 0.0 0.4 1.0 0.8 0.6 0.2 1.5 1.0 1.4 1.2 M, consistent with inhibition of the AP amplification loop. In 6 ch

F ; IC ; 0 | Adv , administered to separate groups ofmice 24, 16,10, 8, 6,or4h = 100%) in rat ( I N Neg nhibitor ( c P efficacy ofefficacy the structurally related compound eg) andpositive control group ( ) os isindicated by thelineslabeled 50 LPS plasma LPS ocular 7 emi Supplementary Fig. 5 P a * = 0.05 = fully blocked C3 deposition on the surface of CD59- of surface the on deposition C3 blocked fully lasma nce online public ** −6 Pos

B EC D 6 (cm/s) = 18.1; (B-A)/(A-B) = 1.19; respective values Oral doseof l/6 mice expressing humanFD. ** 10 mg/kg 81 : 1.35, 9.66 and 7.17) and improved oral bioavail improvedoral and 7.17) and 9.66 1.35, : ata pointsongraph are color coded to time(h)after 6 ca 50 B in vitro in 6 (1, 3, and 10 mg/kg) given 3.5hafter (1,3,and10mg/kg) Time (h) ± a ( B * =0.034 ) blocks *** s.e.m. in a andocular μ a

3 mg/kg l biology M in the AP-induced MAC formation assay formation MACAP-induced the in M ). Such C3-fragment-coated RBCs are com are RBCs C3-fragment-coated Such ). ) andocular 6 n *** 3 Supplementary Table Supplementary 4 potency to compound O =62), relative to plasma 0 6 , blocked hemolysis but generated eryth 6 1 mg/kg nline Methodsand L μ PBS ineachsamplefrom dose–response and a PS-induced systemic andocularAP a 2 M for – 9 ** – a . Compound . c 3 c tion | by A 2 N ; * ). C ( eg was setat1(linelabeled 3d/i P n P C 6 24 <0.05, ** =4–10 female mice pergroup as app 3d/i . NOV www.nature.com/naturechemicalbiology D C AB × 10 × (A-B) ashed linesrepresent the95%

3b levels after one 30 mg/kg 3b levels one30mg/kg after d P Relative ocular C3d/iC3b C % inhibition 100 0.0 0.4 0.8 0.6 0.2 os) are indicated (see 1.0 1.4 o A with 1.2 40 80 60 90 20 50 70 30 10 3b ( 0 i LP : 11

1 6 P Neg 0 S. b Supplementary Fig. 5 inhibited both hemo both inhibited <0.01, *** ( . ) determined 4h 1 n D a 0 =4mice pergroup. 0 , 3 unnett’s posttest. b B 6 Pos 8 ; for compound ) a inhibition,was Oral doseof ( −6

10 mg/kg / D Supplementary Supplementary n c/) 15.3; = (cm/s) 100 ose-dependent LP nM c 3 *** 1 h P S injection. and are sus Fig. 5a Fig. <0.001 1,000 3 mg/kg e Fig. 5 7 m ( 50 PB ** 6 24 16 10 8 6 4 b Fig. 5 value of O i h 10,000 S);

1 mg/kg o nline a . , 2 *** c and and 2 , ). d b 0 2 6 ). ), 9 8 ------: .

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. N sustained inhibition of FDat asystemic level. compoundssmall-molecule are the first inhibitors that would allow systemic by application of a disturbedFD antibody or antibody fragment unfavorably is clearance, renal rapid with level of endogenous FD, the result of a high production rate coupled tion to the inherent short half-life of Fab fragments, the steady-state addi In injection. local to restricted is atrophy, use geographicits of treatment the for trials clinical in currently is activity catalytic FD neutralizing of capable fragment Fab an Although inhibitors. final the to led ultimately and motifs structural different of nation combi the allowed compounds, various the of modes binding the of understanding structural detailed a combinationwith in which, an of screening dedicated subsequent the was inhibitors initial the ofoptimization the to Central FD. of inhibitors small-molecule reversible and tive approach library discoverpotent,selec chemicalto a coupledwith utility of structure-based focused screening and the rational demonstrates drug design here described work The collection. compound find not did we any suitable inhibitor that by high-throughput screening fact of a 1.2 million the by reflected is This discovery. drug for target challenging a family protease S1 the of enzyme this ders self-inhibitory a loop that preventsand access to the non-prime triad sites, ren catalytic the of conformation distorted a by acterized char D, factor latent ofarchitecture site active non-canonical The D erythrocytes was determined by flow cytometry (see ± erythrocytes. no PN neutralizes serumcomplement activity. antibody. from a ( and C classical pathway. Hemolysis was measured by increase inopticaldensity. the alternative pathway andprevent antibody-mediated activation ofthe into acidifiedseruminthepresence ofMg were incubated with eculizumab (see of erythrocytes from healthy donors for compound PNH erythrocytes. Figure 5| nature CH by rationalized be can This chemotype. inhibitor this for observed c c a s.e.m. isshown. urves are representatives of ) I PNH Normal Hemolysis (%) at H erythrocytes disappearfrom thegate intheabsence ofinhibitor, and SC C 100 Significant species specificity for human versus mouse FD was was FD mouse versus human for specificity species Significant C 40 80 60 20

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) m 10 b 5 i - - - - - o . 2 2 organs such as the kidney. Fully effective treatment of may these diseases proteins), predisposes to a number of diseases with local manifestation in C3 convertase (either the directly or by stabilizingneutralization of auto-antibodiescomplement regulatory or proteins, constituent its in tions mutagenetic consequentto AP the dysregulationofsystemic addition, apeutic advantage over the current standard of care in PNH treatment. In deposition on erythrocytes which prevents MAC-mediated intravascular lysis but does not block C3 therapy,anti-C5 receiving patients in apparentmore becomes often sis hemoly Extravascular spleen. and liver the in hemolysis extravascular Kupffer cells, rendering C3-coated subject erythrocytes to destruction by and phagocytes onexpressed 4 andcomplement by3 receptors nized 1, recog are fragments C3 Opsonizing patients. PNH from erythrocytes Additionally,compound erythrocytes. of opsonization C3 inhibited also but lysis pathophysiology. Both compounds not only blocked AP-induced hemo are result the infact of selectively blocking FDproteolytic activity. compoundConsequently, crystallography. X-ray by revealed as FD human and murine of conformations site active distinct the and and codes accession with Bank Data Protein pounds KLK7– human codes. Accession version o the in available are references associated any and Methods M published online24 October 2016 R therapeutic benefit. tion-specific basis, against the exciting prospect for their significant indica an on this, evaluate to and inhibitors, FD of profile safety clinical the define to be developmentwill their in step logical next The complement-relatedof treatment diseases. the pathwayin tive therapeutic versus effect any potential sideeffects. desired the for titration better permit would this feasible, If type. patient’spheno convertthe low-risk toto be high-risk fromstatus would objective therapeutic the where (PNH), mutations somatic mechanisms, regulatory for example, and due to stimulatorygenetic polymorphisms (AMD) or between imbalance an of result the is activation AP where diseases forimportant be may This therapies. antibody-based with that more interventionthan tunablepharmacologic allow principle, in would, inhibitors small-molecule of use the addition, In activation. pathway classical antibody-dependent efficient throughpathway lytic the recruit therefore can and nated vacci adequatelyare patients if true particularly be will This tion. which the lytic pathway is critically important for eliminabacterial a carry reduced risk for infections such as alternative-pathway blockade, when compared to C5 blockade, may specific that possible discovery,is contextit this In risk. infection for drug also but with associated traditionally risks off-target and to evaluate the clinical safety of FD inhibitors, not just for on-target infection bacterial to susceptibility potential utility clinical of oral FDinhibitors for such use. the support findings our and level, systemic a at inhibition AP require clude that the selectivity,and potency conofweterms profile in biochemicalable vivo in we had to generate human-FD KI mice to demonstrate AP inhibition not block AP activation in the serum of C57Bl/6wild-type mice, and 0 eceived 20May 2016; accepted 15 8 e Inhibitors Inhibitors The work presented here offers a new way to target the alterna the target to way new a offers herepresented work The Genetic deficiency of AP components is associated with increased th 5FC . Based on these findings, as well as on the compound’son the aswell as findings, onthese Based . favor o 2 R f the papef the ds , , respectively. 4 7 and and was found to prevent both lysis and opsonization of of opsonization and lysis both prevent to found was

6 in vivo in and and 1 ope, o hmn D n ope wt com with complex in FD human for complex, 6 Atomic coordinates and structure factors for the the for factors structure and coordinatesAtomic , and for the mouse apo-FD are deposited in the the in deposited are apo-FD mouse the for and , r . and and 7 ee cie n n sa ta mmc PNH mimics that assay an in active were 3 ex vivo 1 . Inhibition of FD may therefore offer a ther effects observed with effects observed our inhibitors 3 3 . It will therefore be important be therefore will It . A ugust 2016; 5FA Neisseria meningitidis H , 5FB article E , 5FBI , online 5FC 6 did did , for K 5 ------

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. article 24. 23. 22. 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1. R 6

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33. 32. 31. 30. 29. 28. 27. 26. 25. We thank T. Zoller for the preparation of the proline-based compound library, A requests for materials should addressed to be J.M. or K.A. available online at available inthe Any supplementary information, chemical compound information and source data are A of pape the The authors declare competing interests: financial details accompany the Competing financialinterests work, the ments, and supervised wrote manuscript. the ofments part work. the and J.M., supervised J.E., and K.A.conceived A.S.,R.H. experi O.D. and the S.-M.L.performed produced recombinant proteins. T.G. and designed interpreted pharmacokinetic studies. data.the S.B., B.K. and F.A.K. generated human the mice. FDknock-in B.G. and J.E. assays the performed data.the from A.M.R. using PNH patient erthrocytes and analyzed biochemical assay data. A.S.and J.W. PNH the performed surrogate assay and analyzed ing measurements. affinity N.H.and P.E. conducted SPR experiments. F.C. generated the compounds. S.Rüdisser and P.E. NMRexperiments performed for screening and bind E.L. and J.M. inhibitors. FDstructure-based the designed S.D., C.D. and K.F. synthesized N.O. and A.M.S.solved structures the by X-ray crystallography. U.H., S.Randl, A.V., A ing human assays, whole blood and L.Ferrara for performing APhemolytic the assay. and G.Marsh for pharmacokinetic and bioanalytical support, Erkenez A.De for perform iments, C.Dentel and F. Tritsch for chemical syntheses, U. Argikar, A.Brown, S.Bailey preparation of F. for Zink assistance inpreparing FD and KLK7inhibitor complex S.Kapps crystals, for mice,knock-in N.Buchanan for coordinating maintenance the of mouse the colony, J. Wirsching, T. and Doll A.Isken for assistance their ingenerating human the FD biology ure cknowledgments dditional information uthor Contributions

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2 - 0 - - 8 - - © 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. ua KK fursec-ieie assay. fluorescence-lifetime KLK7 Human (Q Sepharose), followed by an enterokinase cleavage step and SEC. final 50 of concentrationprotein final a at GSSG mM 0.5 and GSH mM 1.25 EDTA, CaCl NaCl,mM mM 10 containing500 cold)urea, M 2 °C (10 buffer 8.0 Tris/HClpH mM 50 with °C 10 soluat bodies of inclusion bilized dilution rapid by refolded was and bodies inclusion of form the in in expressed was enterokinasecleavageKLK7 site. and pro-peptide His-tag, comprising extension N-terminal the after directly Arg. The PCR products were cloned into pET-24 a modified vector (Novagen) by replaced wasTyr180 residue KLK7, ofToautocleavage prevent collection. (aa 37–253) (UniProt; P49862) was amplified by PCR using an in-house cDNA mutant). (Y180R KLK7 human recombinant of Preparation protein from was purified culture cell the medium by ultrafiltration and SEC. FB domain Catalytic pools. cell-line stable(Invitrogen) as FCS containing1% in expressed and using an in-house cDNA collection. The PCR product was cloned human into of pMT–BIP (Asp470–Leu764) FB. complement human recombinant of Preparation H uniformly preparationanalogousof the For °C. 8 at h 6 for beads trypsin-coated of ence pres the in sequence pro-petide the of removal by formed was domain lytic cata chromatographyFD size-exclusion(SEC).by purified andconcentrated 50 of tion and 0.5 mM glutathione disulfide (GSSG), leading to a final protein concentra CaCl mM 10 arginine, M containing0.8 8.5) (pH by rapid dilution of solubilized inclusion atbodies 10 °C with 50 mM Tris/HCl concentration of approximately 5 to 10 mg/mL. Pro-FD refolding was obtained solubilized in 6 M guadinium HCl containing 100 tomM DTT reach a protein Escherichia coli in expressed (Novagen)and pET24 into cloned were products PCR The tion. collec cDNA in-house an using PCR by amplified was (G24-A253) domain human whereas (lung), RNA C57Bl/6 from Cfd Preparation of recombinant murine and human FD catalytic domains. usingdetected goat polyclonal anti-human antibody (R&DSystems; AF1824). clonal anti-mouse C3d (R&D Systems; AF2655) were used. Humanpoly and factorA311) (Quidel, D B was factoranti-human polyclonal a C3d+iC3b, and Ba All other chemicals were of analytical grade. For western blot detection of mouse Pharmacy.US monoclonalaobtained from antibodyeculizumabwas(Soliris) anti-C5 The (59-PE). PE-conjugated Occhiena Valter from and antibody monoclonal (Ab14396) anti-CD59 Abcam from obtained was antibody clonal poly anti-C3Technologies. FITC-conjugated Life from obtained Kit Labeling IgG2amouse using 647 Fluor Alexa Zenon pre-labeled or IgG1mouse 488 were Fluor Alexa Zenon Antibodies Technologies. Life from mIgG2a) aE11; clone neoepitope, (C9 anti-C5b-9 and (A250) Quidel from mIgG1 (neo) C3d UK clone HD1A (mIgG2b) was kindly provided by P. Morgan, University of Cardiff,Anti-CD55 (3010-05).BiotechHRP-conjugated Southern IgG from Antibody (HycultHycultobtainedfromwas anti-ratbiotech; BiotechGoatHM1065) and C3b/iC3b/C3c Anti-mouse (Z-4250). Sigma from obtained was A Zymosan AS. Monoclonals Diatec from ordered custom was deposition MAC of tion protocol published the to neoepitopeantibody (clone conjugatedaE11) phosphatase alkaline to for detec according 2,4-dinitrobenzenesulfonyl-2,7-desmethyl- prepared was reagent fluorecein thiol-scavenging The Hycult. from purchased serum were fromantibodies neoepitope purified Anti-C3a (Eurogentec). were and rabbit in raised been have IgGs neoepitope Materials. O doi:10.1038/nchembio.2208 5.6, pH at buffer citrate sodium mM 50 in temperatureroom at h 1 for tions concentra various atinhibitor with pre-incubated wasconcentration) nM (5 4 N Cl asnitrogen sole the Cl source was used. μ 3 (UniProt; P03953) catalytic domain (G24–T253) was amplified by RT-PCR 5 g/mL. Refolded protein was purified using ion exchangeionchromatographyusing purified proteinRefoldedwas g/mL. LI . Anti-CD59 clone MEM43 (mIgG2a) was purchased from ABCAM, anti- ABCAM, frompurchased was (mIgG2a) MEM43 cloneAnti-CD59 . N E Cobra venom factor (CVF) was purchased from Quidel. Anti-Ba Quidel. from purchased was (CVF) factor venom Cobra μ 15

ME g/mL. After gentle agitation at 10 °C for one day, the protein was proteinday, the one for °C 10 at agitation gentle After g/mL. N-isotope-labeled humanN-isotope-labeled [ FD,mediumwith minimal M9 an (Rosetta) in the form of inclusion bodies. Inclusion bodies were TH Drosophila O DS SL3 cells that were grown in Sf-900 II medium II Sf-900 in grown were that cells SL3 CFB Uirt P05) a apiid y PCR by amplified was P00751) (UniProt; CFD eobnn hmn KLK7 human Recombinant (UniProt; P00746) catalytic P00746) (UniProt; 2 , 1 mM EDTA,GSH, mM mM 1 1 , Escherichiacoli The catalytic domain catalytic The 2 , 0.1 M NH M 0.1 , Human 3 BL21(DE3) 4 . Anti C9 C9 Anti . 4 Cl, 1 mM 1 Cl, Mouse KLK7 15 N] ------

acltd rm ecnae f niiin f D ciiy s fnto of function a as activity FD of inhibition of percentage from calculated IC spectrofluorimeter. microplate a in nm 535 of wavelength emission an and nm 485 of wavelength excitation an at recorded was cence final 200 and to of concentrations Switzerland) added Bubendorf, were (Bachem, 2,4-dinitrobenzenesulfonyl-2,7-desmethyl-fluorecein Z-Lys-thiobenzyl substrate The M containing7.5) MgCl Hepesbuffer(pH mM 1 0.1 in temperature room at h 1 for concentrations various at compound with incubated were concentration) nM (10 domain catalytic FD murine or procedures published to according formed assay.thioesterolysis FD Complement regression analysis. assay as the plot of percentage of inhibition vs. inhibitor used concentration using nonlinear parameters The channel. peak readout were the mono-exponential lifetimes. IC the in counts 1,000 mately approxi yielding s, 1 nm to set 25 was well with per time filter measurement The bandpassbandwidth. nm 450 a through collected was emission The MHz. 10 of frequency repetition selected produca with nm, pulses light 405 picosecond ating laser semiconductor a was source light excitation Switzerland).The Männedorf, (TECAN, reader lifetime fluorescence Evolution Ultra an on conducted were measurementsFluorescence-lifetime BS-#7599). Leu-Gly-Cys(PT14)-Glu-NH Ac-Glu-Phe-Lys-Pro-Ile-Leu-Trp-Arg- substrate the of addition by initiated was reaction enzyme CHAPS.The (w/v) 0.05% andNaCl mM containing150 antibody was added to each well, followed by incubation for 60 min at room at min 60 for incubation by followed well, each to added was antibody neoepitope Anti-C3aPBS–Tween 20. with washed then wereplates assay and saturated by the addition of Starting Block T20 for 5 min at room temperature, PBS–Tweenwasbindingcapacity with free platesRemainingwashedwere 20. 97 with pre-filled MaxiSorp) (NUNC plates protein-binding high-capacity 384-well immunosorbent assay (ELISA). Aliquots of reaction samples were pipetted into enzymaticcleavage the quantifiedenzyme-linked productbyanaswas C3a of inhibitorsvariousenzyme ofcOmplete (Roche, Inhibitor Tablets). Generation excess an of addition by stopped was reaction enzyme temperature, the room in the assay buffer to a final concentration of 1 MgCl mM 10 containing 7.4, 0.05% (w/v) CHAPS. pH The at enzyme reaction PBS was started by in addition temperatureof C3 room diluted at h 1 for concentrations various at compound with incubated was concentration) 1 hat room temperature in0.1MPBS (pH7.4)containing 7.5mMMgCl for concentrations various at compound with incubated was concentration) Ba). (ELISA assay proteolysis FD Human inhibitor concentration. Human FB proteolysis assay (ELISA C3a). (ELISA assay proteolysis FB Human FD activity as of afunction test compound concentration. room temperature. IC (100 substrateperoxidase fluorogenicQuantaBlu with time incubation min 20 after measured was activity HRP 20. PBS–Tween with washing sive exten by removed was antibodies excess and step, former the in as perature Bio-Rad) (1 (172-1019; HRP with labeled anti-rabbitantibodygoat Then, PBS–Tween20. with washing by antibody excess ofremovaltemperature roomand at min 60 for incubation by followed well, each to added was antibody Anti neoepitope 20. Ba PBS–Tween with washed then were plates assay and temperature, capacity was saturated by the addition of Starting Block T20 for 5 min at room binding free Remaining 20. PBS–Tween with washed were plates assay °C, 4 96 with pre-filled were enzyme- samples an reaction pipetted into by 384-well high-capacity of protein-binding quantified plates (NUNC Aliquots MaxiSorp) was (ELISA). product assay cleavage linked-immunosorbent enzymatic the as Ba of buffer solution (pH 9.0) containing 0.15 M NaCl and 40 mM EDTA. Generation NaM 0.1 a ofadditionbystopped was temperature,reaction enzyme the room at time incubation h 1 After nM. 200 ofconcentration final a to added 0.075% (w/v) CHAPS. The preformed CVF–human-FB-substrate complex was μ L/well of coating buffer. After an overnight incubation at 4 °C, assay °C, buffer.4 coatingovernightatincubationof an L/well After μ g/well in PBS–Tween 20) was incubated for 60 min at room tem μ L/well of coating buffer. After an overnight incubation at incubation overnight an After buffer.coating of L/well 50 μ values were calculated from percentage of inhibition of M and 25 and M 2 (0.8 μ μ M; Biosyntan, Berlin, Germany, productGermany, Berlin, Biosyntan, M; M, respectively. The increase in fluores in increase The respectively. M, The FD thioesterolysis assay was per was assay thioesterolysis FD The Recombinant human FD (10 nM (10 FD human Recombinant Human CVF–Bb complex (3 nM (3 complex CVF–BbHuman 1 μ 2 2 Bify rcmiat human recombinant Briefly, . , 1 M NaClCHAPS.M and0.05% 1 , M. After 1 h incubation time at 50 values were calculated from nature CH E MIC 50 aus were values A L BIOLOGY μ 2 2 2 and L) at L) and CO ------3

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. for 1 h at 37 °C. A 0.1% NP40 (Sigma) solution in GVB was used as the 100% the as used was GVB solutionin (Sigma) NP40 0.1% A °C. 37 at h 1 for compoundbufferGVB in (plus concentration)DMSO 1% final and incubated concentration)containing (Quidel) variousdilutions humannormal ofserum Resuspended (2.5 erythrocytes × 10 MgCl mM 5 containing BioProducts) (Boston buffer 10 × 8 at resuspended and PBS cold in washed were Biological) Complement alternate pathway hemolytic assay. for 50%human the WBassay. above described as manner similar a in determined was deposition plement emission wavelengths on a suitable fluorometer. Percentage inhibition of com nm 460 and excitation nm 355 at read were Plates min. 30 for incubated was MgCl mM 2 containing 9.0) (pH Tris/HCl M 0.1 0.25 at Diatec) aE11, (clone antibody monoclonal neoepitope anti-humanC9 mouse alkaline-phosphatase-conjugated an with detected was formation MAC ing. 30 min to allow complement activation. The reaction was terminated by wash forplate.coatedreactionplate°C reactionincubatedtureat the The37 wasto mix the of transfer and temperature room at min 15 for pre-incubation by polypropylene the to added was Dilutedplate serum compound with followed MgCl mM 0.5 supplementedtoBioProducts) (Boston buffer GVB the in 50% to foror classical, BioProducts) (Boston buffer GVB++ in 1% to diluted was (Quidel) serum human Normal plates. V-bottom polypropylene in DMSO in diluted pathwayassay. serially were lectin Compounds the for 9.5) (pH bufferbonate (from mannan 10 to diluted Serotec) (AbD IgM human with coated were Technologies)plates assays. deposition MAC pathway lectin and Classical to calculate IC the added) EDTA mM 25 with WB (50% signal background the subtracting was calculated relative to the positive control (50% WB with assay buffer) after compound the by complementometer.deposition of inhibitionPercentage of appropriatefluor an in emission wavelength nm 460 and excitation nm 355 at read were PlatesEDTA. M 0.2 of volume equal an with stopped was tion MgCl mM 2 containing 9.0) Tris/HCl(pH M 0.1 in mg/mL 0.18 at Fisher) (4-MUP; phate (clone aE11, Diatec) at 0.25 phos alkaline antibody an monoclonal neoepitope with C9 anti-human mouse incubated phatase-conjugated then (Nunc washed, plate was ELISA plate 384-well The a Maxisorp). onto supernatants reaction the coating by detected wasgeneration MAC mM). 25 concentrationEDTAof (final M 0.05 of volume equal an adding by stopped was activation Complement min. 40 for all °C 37 at to proceed to added allowed was which was activation, AP mg/mLinitiate to samples Zymosan 1 of 0.9%). concentration was final wells a at test suspension all (Sigma) in concentration DMSO (final min 15 for compound with pre-incubated was mixture WB 50% the and DMSO, in prepared were dilutions serial Compound activation. AP for concentration) BioProducts)Boston (GVB; containing MgCl mM 2 buffer veronal gelatin of volume equal an in diluted then Bayer), (Refludan, lepirudin anticoagulantthrombin-specific the with anticoagulated was Blood participants. all fromconsent informed withprogram, donor employee blood Novartis WB. the under collected humanwas donors individual from (WB) 50% blood Whole in assay formation (MAC) complex Membrane-attack offunction test compound concentration. IC (100 substrate peroxidase fluorogenic Quantablu with washing extensive byPBS–Tween incubationmeasuredmin withwas after20 activitytime HRP 20. removed was antibody excess and the step, in former as temperaure room at min 60 for incubated was 20) PBS–Tween in Then, goat anti-mouse antibody labeled with HRP (A0168; Sigma) (0.2 PBS–Tween20. with washing by antibody excess of removaltemperature and nature was supernatant the and centrifuged was mixture reaction Theassay. this in control vehicle the as used was DMSO 1% containing GVB and control lysis 50 μ values were calculated from percentage of inhibition of FD activity as a as activity FD of inhibition of percentage from calculated were values /L n abnt bfe (H .; im) o te lsia ptwy or pathway, classical the for Sigma) 9.5; (pH buffer carbonate in g/mL μ ch /L Atr ahn, usrt 4MP Fse) t .8 gm in mg/mL 0.18 at (Fisher) 4-MUP substrate washing, After g/mL. 2 e and 2 mM CaCl mM 2 and mic 2 was added and the plate was developed for 30 min. The reac The min. 30 for developed was plate the and added was 50 Saccharomycescerevisiae a value using GraphPad Prism software. l

biology μ 2 final concentrationspathway final lectin assay. for the g/mL. After washing, 4-methylumbelliferyl phos 6 cells/well) were combined with 10% (final ; Sigma) diluted to 20 to diluted Sigma) ; Rabbit erythrocytes (Lampire 2 μ a add n te plate the and added was 2 ) t om temperature. room at L) and 10 mM EGTAmM and10 (final Black MaxiSorp (Life MaxiSorp Black 2 n 1 m EGTA. mM 10 and μ 7 g/mL in car in g/mL /mL in GVB in /mL μ g/well ------

acetate pH 4.8) and equilibrated against 1 mL reservoir solution. For X-ray data 0.5 compound mM 2 chloride, sodium mM 100 inhibitor of presence 0.5 of volume A method. vapor-diffusion ing-drop Crystallography. value was using determined variable slope (four parameters) fitting. curve IC the and Prism, GraphPad using analyzed was Data 100. × background) OD − max background)/(OD OD − sample (OD as calculated wasInhibition IgG (45 min at room temperature) and addition of QuantaBlu (Pierce; #15169). followedHRP-conjugatedby°C), 37 at h anti-ratgoat(1 step first the in body anti C3b/iC3b/C3c anti-mouserat using by ELISA by determined was sition plates and incubated for 40 min at room zymosan-coatedtemperature. After to washing, C3 depo added was serum of uL 25 temperature, room at min 30 for pre-incubation EDTA.After with EGTAreplacing by determined was ity activ background and compound, of absence the in determined was sition 0.1% gelatin, 20 mM EGTA, 4.2 mM Hepes, pH = 7.3–7.4). Maximal C3 depo CaCl mM (0.15 buffer gelatin of addition by 50% to diluted and mice C57Bl/6 knock-in FD human or C57Bl/6 wild-type assay.cleavage C3 Mouse lysis −OD vehicle) ×100%. and percent hemolysis was calculated as: (OD sample − OD vehicle)/(OD 100% wavelength nm 415 at measured was density Optical plate. new a to removed was solved by molecular replacement using the program MOLREP program the using replacement molecular by solved was proc were data andDENZOSCALEPACKwith andscaled diffraction essed The detector. CCD 225 MAR a with equipped were collected from a single atcrystal the Swiss Light Source, beamline X10SA, data nitrogen.diffraction X-rayliquid in wereflash-frozen crystals collection, integrationaromatic the of by determined was buffer in compounds of concentration The D010ES). nr. DMSO- and 71376), D 10% nr. catalogue (Fluka NaCl mM 100 DLM-1814-5), 98% (CIL), formly 100 contained samples NMR The suppression. water for sequence used. was head probe cooled 1 cryogenically a with equipped spectrometer NMR spectroscopy. ( PyMOL marized in with 10% (v/v) added solution glycerol. All data reservoir collection and the refinement in statistics are sum cryo-cooled were Crystals solution. reservoir pH 7.0, 30% v/v Jeffamine ED-2001 pH 7.0) and equilibrated against 200 pH 8.0, 100 mM NaCl) was mixed with 1 1 A vapor-diffusion method. grams Refmac Structureswereprogram built usingthe COOT coordinatesprogram MOLREP the andcode PDBof respectively.The structures were solved by molecular replacement using the XSCALE and XDS with scaled and processed were data diffraction The equipped with a Saturn 92 CCD detector for a crystal of FD in complex with with complex in FD of crystals for detector Swiss CCD 225 MAR a the with at equipped X10SA, beamline crystals Source, Light single from collected were data diffraction X-ray ant. liquidnitrogenin and afteradditionflash-frozen ofglycerol cryoprotectas DMSO for DMSOfor90% in torsolutionstockmM (100 1 against mL reservoir solution.equilibrated wereCrystals soaked by and the addition of 0.5 8.0) pH Tris mM 100 3350, PEG 25% or 7.5 pH 1 with mixed was A 1 coordinates of PDB code H– Human FD was crystallized by the hanging-drop vapor-diffusion method. method. vapor-diffusion hanging-drop by the Human crystallized was FD μ 15 μ L reservoir solution (35% PEG 3350, 200 mM CaCl mM 200 3350, PEG (35% solution reservoir L N HSQC were recorded at 310 K using flip-back pulses L FD solution (18–18.3 mg/mL FD, 10 mM Tris pH 7.0, 100 mM NaCl) NaCl) mM 100 Tris mM 7.0, FD, 10 pH mg/mL (18–18.3 solution FD L 2 O (CIL DLM-2259-1). CompoundDLM-2259-1).solution stock(CIL prepared O was 90% in 15 D -aee F, 0 M Tris–D mM 50 FD, N-labeled http://www.pymol.or 6 /10% D 2 Supplementary TableSupplementary 3 ) to the crystal containing drops for 45 min (2 h for compound 4 0 and BUSTER L7 n ope with complex in KLK7 2 O (v/v) (DMSO- μ A Bruker Avance II 500 MHz and Avance III HD 800 MHz L reservoir solution (22% PEG 3350, 100 mM HEPES HEPES mM 100 3350, PEG (22% solution reservoir L 1 (24.6 mg/mL KLK7, 50 mM sodium acetate pH 5.6, pH acetate sodium mM 50 KLK7, mg/mL (24.6 1SP Compound was titrated into serum obtained from obtained serum into titrated was Compound 1 H signals and referencingandinternalstandard signals the toH J 4 as asearch model. 1 μ . . Mouse by FD the was sitting-drop crystallized g L FD solution (14.4 mg/mL FD, 50 mM TrismM FD,50 mg/mL (14.4 solution FD L ) 4 2 2 . and and D . Images were generated using the program 6 : Eurisotop, St. Aubin Cedex, France, cat. 4 11 , and a Rigaku FRE rotating anode rotating FRE Rigaku a and , μ 1 Cmrde stp Laboratories Isotope (Cambridge L reservoir solution (0.1 M HEPES a c-rsalzd y h hang the by co-crystallized was 2 1 , 141 mM NaCl, 4.5 mM MgCl mM 4.5 NaCl, mM 141 , , 1.8% DMSO) was mixed with mixed was DMSO) 1.8% ,

4 3 3 9 6 and and and refined using the pro usingthe andrefined , respectively., structure The doi:10.1038/nchembio.2208 μ L KLK7 solution in the the in solution KLK7 L 1BI

6 , and 10 mM in 90%in mM and10 , 4 O 2 3 , 100 mM sodium sodium mM 100 , and a watergate as search model. model. search as μ 3 7 L inhibi μ and the the and uni M 44, μ 6 3 6 50 4 L 8 2 ------) 5 , , .

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. torsional angle for N-C tonated (positive charge delocalized). The covalent ligand was removed and the using Epik NY). All water molecules were removed, the protonation states were set York,NewPreparationWizard (Schrödinger,ProteinatLLC,Maestro the pH using 7.4 generated was conformation site active modified This FD. human of Å) (1.8 p with Pipeline Pilot ( (MW carboxamideswasgenerated commercial offrom selected 3,286 amines pool a of library virtual FD.A humanfor specific moremotifs ing compound of complex crystal the in observed interactions ligand–enzyme key and pose center scaffold. proline on based design library Structure-based methods: Computational T200 Evaluation Software using aLangmuir single-site binding model. measured once after the final compound injection. Data was fitted with Biacore the dissociation of the protein–ligand complex. Dissociation of the complex was for allowing withoutsuccessively injected concentrationswereincreasing five (SCK): kinetics cycle single (b) and concentration,following the of injection the before dissociation for waiting and solution compound injecting by tion ics: an independent association–dissociation cycle was run for each concentra used to determine the kinetic parameters of the interaction: (a) standard kinet least 1,200 s (parameters identical within one experiment). Two were methods injectedfor ats 60 rateflow a of to30 60 compoundof dilutions serial Threefold run. were experiments independent several FB, and To determine parameters kinetic for bindingthe of compound (RU). units response 5,000 and 3,000 between ranging levels immobilization with Different chipsweremin. used for 5 aqueousNaOH8.5) solution in (pH hydrochloride ethanolamine M 1 a injecting by deactivated were groups tive different onto reac remainingprotein, immobilization the of injection formin 5 a After 6.0 chip. the of cells pH flow HEPES mM 20 in to diluted mg/mL were 0.05 proteins Both water. in solution salt 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) mM 390 a and solution mM 100 a of mixture (v/v) 1:1 a of injection min 5 a by activated was sensor-chip surface BR-1000-50).The Kit; Coupling Healthcare(Amine GE immobilizationfrompurchased were the for Reagents (GE Healthcare) at a flow rate of 10 Human FD and FB were immobilized covalently to a Series S Sensor Chip CM5 buffer. running supplementedTweenas 7.4) 0.05% (pH used with PBS was 20 ments were performed at 25 °C assay. affinity using binding a spectroscopy Biacore(SPR) resonance T200 Plasmon instrument (GE Healthcare). of the WaterLOGSY signal relative to the unbound compound. tration of 800 were screened in mixtures of eight, which results in a total compound concen 296 K, and by using excitation sculpting for water suppression at time s and 1.6 mixing scans 256 with were recorded Spectra concentration. ducted by WaterLOGSY at 10 small molecules for their potential interaction with latent human FD was con Statistical forComputing. (2014); Environment and Language A R: Team. Core (R protocol published a to according accomplished was fitting Data p.p.m.). million, per parts (in protein free and bound the between difference shift chemical the is Where fitting chemicalto the shift equation: the by complexand 1:1 a formationof the assuming by calculated constantswere suppressionwaterfor excitationsculpting using acquired were (2039-96-5), CAS One-dimensional DLM-32). Laboratories; Isotope Cambridge 2,2-dimethyl-2-silapentane-5-sulfonate, (sodium DSS doi:10.1038/nchembio.2208 hydrogen bond interaction of the urea carbonyl of the proline ligand with the the with prolineligand the ofcarbonyl urea the ofinteractionhydrogen bond KLK7–inhibitor the in observed as ipeline-pilot ≤ P 350; no undesirable functional groups).Compounds undesirablefunctional wereenumerated no 350; ΔΔ is the protein concentration, dd 4 7 1 =+ , and the imidazole of the catalytic His57 residue was manually pro bound to human KLK7, and aimed at identifying S1 and S2 / 6 ) and then docked into the adaptedinto the docked then and ) μ were prepared ranging from 0.4 to 900 nM. Each solution was Each nM. wereprepared900 to rangingfrom0.4 The library design concept was based on the prime-site binding M per sample. A binding hit was defined by a change-of-sign boun http://accelrys.com df - http://www.r- re α ed (( -C PL β -OG of-OG the side Ser195 chain was set to μ M protein concentation and 100 +− KP project.or μ )( L L/min using an amine-coupling protocol.

is the ligand concentration and 1 /products/collaborat rsa cmlx t alw o direct for allow to complex, crystal () μ L/min withL/min dissociationa of time at ++ g LK ). Screening of a selected set of set selected ofa Screening ). N -hydroxysuccinimide (NHS) -hydroxysuccinimide 1DI d 24 C − PDB crystal structure crystal PDB PL ive-science/biovia- N )) 05 -urea . 6 4 4 μ 6 6 1 to human FD . Compounds . Compounds . Dissociation . M compound H NMR data NMR H /( SPR experi SPR 2 P L X Δ -proline- ) 1 δ = 170°, ′ bound-free bind ------

o ae iet otc wt famns ht ee oetal bnig into into GLIDE binding with docked then potentially was library fragment weresame The pocket. S1 that the fragments with contact direct make to lographic location and was moved to an outward position, allowing the Asp189 crystal its from displaced manually was chain side Arg218 The substrate. FB for FD to specifically recognize an arginine residue at the P1 cleavage site of the conformationmanuallyaccountingbysiteS1 constructed active was FD open more- a ofcomputational model a Furthermore, pocket. S1 the ofbottom the at Arg218 with bridge salt a forms Asp189 which in and above, described as the from adapted was which FD, of formation con site active ligand-free the into GLIDE using docked were Compounds ( bond rotatable and groups acceptor/donor hydrogen bond of number weight, molecular criteria including filter properties of molecular set for a applying by collection compound Novartis the from WaterLOGSY.by screening Computational methods: Generation of a fragment focused library for NMR compound testing FDthioesterolysis inthe assay. compound for described as protocol synthesis similar a using of 20 proline-based analogs (out of 48 possible combinations) were synthesized the S1 pocket and 4 residues suitably positioned in the S2 filling motifs structural 12 of selection the to led compoundstop-scoring the of inspection Visualconstraint. a as Leu41, of carbonyl the and/or Gly193 of hydrogen bond interaction of ligands in their docking pose, either with the NH one least at offormation the defining by GLIDE with performed was scoring NH of Gly193 (referred to as the ‘oxyanion hole’; to prevent antibody-mediated activation of the classical complementpathway,classical the of activation antibody-mediatedprevent to addition of Mg tee (Ethikkommission Nordwest- und Zentralschweiz). Serum was prepared by Swiss Human Research Act and with approval of responsiblethe commit ethic through the Novartis Tissue Donor Program (TRI0128) in accordance with the collected consentandinformed volunteershealthyprovidedunder werefrom assay. erythrocyte human PNH Surrogate CCAGGAGAACCTGCACCTTC. CCTACCCTCCTCAGCAGCACwere mice and FD humanized the of typing geno the for used Primers Mice. Nomenclaturefor Genetic Standardized on B6–Cfd strain crossing line Cre-recombinase-expressing by a done with mice was the cassette genotyped. resistance were neomycin offspring floxed the black of and Removal females C57Bl/6 with crossed were males Chimeric females. foster CB6F1 pseudopregnant into transferred then were which blastocysts, intoBALB/Chost injectionfor used wasclone cell ES recombination was confirmed by Southern blot and a positive targeted C57Bl/6 CTATCGCCTTCTTGACGA and CCACCAGCCTGCGTATGT. Homologous isolated and genotyped by PCR for homologous recombination with the primers #10131-019). Ten days after transfection, 300 G418-resistant ES cell clones were (Invitrogen;geneticin mg/ml 0.2 using resistanceneomycin for selected were vector.targeting TransfectedFD cells ScaI-linearized ES electroporationof by FD targeting vector. C57Bl/6 mouse embryonic stem (ES) cells were transfected 3 accession number the vector pRAY-2 into containing a floxed neomycin subcloned resistance cassette (GenBank was fragment chimeric mouse–human resulting The signal. ID mouse the gene (Ensembl DNA genomic mouse ENSMU C57Bl/6 from (PCR) reaction chain polymerase by amplified was 2 exon from nucleotides 5 and 1, intron mice). Generation of humanized complement FD knock-in mice (B6–Cfd screening by WaterLOGSY. NMR for total) in fragments (225 selected finally representaclusterwere each from tives and others), ionic example, dipole–dipole, (for bonding, protein hydrogen the charge–charge, with interactions clustered of type inspected, the visually to according were experiments docking independent two the proteinfromconformation. open identified top-scoredfragments the The ′ homology fragment downstreamfragmenthomology neomycin,ofcomplement the resultingin h mouse The SG0000006178 Cfd tm1.1(CFD)Npa 2+ 3 –EGTA (8 mM EGTA and 2.85 mM MgCl ′ UTR containing a stop codon as well as a poly-adenylation a as well as codon stop a containing UTR U6312 Cfd eoi sqec cnann te rmtr eo 1, exon promoter, the containing sequence genomic 0 nmd codn t te nentoa Committee International the to according named , ) and was fused to the human 0 ) followed by subsequent cloning of a PCR-amplified A set of some 52,000 fragments was assembled was fragments 52,000 some of set A eu ad eaiie blood heparinized and Serum Fig. 1 1DI nature CH 48, 4 C 9 upeetr Fg 2 Fig. Supplementary n rsle i mouse in resulted and CFD b D cytl structure crystal PDB ). Ligand docking and ′ pocket of FD. A total 2 final concentration) cDNA followed by E MIC

2 , followed by followed , A L BIOLOGY tm1.1(CFD)Npa ). - - - - -

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. RBCs (% PNH post/% PNH pre) was normalized, based on the determinationon the pre)PNH normalized,based waspost/% PNH (% RBCs PNH surviving of proportion The RBCs. PNH of percentage (post) cubation post-in and (pre) baseline comparing by measured was hemolysis brief, In Occhiena). Valter (59-PE; antibody monoclonal anti-CD59 PE-conjugated a and Abcam) (Ab14396; antibody polyclonal anti-C3FITC-conjugated a with pellet erythrocyte the of staining after cytometry flow by quantified was lysis hemo (AcNHS), NHS acidified in °C 37 at time incubation h 24 After tion. complementconcentrationsdifferentactiva beforeat tubes the to added was N Compound6.9. and 6.7 0.1 between value a dropto pH a resultedin which HCl), of (1:20 HCl using acidification by achieved was activation AP used. were range normal the in C3 plasma with all subjects three least at from sera final hematocrits of 2%. In order to reduce inter-experiment variability, pooled with incubatedMg subsequently were and saline in washings three after blood peripheral from obtainedwere Erythrocytes Helsinki.of Declaration the with accordance in conducted was study This IRB. local the by approved as sent con informed following and proceduresstandard to accordingvenipuncture Vacutainerafter Pharmingen)EDTA standard(BD serum tubesandin lected col was Blood experiments. all for granulocytes) on >50% erythrocytes, on same (>10% population the cell PNH from large a drawn exhibited who patients, serially untreated three was blood from consistency, and For sera) patients. normal PNH ABO-matched for source a (as volunteers healthy vivo Ex as possible. muchasoutgatedalways was debris The FlowJosoftwareX. the with lyzed for compounds IC reported The parameters). (four slope variable − response vs. log(inhibitor) (BD Biosciences). FACS acquisition. Analysis was performed with an LSRFortessa Cell Analyzer dark. After staining, RBCs were washed with FACS buffer and filtered before (0.5 antibodies Zenon-labeled with incubated were RBCs protocol. manufacturer’s the following Kits Labeling at min 7 to 5 for 4 °C. The r.p.m. antibodies anti-C3d(neo) and anti-C5b-9 were11,000 labeled with Zenon at out carried were steps centrifugation All wereplate transferred into 1.5 mL Eppendorf tubes and 96-deep-well washed with FACS buffer.incubated the of supernatant and cells blood red ments, (100% hemolysis). Averages of control the quadruplets were calculated. maximim as (water) control positive the with measured absorbance the and anceEDTA-treatedin (negativeserum control) baselineas(i.e., hemolysis) 0% Reader.The relative percentage of hemolysis wasusing determined absorb the Microplate 340PC SpectraMax a using measured was wavelength nm 405 at of the supernatant was transferred into a 96-well microplate and the absorbance was added and the plate was centrifuged at 4,000 r.p.m. for 5 min. Then, 100 tion. To terminate the reaction, 200 was sealed with a plastic adhesive film and incubated for 1 h at 37 °C with agita plate Therespectively). lysis, minimal and lysis maximal (forcontrols as tion fied serum. wereErythrocytes incubated in pure water or in 10 mM EDTA solu per RBCs well were plated in a 96-deep-well plate containing 240 MgCl mM 5.7 PIPES, mM 10 gelatin, 0.1% NaCl, CaCl mM (0.15 PIPES-containingGVBR in washing After 7.5). pH CaCl mM (0.15 10 × (2 agitation tion of 10 donorsamewerebyaddi masked the from (RBCs) cells blood phenotype,red PNH-like a mimic to parallel, In min. 10 for ice on incubatedwere and serum compoundof dilutions Serial solution. HCl aqueous of addition by 6.4 pH to acidified and nature finally This percentagereciprocal the the ofsurvival. ofas calculated then was of residual normal RBCs (dividing by % N post/% N pre). The rate of hemolysis 50 IC For fluorescence-activated cell sorting (FACS) analysis of complement frag 2+ is an average of a total of 11, 2, and 7 experiments performed in duplicate splmne sr fo AOmthd elh idvdas NS at (NHS) individuals healthy ABO-matched from sera -supplemented 50 aus ee acltd ih rpPd rs uig h equation: the using Prism GraphPad with calculated were values PNH patient erythrocyte assay. erythrocyte patient PNH ch μ e g/mL anti-CD55 and 30 mic %

6 2 hemolysi a , , 145 mM NaCl, 0.1% gelatin, 4.2 mM HEPES, 5.7 mM MgCl mM 5.7 HEPES,NaCl, gelatin,mM mM 0.1% 4.2 145 ,

6 9

l 7 el/L i HPScnann GBelcmn (GVBR) GVBreplacement HEPES-containing in cells/mL) ,

biology , or anti-C5 eculizumab were prepared directly in acidified preparedwereanti-C5ineculizumab directlyor ,

7 ,and eculizumab, respectively. The FACSdata was ana s = () samp le μ valu μ L/well of GVBR containing 15 mM EDTA g/mL anti-CD59 for 30 min at 37 °C with averag μ g per stain) for 30 min at 4 °C in the the in °C 4 at min 30 for stain) per g ea Peripheral blood was collected from collected was blood Peripheral − em verage aximum ba 2 , pH 6.4), 2 × 10 × 2 6.4), pH , seline * 100 μ L/well acidi 2 , 145 mM 145 , 7 masked μ L 2 ------,

in 100 in from (LPS) lipopolysaccharide injection 7.5 h before the end of the study. the See of end the before h 7.5 injection intraperitonealby LPS given weregroups All 0). = (time study the of end the were given one dose of compound by oral gavage at 24, 16, 10, 8, 6, or 4 h before study.point) time For duration(groupedstudies,groupsper mice action ofof vehicle) was administered by oral gavage 3.5 h later, or 4 h before the end of the (orcompoundand atdoses various0), = study (time ofbefore end h 7.5 tered adminis was PBS) (or LPS studies, dose–response For prepared. was water, in 80 Tween 0.5% and methylcellulose 0.5% using formulation suspension a orwater, in solutol 10% and glycol propylene 20% in formulation solution a vehicleanddosingalonebygavage. oral For compound administration, either by drug) the oral gavage. Positive control animals received intraperitoneal LPS and dosing vehicle alone (consisting of the formulations below,detailed minus 100 of injection intraperitoneal an received opeet ciiy a idcd y nrprtna ijcin f 50 of injection intraperitoneal by induced was g. activity 20 approximatelyComplement weighing mice 7-week-old in performed were Studies ance with the policy of the NIBR Cambridge Animal Care and Use Committee. sterile water ad libitum. Protocols, handling and care of the mice were in accord 12 h light/dark cycle. Mice were fed with standard rodent laboratory chow and a on facility pathogen-free a in housed were Mice experiments. for in-house broughtwere animals old 6-week female NY,(GermantownandFarms USA) B6–Cfd vivo In analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. culated with GraphPad Prism was using and one-way determined significance resultindividualmean the forpatient each averagedwas cal wasand s.e.m. individual patients (2 to 14 experiments were performed per patient). Finally, percent hemolysis ofat PNH the erythrocytes indicated conditions for three the presence of 1 to bind both C3b and its degradation fragments iC3b and C3dg the hemolytic assay. The anti-C3 used in this study (Ab14396) has been shown activation in the presence of compound AP to exposed erythrocytes on fragments C3 of deposition the evaluating by ×(%Npre/%PNH Npost) post/% PNH pre) ×100. leads to the formula previously published by Ferreira buffer every buffer10 min every and then were incubated for 1 h at room temperature with kDa), the changingby TBST (68 with times three washed wereBlots iC3b kDa). (41 C3d and C3b, fragments cleavage C3 the kDa), (120 chain alpha C3 body recognized any mouse-C3d-containing proteins, including the full-length buffer.blocking in antiantibodydilutedby1:2,000 first The primary Quidel) (A311; B anti-factor or Systems) goat D & either R (AF2655; with C3d anti-mousepolyclonal °C 4 at overnight probed and min 30 for (Odyssey) buffer blocking with incubated was membrane nitrocellulose The min. 7 for (iBlot) blotting semi-dry NOVEX/Invitrogen)by Nitrocellulose; Transfer Stacks Gel (iBlot membranes nitrocellulose onto transferred then NOVEX/Invitrogen), retically on denaturing SDS polyacrylamide gels (NuPAGE 4–12% Bis–Tris gel; (~40 extract analysis. blot western and gel Protein to determine protein the concentration of respective the samples. used was Pierce) 1976; Bradford(BradfordassayM The °C. −80 storedatand plate 96-well a or tube new a either forto transferred Supernatantswere min. °C 10 4 at r.p.m. 11,000 at centrifugation by pelleted was debris tissue and (Qiagen) TissueLyserII with lysed then were tissues Eye tube. sample each to EDTA-free tablet (Roche Diagnostics), 1 mM PMSF (Sigma) and 20 mM PNPP cocktail inhibitorprotease complementmini Technology),1× Signaling (Cell were extracts 200 addingprepared byEye °C. −80 at stored and ice, dry on quickly frozen (Fisher), tubes microcentrifuge mL 2 in collected were (Becton lens without Eyes tubes Dickinson). EDTA microtainer in collected was Plasma times. indicated extracts. eye of preparation and collection Tissue schematic depiction of study the designs. The same anti-C3 and anti-CD59 staining also served to assess opsonization μ tm1.1(CFD)Npa tde: nml ad P-nue cmlmn activation. complement LPS-induced and Animals studies: L sterile PBS per animal. In these studies, negative-control animals negative-control studies, these In animal. per PBS sterile L μ g protein) or 0.5 or protein) g mice or wild type C57Bl/6 mice weremaintainedTaconicatmice C57Bl/6 type wild or mice μ M compound μ L of cell-lysis buffer consisting of 1× cell lysis buffer lysis cell 1× consistingof buffer cell-lysis of L μ Salmonella

7 L sampled plasma was resolved electropho resolved was plasma sampled L was calculated by determining the average qa aons f ihr y protein eye either of amounts Equal

7 using flow atcytometry the end of yhmru (im) dissolved (Sigma) typhimurium μ L sterile normal saline solution, saline normal sterile L Supplementary Figure 5 Figure Supplementary doi:10.1038/nchembio.2208 Mice were euthanized at euthanized were Mice et al. 5 0 : % lysis = 100 − (% 4 9 . Inhibition in μ for a for of g - - - - -

© 2016 Macmillan Publihers Limited, part of Springer Nature. All rights reserved. with PCR strip cap and stored frozen (–20 °C) for parentcompoundanalysis.for °C) (–20 frozenstored and cap strip PCR with r.p.m.resultantand the 3,000 plate,96-welltransferredplasma wasPCR a to capped at centrifuged was Blood tubes. EDTA a h to 24 transferred and and 7, 4, post-dose 2, 1, 0.5, only), (p.o. min 15 only), dose (i.v. min 5 at tail the Approximatelyanimal). 50 per mL (10 gavage oral by administered was which compound of solution mg/mL 0.1 a preparationof the for used was vehicle same The vein. jugular the of cannula solutol intothe injection20% by dosed wereanimals and injectionwaterforsolution, and a of 25% PG, 10% EtOH, 5% containing solution mg/mL 1.0 a as prepared was formulationi.v. The respectively. dose, weight) body kg per mg (p.o.)(1 peroral single a and weight) body kg per mgintravenous (i.v.)(1 ( rats Dawley Sprague male old week 6–10 in mined studies. Pharmacokinetic GraphPad Prism. cacious concentration (EC vidual plasma Ba inhibition results, to determine the indi the with conjunction in used, were data exposure These studies. action of duration and dose–response both from point time terminal the at samples mass spectrometry (LC–MS), plasma levels of chromatographyto Usingcoupledliquid twice. least at conducted was study) of type Each 100. × group) vehicle + LPS in C3d+iC3b or Ba of value average / compound) and LPS of presence in C3d+iC3b or Ba of (value – (1 = inhibition % as: culated cal was inhibition percent the values, baseline-subtracted the Using group. treated LPS each for values respective the from groups treated PBS+vehicle the in measured levels C3d+iC3b or Ba of values baseline the subtracting by analyzed was Data group.control positive vehicle-treated + LPS the to pared Dunnett’susingsons * where post-test, multiplecompariforcorrection byfollowed variance, of one-wayanalysis by were compared and considered statistically significant when mean and methods Statistical Prism using (GraphPad) software. curve standard the from extrapolated was animal each in tion captured using Odyssey imager and software (Li-Cor). Serum FD concentra was protein FD the of intensity fluorescent The kDa). (~25 band protein FD Rockland) was used as the secondary antibody to visualize the expected humaninfrared An °C. 4 at fluorescently conjugatedovernight IRDye 800CW donkey anti-goat IgG (605-731-125; antibody R&D) (AF1824; anti-FD an with and were incubated (Li-Cor) buffer blocking Odyssey the using were blocked described standard.membranes the The as serve togel same(preparation the in loaded above)was protein FD human recombinant diluted serially A mice) were resolved on SDS gels membranes. and to transferred nitrocellulose using quantified were (Bio-RAD), markers software.Odyssey weight molecular relative positions protein their on to based assigned band, kDa) (33 Ba the or bands, kDa) (68 iC3b and kDa) (41 C3d in intensity fluorescent average The blots. western Odyssey on antigen the of amount the to proportional directly were signals Fluorescent (LI-COR). imager Odyssey an with detected were signals fluorescent infrared and above described as again washed were Blots buffer. blocking in 1:5,000 by diluted antibody secondary IgG Rockland) (605-731-125; anti-goat donkey 800CW IRDye conjugated fluorescently infrared the doi:10.1038/nchembio.2208 Plasma samples (0.5 ± s.e.m. Groupss.e.m. indicated.asconsisted mice Mean10 toof 4 differences in vivo in μ L each) from human FD knock-in mice (B6–Cfd in vivo in The pharmacokinetics of compound of pharmacokinetics The 50 experiment (dose–response or duration of action of duration or (dose–response experiment ) for 6 data analyses. data using nonlinear regression curve fitting with P μ < 0.05, ** 0.05, < L of whole blood was collected from collected was blood whole of L

6 were determined in individual Data are presented as group as presented are Data P < 0.01, *** 0.01, < in vivo n = 2) following a single a following 2) = half-maximal effi P < 0.05 measured P < 0.001 com 0.001 < 7 were deter were tm1.1(CFD)Npa ------

37. 36. 35. 34. fromchased Pharsight Corporation (St. Louis, MO). WinNonlinnon-compartmentalusing methods (Enterprise,Version pur 5.2) ( tion ( concentration initial Extrapolated rule. trapezoidal linear the using calculated were (AUC), curve 50 of (125 using a MAC-MOD ACE C18 column (Mac-Mod Analytical), the supernatant analyses HPLC–MS/MSFor °C. 4 atr.p.m. g) 4,000 at(2,800 min 5 forgation centrifu and min 10 for vortexing by followed (glyburide), standard internal (150 acetonitrile adding by de-proteinated (25 Aliquots the plasma. from in plasma compound of quantify to used and were developed methods (HPLC)-MS/MS chromatography liquid performance High 48. 47. 46. 45. 44. 43. 42. 41. 40. 39. 38. 50. 49.

replacement. mode. oscillation (2002). solutions. and pitfalls proteins: fusion immunoglobulinrecombinant using domains protein specific for antibodies Engl. Ed. Int.Chem. assays. enzyme Ellman’sthiol-quantification to in alternativesreagent Des. molecules. drug-like for generationprotonation state (1995). 275–279 gradients. field pulsed and waveforms arbitrary using A Reson. Magn.J. suppressionfor (1992). 661–665 solutions. aqueous of spectroscopy NMR single-quantum saturation. water avoids that scheme detection (FHSQC) HSQC fast new a using delays interscan short atprotons exchanging of spectra HSQC of sensitivity (2004). 2210–2221 BUSTER-TNT.in likelihood Crystallogr. method. maximum-likelihood the by structures Coot. of (1993). constants. cell and symmetry unknown initially 2190–2192 (2007). 2190–2192 erythrocytes. PNH of survival the for critical is complementfrom mouse the by mediated expression cells. of deletionubiquitous Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular for automatedprogram Teplyakov,an & MOLREP: Vagin,A. A. in collected data diffraction X-rayMinor, W.of & Processing Z. Otwinowski, monoclonal B.P.Morgan, of generation & Efficient D.M.Lublin, C.L., Harris, H. Maeda, Schwenk, F., Baron, U. & Rajewsky, K. A Rajewsky,K. U.F.,& Baron,Schwenk, Shelley,J.C. sculpting Excitation Waterworks.A.J. suppressionthat Shaka, T.L.Hwang,& V.Saudek,water Gradient-tailored & R. Sklenar,V.,Leppik, M., Piotto, V.Sklenár,V.forSaudek, excitation & Gradient-tailored M., Piotto, P.C.M.Zijl, Improvedvan & M.O.Johnson, C., Abeygunawardana, S., Mori, 2002). System (Schrödinger,Graphics LLC, V.1.7.6.5. Molecular PyMOL The E. Blanc, macromolecular of Refinement E.J. Dodson, & Murshudov,Vagin,A.A. G.N., development andFeatures K. W.G.Cowtan, Emsley,P.,Scott, & B., Lohkamp, of crystals from data diffraction W.rotation Kabsch,of Automaticprocessing Ferreira, V.P. & Pangburn, M.K. Factor H mediated cell surface protection surface cell mediated V.P.H Ferreira,Factor M.K. Pangburn, & transgene Conditional B. Kinzel, & Müller,M. U.,J.A.,Gasser,Jägle, μ V μ L) of each well was transferred to a new plate and diluted by addition by diluted and plate new a to transferred was well each of L) L of water. Pharmacokinetic parameters, including the area under the the under area the including parameters,water. Pharmacokinetic of L

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