Novel Peptide Inhibitors of Angiotensin-converting Enzyme 2
2003; Elsevier BV; Volume: 278; Issue: 18 Linguagem: Inglês
10.1074/jbc.m212934200
ISSN1083-351X
AutoresLili Huang, Daniel J. Sexton, Kirsten Skogerson, Mary Devlin, Rodger Smith, Indra Sanyal, Tom J. Parry, Rachel B. Kent, Jasmin Enright, Qilong Wu, Greg Conley, Daniel B. DeOliveira, Lee A. Morganelli, Matthew D. Ducar, Charles R. Wescott, Robert C. Ladner,
Tópico(s)Coagulation, Bradykinin, Polyphosphates, and Angioedema
ResumoAngiotensin-converting enzyme 2 (ACE2), a recently identified human homolog of ACE, is a novel metallocarboxypeptidase with specificity, tissue distribution, and function distinct from those of ACE. ACE2 may play a unique role in the renin-angiotensin system and mediate cardiovascular and renal function. Here we report the discovery of ACE2 peptide inhibitors through selection of constrained peptide libraries displayed on phage. Six constrained peptide libraries were constructed and selected against FLAG-tagged ACE2 target. ACE2 peptide binders were identified and classified into five groups, based on their effects on ACE2 activity. Peptides from the first three classes exhibited none, weak, or moderate inhibition on ACE2. Peptides from the fourth class exhibited strong inhibition, with equilibrium inhibition constants (K i values) from 0.38 to 1.7 μm. Peptides from the fifth class exhibited very strong inhibition, withK i values <0.14 μm. The most potent inhibitor, DX600, had a K i of 2.8 nm. Steady-state enzyme kinetic analysis showed that these potent ACE2 inhibitors exhibited a mixed competitive and non-competitive type of inhibition. They were not hydrolyzed by ACE2. Furthermore, they did not inhibit ACE activity, and thus were specific to ACE2. Finally, they also inhibited ACE2 activity toward its natural substrate angiotensin I, suggesting that they would be functional in vivo. As novel ACE2-specific peptide inhibitors, they should be useful in elucidation of ACE2 in vivo function, thus contributing to our better understanding of the biology of cardiovascular regulation. Our results also demonstrate that library selection by phage display technology can be a rapid and efficient way to discover potent and specific protease inhibitors. Angiotensin-converting enzyme 2 (ACE2), a recently identified human homolog of ACE, is a novel metallocarboxypeptidase with specificity, tissue distribution, and function distinct from those of ACE. ACE2 may play a unique role in the renin-angiotensin system and mediate cardiovascular and renal function. Here we report the discovery of ACE2 peptide inhibitors through selection of constrained peptide libraries displayed on phage. Six constrained peptide libraries were constructed and selected against FLAG-tagged ACE2 target. ACE2 peptide binders were identified and classified into five groups, based on their effects on ACE2 activity. Peptides from the first three classes exhibited none, weak, or moderate inhibition on ACE2. Peptides from the fourth class exhibited strong inhibition, with equilibrium inhibition constants (K i values) from 0.38 to 1.7 μm. Peptides from the fifth class exhibited very strong inhibition, withK i values <0.14 μm. The most potent inhibitor, DX600, had a K i of 2.8 nm. Steady-state enzyme kinetic analysis showed that these potent ACE2 inhibitors exhibited a mixed competitive and non-competitive type of inhibition. They were not hydrolyzed by ACE2. Furthermore, they did not inhibit ACE activity, and thus were specific to ACE2. Finally, they also inhibited ACE2 activity toward its natural substrate angiotensin I, suggesting that they would be functional in vivo. As novel ACE2-specific peptide inhibitors, they should be useful in elucidation of ACE2 in vivo function, thus contributing to our better understanding of the biology of cardiovascular regulation. Our results also demonstrate that library selection by phage display technology can be a rapid and efficient way to discover potent and specific protease inhibitors. angiotensin-converting enzyme angiotensin I angiotensin II angiotensin-converting enzyme homolog angiotensin-(1–9) angiotensin-(1–7) variegated DNA polymerase chain reaction phosphate-buffered saline enzyme-linked immunosorbent assay dimethyl sulfoxide One major control mechanism for blood pressure homeostasis is the renin-angiotensin system, in which angiotensin-converting enzyme (ACE)1 is a vital player. ACE, a zinc metallopeptidase, promotes blood pressure elevation at least in part by cleaving the inactive angiotensin I (Ang I) to the vasoconstrictor Ang II (1Skeggs L.T. Kahn J.R. Shumway N.P. J. Exp. Med. 1955; 103: 295-299Crossref Scopus (715) Google Scholar) and inactivating the vasodilator bradykinin by cleavage (2Dorer F.E. Kahn J.R. Lentz K.E. Levine M. Skeggs L.T. Circ. Res. 1974; 34: 824-827Crossref PubMed Scopus (169) Google Scholar). Its role in regulating blood pressure and renal function is underscored by the effective clinical use of ACE inhibitors in the treatment of hypertension and other cardiovascular diseases. ACE2 is a recently identified human homolog of ACE (3Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar, 4Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar). It contains a single zinc-binding catalytic domain, which is 42% identical to the human ACE active domain. Genomic structure comparison suggests that ACE2 and ACE genes arose by duplication of a common ancestor (3Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar). Although both ACE2 and ACE are zinc metallopeptidases and angiotensin-converting enzymes with a membrane-associated and a secreted form, many differences exist between these two enzymes (for reviews, see Refs. 5Turner A.J. Hooper N.M. Trends Pharmacol. Sci. 2002; 23: 177-183Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar and 6Eriksson U. Danilczyk U. Penninger J.M. Curr. Biol. 2002; 12: R745-R752Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). First, they are different in enzymatic activity; ACE2 is a carboxypeptidase, removing the C-terminal residue from the decapeptide Ang I to form angiotensin-(1–9) (Ang-(1–9)) (3Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar,4Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar), whereas ACE is a dipeptidase, cleaving the C-terminal dipeptide from Ang I to form the octapeptide Ang II. Second, ACE2 and ACE have different substrate specificities; ACE2 cleaves Ang I, Ang II, apelin-13, apelin-36, dynorphin A-(1–13), and des-Arg bradykinin (3Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar, 7Vickers C. Hales P. Kaushik V. Dick L. Gavin J. Tang J. Godbout K. Parsons T. Baronas E. Hsieh F. Acton S. Patane M. Nichols A. Tummino P. J. Biol. Chem. 2002; 277: 14838-14843Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar); ACE cleaves Ang I, Ang-(1–9), bradykinin, and many other bioactive peptides such as substance P, neurotensin, and enkephalin (8Corvol P. Williams T.A. Soubrier F. Methods Enzymol. 1995; 248: 283-305Crossref PubMed Scopus (227) Google Scholar). Another difference between these two enzymes is the inhibitor specificity; ACE2 cannot be inhibited by ACE inhibitors (3Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar, 4Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar). Finally, a difference in tissue expression has been observed; ACE2 is primarily expressed in the heart, kidney and testis, whereas ACE is more ubiquitously expressed in tissues including heart, lung, kidney, colon, small intestine, ovary, testis, prostate, liver, skeletal muscle, pancreas, and thyroid (3Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar, 4Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar). One in vitro function of ACE2 is the catalysis of Ang I to Ang-(1–9) (3Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar, 4Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. J. Biol. Chem. 2000; 275: 33238-33243Abstract Full Text Full Text PDF PubMed Scopus (1656) Google Scholar). In vivo detection of Ang-(1–9) in rat and human plasma has been described, and the levels are twice that of Ang II (9Oparil S. Tregear G.W. Koerner T. Barnes B.A. Haber E. Circ. Res. 1971; 29: 682-690Crossref PubMed Scopus (38) Google Scholar, 10Johnson H. Kourtis S. Waters J. Drummer O.H. Peptides (Elmsford). 1989; 10: 489-492Crossref PubMed Scopus (18) Google Scholar, 11Drummer O.H. Kourtis S. Johnson H. Biochem. Pharmacol. 1990; 39: 513-518Crossref PubMed Scopus (24) Google Scholar). Although Ang-(1–9), itself catabolized by ACE, is considered a competitive inhibitor of ACE (3Donoghue M. Hsieh F. Baronas E. Godbout K. Gosselin M. Stagliano N. Donovan M. Woolf B. Robison K. Jeyaseelan R. Breitbart R.E. Acton S. Circ. Res. 2000; 87: E1-E9Crossref PubMed Google Scholar, 12Snyder R.A. Wintroub B.U. Biochim. Biophys. Acta. 1986; 871: 1-5Crossref PubMed Scopus (38) Google Scholar), it has been demonstrated to have weak pressor effects in anesthetized rats and dogs, and weak vasoconstricting activity in isolated rat aorta (9Oparil S. Tregear G.W. Koerner T. Barnes B.A. Haber E. Circ. Res. 1971; 29: 682-690Crossref PubMed Scopus (38) Google Scholar). A recent study 2T. J. Parry, R. Tallarida, R. Schulingkamp, D. Keleti, L. Huang, L. Sekut, R. Smith, V. Albert, N. Nguyen, D. Chinchilla, D. Parmelee, Y. Li, H. Lin, S. Strawn, J. Porter, M. C. Barber, M. Valmonte, T. Coleman, S. Ruben, and I. Sanyal, unpublished results. with Ang-(1–9) also indicates that Ang-(1–9) is a pressor agent that potentiates Ang II-mediated vasoconstriction in isolated rat aortic rings and pressor effects in the awake rat. ACE2 also cleaves Ang II to produce Ang-(1–7). Ang-(1–7) is proposed to be a vasodilator in animal studies (13Ren Y. Garvin J.L. Carretero O.A. Hypertension. 2002; 39: 799-802Crossref PubMed Scopus (183) Google Scholar, 14Lemos V.S. Cortes S.F. Silva D.M. Campagnole-Santos M.J. Santos R.A. Br. J. Pharmacol. 2002; 135: 1743-1748Crossref PubMed Scopus (36) Google Scholar). However, its significance in humans is still controversial (6Eriksson U. Danilczyk U. Penninger J.M. Curr. Biol. 2002; 12: R745-R752Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Unlike ACE, ACE2 does not cleave bradykinin. However, ACE2 cleaves and inactivates des-Arg bradykinin, a local vasodilator functioning through binding to the B1 receptor expressed when inflammation or tissue damage occurs (15Marceau F. Bachvarov D.R. Clin. Rev. Allergy Immunol. 1998; 16: 385-401Crossref PubMed Scopus (136) Google Scholar). In contrast, bradykinin, cleaved and inactivated by ACE, functions as a systemic vasodilator through binding to the B2 receptor (15Marceau F. Bachvarov D.R. Clin. Rev. Allergy Immunol. 1998; 16: 385-401Crossref PubMed Scopus (136) Google Scholar). Based on the potential in vivo functions of Ang-(1–9) and des-Arg bradykinin, it is tempting to speculate that ACE2 plays a role in the regulation of vasomotor tone and blood pressure at least in part through cleavage of Ang I and des-Arg bradykinin. However, a recent knock-out mice study demonstrates that disruption of ACE2 in mice does not alter blood pressure and renal function but leads to increased levels of Ang II, up-regulation of hypoxia-induced genes, and decreased cardiac contractility that can be rescued by a second mutation causing ACE deficiency (16Crackower M.A. Sarao R. Oudit G.Y. Yagil C. Kozieradzki I. Scanga S.E. Oliveira-dos-Santos A.J. da Costa J. Zhang L. Pei Y. Scholey J. Ferrario C.M. Manoukian A.S. Chappell M.C. Backx P.H. Yagil Y. Penninger J.M. Nature. 2002; 417: 822-828Crossref PubMed Scopus (1412) Google Scholar). Thus, ACE2 appears to be essential for regulating heart function in vivo. However, its role in blood pressure regulation remains unclear. Animal studies with specific ACE2 inhibitors should provide more information to our understanding of the physiological roles of ACE2 in cardiovascular regulation. Here we described the discovery of novel ACE2 peptide inhibitors through selection of constrained peptides from libraries displayed on filamentous phage. We discovered very potent ACE2 peptide inhibitors with K i values as low as 2.8 nm. These peptides were stable inhibitors, not hydrolyzed by ACE2, and were specific for ACE2. As novel ACE2-specific peptide inhibitors, they should be useful in elucidation of ACE2 in vivofunction. Biotinylated anti-FLAG M2 monoclonal antibody, FLAG peptide, ACE, angiotensin I, NAD, resazurin, diaphorase, and captopril were purchased from Sigma. Horseradish peroxidase-conjugated anti-M13 antibody was purchased from Amersham Biosciences. Tetramethylbenzidine peroxidase substrate solution was purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD). Ser-Mag streptavidin magnetic beads were purchased from Seradyn (Ramsey, MN). Fluorogenic ACE2 substrate M-2195 was purchased from Bachem (King of Prussia, PA). Leucine dehydrogenase was purchased fromCalbiochem. Teprotide (pyro-Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro), also known as bradykinin potentiating factor SQ20881, was purchased from ICN Pharmaceuticals (Costa Mesa, CA). The cDNA encoding the extracellular domain of ACE2 was cloned into a baculovirus transfer vector pA2. A recombinant baculovirus was generated by transfecting Sf9 cells with the ACE2 expression vector. ACE2 protein (∼85 kDa) was purified from conditioned media of Sf9 cells that had been infected with the recombinant baculovirus. FLAG-tagged ACE2 was purified by affinity purification from the supernatant of 293 cells that had been transiently transfected with a mammalian expression vector expressing the FLAG-ACE2 protein. The disulfide-constrained loop peptide libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, and TN12/1, were constructed in MANP, a derivative of M13mp18. This vector has the LacZ complementation system removed, a bla(Amp R) gene, and a modified junction between signal sequence and coding region of gene III. Two unique restriction sites, NcoI and PstI, were introduced into this modified junction for easy directional cloning. The variegated DNA (vgDNA) flanked by constant sequences was synthesized by MorphoSys (Munich, Germany) using TRIM technology. In TRIM, one can add preformed trinucleotides allowing complete control of what relative abundance of each amino acid type is allowed at each variegated position. The vgDNA was PCR-amplified using a top strand primer containing anNcoI site and a bottom-strand primer containing aPstI site, cleaved with NcoI and PstI, and ligated into similarly cleaved MANP vector. The peptides encoded by vgDNA and the flanking sequences of each library are shown in TableI. The TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, and TN12/1 libraries encode peptide loops of 6, 7, 8, 9, 10, and 12 amino acids (counting the cysteines), respectively.Table IInsert and flanking sequences in constrained loop libraries Open table in a new tab Two linear libraries Ph.D.-7 and Ph.D.-12 were obtained from New England Biolabs (Beverly, MA). Ph.D.-7 has 7 variable residues (XXXXXXXGGGSAET), whereas Ph.D.-12 has 12 variable residues (XXXGGGSAET). Peptides from the six constrained loop libraries and two linear libraries were selected using FLAG-ACE2 as the target, which can be immobilized to streptavidin-coated magnetic beads via a biotinylated anti-FLAG antibody. To remove binders to streptavidin beads, anti-FLAG antibody, and FLAG peptide, the libraries were depleted 5 times by binding to FLAG peptide/biotinylated anti-FLAG antibody-immobilized beads before selection against the target. The depleted libraries were incubated with 6 μg of FLAG-ACE2 in 300 μl of phosphate-buffered saline (PBS) for 1 h and then incubated with biotinylated anti-FLAG antibody-immobilized beads for 1 h. The beads were washed 7 times with PBS, 0.1% Tween 20 (PBST) to remove unbound phage. The bound phage were then eluted with FLAG peptide (100 μg/ml) in Tris buffer (10 mm Tris-Cl, 150 mm NaCl, pH 7.5) for 30 min. Eluted phage were amplified and underwent two more similar rounds of selection and amplification. In round 1, the six constrained peptide libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, and TN12/1, were selected separately. To accelerate the selection procedures, in the subsequent rounds of selection, these six libraries were combined into two pools: pool A composed of TN6/6, TN7/4, and TN8/9, and pool B composed of TN9/4, TN10/9, and TN12/1. The two linear peptide libraries, Ph.D.-7 and Ph.D.-12, were combined as Ph.D.-7/12, from the beginning of selection. Phage enriched from the third round of selection were screened by phage ELISA for ACE2 binding. Immulon 2 96-well plates were coated with streptavidin for 1 h at 37 °C and subsequently coated with biotinylated anti-FLAG antibody for 1 h at room temperature. Half of the plates were further coated with FLAG-ACE2 as the target plates, and the other half were coated with FLAG peptide as the background plates. The amount of each protein or peptide coated was 100 ng per well. The coated plates were then incubated for 1 h with 1:2 diluted overnight phage cultures that were made by inoculating phage from individual plaques into bacteria cells. After washing 7 times with PBST, the plates were incubated with horseradish peroxidase-conjugated anti-M13 antibody for 1 h, washed 5 times, developed with tetramethylbenzidine solutions, and read at 630 nm with an ELISA plate reader. DNA sequences encoding displayed peptides of positive phage binders were amplified by PCR and sequenced by automatic sequencing. Based on the motif sequences identified by sequence cluster analysis, representative peptides from each motif were synthesized. The crude peptides were ordered from Sigma. The peptides were then cleaved from resin with trifluoroacetic acid, purified using reverse phase-high pressure liquid chromatography, oxidized, and lyophilized. The purity of each oxidized peptide was greater than 90%. The peptides were dissolved in dimethyl sulfoxide (Me2SO) at a stock concentration of 25 mm, aliquoted, and stored at −20 °C. Peptide concentrations were quantified by extinction coefficient. The enzymatic activity of ACE2 was assayed using a fluorogenic substrate, M-2195, 7-methoxycoumarin-Tyr-Val-Ala-Asp-Ala-Pro-Lys(2,4-dinitrophenyl)- OH. Cleavage of this substrate at the C-terminal Lys residue by ACE2 removes the 2,4-dinitrophenyl moiety that quenches the fluorescence of the 7-methoxycoumarin moiety, thus resulting in increased fluorescence. For the initial screen of inhibitors from ACE2 binders, each peptide was incubated with 20 nm ACE2 for 10 min at room temperature at a concentration of either 100 μm or 2 mm in 100 mm Tris-HCl, pH 7.4, 0.1% Tween. The amount of Me2SO was kept the same in each sample. Following incubation, the substrate M-2195 was added to achieve a final concentration of 50 μm, and the plates were read immediately on a SpectraMAX Gemini fluorescence spectrophotometer at an excitation wavelength of 328 nm and an emission wavelength of 392 nm. Fluorescence was monitored at 36-s intervals for 15 min. For IC50 determination, increasing concentrations of peptide inhibitors (0–100 μm) were incubated with 20 nm ACE2 prior to the addition of the substrate M-2195 (50 μm). For K i determination, 7 nm ACE2 was incubated with the peptide inhibitors ranging from 0.1- to 5-fold, the IC50 value of individual inhibitor. The concentrations of added substrate M-2195 ranged from 14 to 50 μm. The inhibition assay of ACE was carried out essentially in the same way as that of ACE2 with the same substrate M-2195. ACE2 activity toward its natural substrate was measured by an assay based on a spectrofluorometric enzyme-coupled system. ACE2 hydrolyzes Ang I (NH2-DRVYIHPFHL-COOH) to produce Ang-(1–9) (NH2-DRVYIHPFH-COOH) and leucine. The released leucine can then be monitored by the activity of leucine dehydrogenase with concomitant conversion of NAD+ to NADH. The production of NADH is coupled to the diaphorase-catalyzed reduction of resazurine to resorufin, which can be monitored on a fluorescence reader. In the inhibition assay, ACE2 was incubated with the peptide inhibitor or ACE inhibitor for 10 min at room temperature in reaction buffer consisting of 100 mm Tris, pH 8, 0.01% Tween, 4 mm NAD, 25 μm resazurin, 0.1 unit/ml leucine dehydrogenase, and 0.1 unit/ml diaphorase. The amount of Me2SO was kept the same in each sample. The substrate Ang I was subsequently added, and following an additional 30 min of incubation, the plate was read on a SpectraMAX Gemini fluorescence spectrophotometer at an excitation wavelength of 565 nm and an emission wavelength of 585 nm. Fluorescence was monitored at 1-min intervals for 2 h. The IC50 and K i values were determined similarly as described for the assay with the synthetic substrate. The binding affinities of the selected peptides for ACE2 were measured using a BIAcore 3000. ACE2 (250 nm) in 50 mm acetate, pH 4.0, was coupled to the dextran surface of a CM5 sensor chip by the standardN-hydroxysuccinimide/1-ethyl-3-(dimethylaminopropyl)-carbodiimide coupling procedure to a ligand density of 5425 response units. A flow cell containing blocked dextran was used as a control. Experiments were performed in 100 mm Tris, pH 8.0, plus 0.01% Tween 20. Serially diluted peptide solutions (500, 250, 125, 62.5, and 31.3 nm) were injected at 20 μl/min for 2 min using the kinject function. Following a 3-min dissociation, the surface was regenerated with a quick inject of 1 m NaCl for 25 s at 50 μl/min. Sensorgrams were fit by global analysis using the BIAevaluation software 3.1 for a Langmuir 1:1 interaction. The equilibrium dissociation constants (K d ) were calculated from kinetic rate constants (K d =k off/k on). Six constrained loop peptide libraries, TN6/6, TN7/4, TN8/9, TN9/4, TN10/9, and TN12/1, and two linear peptide libraries Ph.D.-7 and Ph.D.-12, were used for selection against FLAG-ACE2 target. After incubation with libraries in solution, the target was immobilized to streptavidin-coated magnetic beads via biotinylated anti-FLAG antibody. The bound phage were eluted with FLAG peptide. After three rounds of selection, the fraction of input, which was calculated as the total amount of output phage divided by the total amount of input phage, increased from 10−6 to 10−5 at the first round to 10−2 to 10−1 by the third round. To identify positive phage binders, the eluted phage from the third round of selection were screened by ELISA. The target plates were sequentially coated with streptavidin, anti-FLAG antibody, and FLAG-ACE2, and the background plates were sequentially coated with streptavidin, anti-FLAG antibody, and FLAG peptide. Phage ELISA of selected isolates (n = 1916) from constrained peptide libraries showed that ∼32% of the isolates were ACE2 binders, with target to background signal ratios ≥2. ELISA of selected phage isolates (n = 144) from the linear libraries, however, showed no binders. The ELISA positive isolates (n = 613) from constrained libraries were sequenced. The amino acid sequences of the encoded peptides were analyzed for shared motifs and, as shown in Fig.1, 10 major motifs were found. Some clusters were found in multiple libraries, some were found exclusively in one library. For example, theALFCV(D/E)F andR XXX RDSRC motifs were found in both TN6/6 and TN10/9 libraries (Fig. 1, A and D); the (F/Y)C(F/L/I)(D/E)F motif, similar to theALFCV(D/E)F motif, was found in TN8/9 and TN10/9 libraries (Fig. 1 B); theD XCX TW XX PC motif was found in TN7/4 and TN8/9 libraries (Fig. 1 I); and the CF(D/E)W(E/D) motif was identified in the TN7/4, TN8/9, and TN12/1 libraries (Fig. 1 F). Whereas the(D/E)C(E/D)W XX (F/W) and CX P X R XX PW XXC motifs were found only in the TN12/1 library (Fig. 1, C andJ), the CX T X DCV motif in the TN6/6 library (Fig. 1 E) and the(Y/W)E XCH(W/Y) X P andKECKFGY XXCL X Wmotifs were found in the TN8/9 library (Fig. 1, G andH). Based on these consensus motifs and the number of isolates occurring per sequence, 23 peptides representing these 10 motifs were synthesized. The 23 peptides synthesized as ACE2 peptide binders were further screened for ACE2 inhibitors by assays using fluorogenic substrate M-2195. For initial screening of inhibitors, ACE2 (20 nm) was incubated with each peptide at 100 μmprior to the addition of 50 μm substrate. Based on their effects on ACE2 enzyme activity, the peptides were classified into 5 groups with none (−), weak (+), moderate (++), strong (+++), and very strong (++++) inhibition, respectively (TableII). Peptides from the first group show no inhibition on ACE2 activity at a peptide concentration of 100 μm. Some of these peptides were tested at concentrations up to 2 mm, and yet showed no inhibition on ACE2 (data not shown). Peptides from the second group showed weak inhibition on ACE2, exhibiting 20–60% inhibition at 100 μm. These peptides could have K i values no lower than about 50 μm. Peptides from the third group showed moderate inhibition, exhibiting about 80% inhibition at 100 μm; these peptides could have K i of ∼25 μm. Peptides from the fourth group showed strong inhibition, exhibiting about 99% inhibition at 100 μm;K i could be around 1 μm. Peptides from the fifth group showed very strong inhibition, exhibiting complete inhibition at 100 μm; K i could be <1 μm. Peptides with strong and very strong inhibition were further analyzed to determine their K i values. The strong inhibitors have K i values ranging from 0.38 to 1.7 μm, whereas the very strong inhibitors haveK i values ranging from 3 to 139 nm(Table II).Table IISequences of synthesized peptides and their effects on ACE2 activityPeptideLibrarySequence2-aSequence; Ac- denotes N-terminal acetylation; -NH2 denotes C-terminal amidation.Inhibition2-bInhibition, −, no inhibition on ACE2 activity at concentrations up to 100 μm; +, weak inhibition (20–60% inhibition at 100 μm); ++, moderate inhibition (at least 80% inhibition at 100 μm, with IC50 values of about 30 μm); +++, strong inhibition (about 99% inhibition at 100 μm, with IC50 values of 0.4–1 μm); ++++, very strong inhibition (complete inhibition at 100 μm, with IC50 values <140 nm).IC502-cIC50, determined by inhibition assays with 20 nm ACE2, peptides ranging from 0 to 100 μmand 50 μm substrate M-2195.K i 2-dK i, determined by enzyme kinetic analysis with 7 nm ACE2, peptide inhibitors ranging from 0.1 to 5-fold the IC50 value of individual inhibitor and the substrate M-2195 ranging from 14 to 50 μm.k on2-ek on, k off, andK d, determined by BIAcore-based kinetic analysis of peptide binding directly to ACE2 coupled to a CM5 sensor chip at a ligand density of 5425 response units, with the injection of 0–500 nM peptides. χ2 values for all measurements were less than 0.4, indicating a close fit.k off2-ek on, k off, andK d, determined by BIAcore-based kinetic analysis of peptide binding directly to ACE2 coupled to a CM5 sensor chip at a ligand density of 5425 response units, with the injection of 0–500 nM peptides. χ2 values for all measurements were less than 0.4, indicating a close fit.K d 2-ek on, k off, andK d, determined by BIAcore-based kinetic analysis of peptide binding directly to ACE2 coupled to a CM5 sensor chip at a ligand density of 5425 response units, with the injection of 0–500 nM peptides. χ2 values for all measurements were less than 0.4, indicating a close fit.nm1/Ms1/snmDX500TN6/6Ac-GSNRECHALFCMDFAPGEGGG-NH2+NDNDNDNDNDDX501TN6/6Ac-GSSPTCRALFCVDFAPGEGGG-NH2+NDNDNDNDNDDX502TN6/6Ac-GSLEMCEALFCVEFAPGEGGG-NH2−NDNDNDNDNDDX507TN10/9Ac-GSNDYCTVFTGALFCLDFAPEGGG-NH2−NDNDNDNDNDDX514TN10/9Ac-GSPNQCGVDIWALFCVDFAPEGGGK-NH2+NDNDNDNDNDDX504TN8/9Ac-AGEGNCFLIGPWCFEFGTEGGG-NH2−NDNDNDNDNDDX508TN10/9Ac-GSYDNCLGLANLNFCFDFAPEGGG-NH2+NDNDNDNDNDDX510TN12/1Ac-GDDDDCGWIGFANFHLCLHGDPEGGG-NH2−NDNDNDNDNDDX511TN12/1Ac-GDPFECDWGPWTLEMLCGPPDPEGGG-NH2+NDNDNDNDNDDX524TN6/6Ac-GSRIGCRDSRCNWWAPGEGGG-NH2+++600540NDNDNDDX525TN6/6Ac-GSRGFCRDSSCSFPAPGEGGG-NH2+++1.0 × 1031.7 × 103NDNDNDDX526TN6/6Ac-GSWPTCLTMDCVYNAPGEGGG-NH2+NDNDNDNDNDDX527TN7/4Ac-AGWVLCFEWEDCDEKGTEGGG-NH2−NDNDNDNDNDDX528TN8/9Ac-AGVYFCFDWEQDCDEMGTEGGG-NH2−NDNDNDNDNDDX529TN8/9Ac-AGWEVCHWAPMMCKHGGTEGGG-NH2+++400380NDNDNDDX530TN8/9Ac-AGQKECKFGYPHCLPWGTEGGG-NH2++3.0 × 104NDNDNDNDDX531TN8/9Ac-AGSDWCGTWNNPCFHQGTEGGG-NH2+++500540NDNDNDDX512TN12/1Ac-GDRLHCKPQRQSPWMKCQHLDPEGGG-NH2++++ 601391.4 × 1051.4 × 10−2 96DX513TN12/1Ac-GDLHACRPVRGDPWWACTLGDPEGGG-NH2++++ 901262.4 × 1044.0 × 10−3170DX599TN12/1Ac-GDRYLCLPQRDKPWKFCNWFDPEGGG-NH2++++114 46.52.3 × 1051.1 × 10−248.6DX600TN12/1Ac-GDYSHCSPLRYYPWWKCTYPDPEGGG-NH2++++10.12.84.3 × 1044.6 × 10−410.8DX601TN12/1Ac-GDGFTCSPIRMFPWFRCDLGDPEGGG-NH2++++56.8 30.99.9 × 1045.6 × 10−356.3DX602TN12/1Ac-GDFSPCKALRHSPWWVCPSGDPEGGG-NH2++++127.5121.21.0 × 1057.7 × 10−374.4ND, not determined.2-a Sequence; Ac- denotes N-terminal acetylation; -NH2 denotes C-terminal amidation.2-b Inhibition, −, no inhibition on ACE2 activity at c
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