3551 SVSVGMKPSPRP 1 Phage display (common panning) 5 PMID:31812636 Recombinant envelope (E) protein TrX Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA After five rounds of bio-panning, phages showing high binding activity to the recombinant E protein based on an indirect enzyme-linked immunosorbent assay (ELISA) were selected (data not shown). The Ph.D.-12™ Phage Display Peptide Library (New England Bio labs, USA) was exposed to plates coated with purified recombinant protein Trx. Unbound phages were washed away, and specifically bound phages were eluted and amplified for a second set of screening. After five rounds of bio-panning, phages showing high binding activity to the recombinant E protein based on an indirect enzyme-linked immunosorbent assay (ELISA) were selected then sequenced. 3552 AGDVPRGWTSSS HTPAATLHPVFL TPSQSMGWDSSA THNKVQQ NTTLNGL PETWTHW TNHOKTW DYRVQMA HQSQSRM PTKWSAT PNSVKAQ GLELREK 12 Phage display (in vivo) 3 PMID:31589023 C57bl/6 mice heart Not determined. Not determined. Not determined. Ph.D.-12 and Ph.D.-C7C phage display library pool For library screening, Ph.D.−12 and C7C phage library (New England Biolabs, USA) were mixed, 1.5e13 pfu/mL, and injected intraperitoneally (IP) into C57bl/6 mice (100 μl, n=2 per group). Prior to tissue harvest, mice were euthanized and perfused with 20 mL saline to ensure complete tissue exsanguination. Spinal cord, brain, and cardiac tissue was harvested and flash-frozen in DMEM (Invitrogen, Inc.) with 1% BSA (Fisher Scientific). After homogenization, tissue homogenates were centrifuged and resuspended in 1% Triton-X 100 (Sigma-Aldrich, Inc.) and passed through a 25-gauge needle to ensure cell lysis. Phage titer was quantified and spinal- and brain-tissue homogenate was amplified for subsequent screens. The series of TACL peptides were synthesized and tested for their ability to deliver a model enzyme (NeutrAvidin-horseradish peroxidase fusion) to the brain and spinal cord. Three TACL-peptides facilitated significant active enzyme delivery into the CNS, with limited accumulation in off-target organs. Peptide structure and serum stability is increased when internal cysteine residues are cyclized by perfluoroarylation with decafluorobiphenyl, which increased delivery to the CNS further. TACL-peptide was demonstrated to localize in parasympathetic ganglia neurons in addition to neuronal structures in the hindbrain and spinal cord. By targeting uptake into ANS neurons, we demonstrate the potential for TACL-peptides to bypass the blood-brain barrier and deliver therapeutics into the brain and spinal cord. 3553 NIVCPNEHPRCS THNKVQQ DYRVQMA PSHLTKM SQKNFTH PLSKGKL 6 Phage display (in vivo) 3 PMID:31589023 C57bl/6 mice brain Not determined. Not determined. Not determined. Ph.D.-12 and Ph.D.-C7C phage display library pool For library screening, Ph.D.−12 and C7C phage library (New England Biolabs, USA) were mixed, 1.5e13 pfu/mL, and injected intraperitoneally (IP) into C57bl/6 mice (100 μl, n=2 per group). Prior to tissue harvest, mice were euthanized and perfused with 20 mL saline to ensure complete tissue exsanguination. Spinal cord, brain, and cardiac tissue was harvested and flash-frozen in DMEM (Invitrogen, Inc.) with 1% BSA (Fisher Scientific). After homogenization, tissue homogenates were centrifuged and resuspended in 1% Triton-X 100 (Sigma-Aldrich, Inc.) and passed through a 25-gauge needle to ensure cell lysis. Phage titer was quantified and spinal- and brain-tissue homogenate was amplified for subsequent screens. The series of TACL peptides were synthesized and tested for their ability to deliver a model enzyme (NeutrAvidin-horseradish peroxidase fusion) to the brain and spinal cord. Three TACL-peptides facilitated significant active enzyme delivery into the CNS, with limited accumulation in off-target organs. Peptide structure and serum stability is increased when internal cysteine residues are cyclized by perfluoroarylation with decafluorobiphenyl, which increased delivery to the CNS further. TACL-peptide was demonstrated to localize in parasympathetic ganglia neurons in addition to neuronal structures in the hindbrain and spinal cord. By targeting uptake into ANS neurons, we demonstrate the potential for TACL-peptides to bypass the blood-brain barrier and deliver therapeutics into the brain and spinal cord. 3554 GFPSVRDLSPLR WATLDLGPQPYS PETWTHW HQSQSRM PLSKGKL NTSPLNT PTKWSAT ASGFTAT PNSVKAQ TMSTTQV GLELREK 11 Phage display (in vivo) 3 PMID:31589023 C57bl/6 mice spinal cord Not determined. Not determined. Not determined. Ph.D.-12 and Ph.D.-C7C phage display library pool For library screening, Ph.D.−12 and C7C phage library (New England Biolabs, USA) were mixed, 1.5e13 pfu/mL, and injected intraperitoneally (IP) into C57bl/6 mice (100 μl, n=2 per group). Prior to tissue harvest, mice were euthanized and perfused with 20 mL saline to ensure complete tissue exsanguination. Spinal cord, brain, and cardiac tissue was harvested and flash-frozen in DMEM (Invitrogen, Inc.) with 1% BSA (Fisher Scientific). After homogenization, tissue homogenates were centrifuged and resuspended in 1% Triton-X 100 (Sigma-Aldrich, Inc.) and passed through a 25-gauge needle to ensure cell lysis. Phage titer was quantified and spinal- and brain-tissue homogenate was amplified for subsequent screens. The series of TACL peptides were synthesized and tested for their ability to deliver a model enzyme (NeutrAvidin-horseradish peroxidase fusion) to the brain and spinal cord. Three TACL-peptides facilitated significant active enzyme delivery into the CNS, with limited accumulation in off-target organs. Peptide structure and serum stability is increased when internal cysteine residues are cyclized by perfluoroarylation with decafluorobiphenyl, which increased delivery to the CNS further. TACL-peptide was demonstrated to localize in parasympathetic ganglia neurons in addition to neuronal structures in the hindbrain and spinal cord. By targeting uptake into ANS neurons, we demonstrate the potential for TACL-peptides to bypass the blood-brain barrier and deliver therapeutics into the brain and spinal cord. 3555 CWRDYLI(10)[8.2±0.8] CQWFSHR(8)[6.9±0.9] CGTWLKF(7)[NA] 3 Phage display (subtractive panning) 3 PMID:31398275 Tobacco Etch Virus protease (TEV protease) Not determined. Not determined. Not determined. pADLg3-TGC-(NNK)6-TAG phagemid library Fluorescence polarization We incubated a 25 nM 5-FAM-conjugated cyclic peptide and different concentrations of a target protein (160 nM to 160 µM) at black 96-well plates in a 200 µL total volume that was adjusted by adding the PBS buffer and then measured fluorescence polarization in a microplate reader at Ex/Em = 490 nm/520 nm. The Kd (μM) value was determined and shown. In the selection, in order to remove individuals capable of non-specific binding we incubated the phage library with only streptavidin magnetic beads for every round of selection, collected the unbound phages, and then subjected them to bind protein-binding streptavidin magnetic beads. After the third round, we sequenced 25 phage clones that converged to only three peptide sequences, CWRDYLI-AcrK, CQWFSHR-AcrK, and CGTWLKF-AcrK. The results indicated that both CWRDYLI-Ark and CQWFSHR-Ark bind to TEV protease with a single digit μM dissociation constant and both cyclic peptides bind to TEV protease significantly better than their linear counterpart (>6-fold). 3556 CQSLWMN(8)[7.1±0.7,9.7±0.7] CKHSLWV(2)[NA, NA] CLSDCRV(1)[NA, NA] 3 Phage display (subtractive panning) 3 PMID:31398275 Histone deacetylase 8, HD8 Not determined. Not determined. Not determined. pADLg3-TGC-(NNK)6-TAG phagemid library Fluorescence polarization We incubated a 25 nM 5-FAM-conjugated cyclic peptide and different concentrations of a target protein (160 nM to 160 µM) at black 96-well plates in a 200 µL total volume that was adjusted by adding the PBS buffer and then measured fluorescence polarization in a microplate reader at Ex/Em = 490 nm/520 nm. The Kd (μM) value was determined and shown in the firt column of the affinity values. In addition, IC50 (μM) values of selected cyclic peptides when binding to their protein targets were also determined and shown in the second column of the affinity values. In the selection, in order to remove individuals capable of non-specific binding we incubated the phage library with only streptavidin magnetic beads for every round of selection, collected the unbound phages, and then subjected them to bind protein-binding streptavidin magnetic beads. All selected cyclic peptide ligands showed 4 to 6-fold stronger affinity to their protein targets than their linear counterparts. 3557 LLADTTHHRPWT[0.472097±0.001114,0.32025] HATGTHGLSLSH[0.074187±0.001120,0.118277±0.001120] TSGYLHLRGRWR[0.03526,0.0615] 3 Phage display (subtractive panning) 4 PMID:31786302 C-terminal domain of CCN family member 2 Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA Positive phage clones were adsorbed to PKW-CTGF- and TrxA-CTGF-coated microplates, and the optical density at 450 nm (OD450) was directly determined using ELISA. OD450 values were reproduced from Figure 2A and shown. The first column of affinity values was the binding affinity of phage clones to PKW-CTGF, and the second column the binding affinity of phage clones to TrxA-CTGF. Human TrxA-CTGF/C and TrxA dissolved in sodium bicarbonate (NaHCO3, pH 8.6) were coated onto 96-well plates and incubated overnight at 4 °C. After blocking with 0.5% (w/v) bovine serum albumin (BSA), the wells were washed with TBST 6 times. The random peptide library was added to the TrxA-coated plate and incubated at 37 °C for 1 h. Supernatants were then added to the TrxA-CTGF/C-coated plate for 1 h at 37 °C. The plates were then washed six times with TBST and eluted with 0.2 M glycine/HCl (pH 2.2) containing 0.5% BSA, then immediately neutralized with Tris/HCl (pH 9.1). Next, phages were inoculated into LB medium with 10% E2738 at 37 °C for 4.5 h and centrifuged at 30,000 ×g for 15 min at 4 °C, the supernatants added to 1/6 volume PEG8000/NaCl at 4 °C overnight. Four rounds of selection were performed as follows: the supernatants were centrifuged at 12,000 ×g for 15 min and suspended in 0.5% Tween20-TBST; in the second round washed 10 times 0.5% Tween20-TBST; in the third round, washed 15 times with 1% Tween20-TBST; and, finally, in the fourth round, washed 20 times with 2% Tween20-TBST. 810A (LLADTTHHRPWT) was developed as a novel peptide, which binds to the C-terminal domain of connective tissue growth factor (CTGF). It could effectively inhibit the proliferation, migration, and expression of TGF-β and α-SMA in cells pretreated with CTGF. In in vivo experiments using an animal model of BLM-induced pulmonary fibrosis, the peptide demonstrated similar effects, which effectively reduced the onset and development of pulmonary fibrosis in mice. 3558 WEYDRYRGWHIG(2) RWPPHFEWHFDD(1) 2 Phage display (common panning) 4 PMID:32176482 Anti-F1 capsular antigen monoclonal antibody (YPF19) and human sera pool IgG of Alzheimer’s disease (AD) patients F1 capsule antigen Not determined. Not determined. pVIII-12aa phage display library (X12) ELISA Immunoscreening:positive spots on the immunoblots were detected using the Stable DAB chromogen system (Life Technologies, Monza, Italy). ELISA:the ELISA signal was measured at 450 nm using Labsystem Multiskan Bichromatic. Briefly, in the first round of selection, 500 μL of DYN−mAb YPF19 were incubated with 100 μL of each of the four phage libraries with a titer of e12 for 3−4 h at room temperature under mild stirring. The beads were washed 3 times in PBS−0.05% Tween 20 and separated with a magnetic device for 1−2 min to eliminate supernatants containing phage that did not bind the target present on the functionalized beads. Selected phage clones were eluted from antibodies with 500 μL of eluting buffer, 0.2 M of glycine-HCl (pH 2.2) + 0.1% BSA, neutralized immediately with 1 M Tris−HCl, pH 9.6. The enriched phage pools were amplified by infecting TG1 E. coli, purified twice by PEG precipitation, titrated, and used as the input for further panning. Biopanning affinity selection was repeated in the second round against DYN−IgG-AD, then in the third round against DYN−mAb YPF19 again, and finally a fourth selection round was carried out as the second one. Peptide 12III1(RWPPHFEWHFDD), was found to be able to prevent in vitro Aβ1-42-induced cytotoxicity in SH-SY5Y cells, as well as to promote disaggregation of preformed fibrils, to a greater extent with respect to wild-type phage (pC89). IgG levels detected by 12III1 provided a significant level of discrimination between diseased and nondemented subjects, as well as a good correlation with the state progression of the disease. These results give significant impact in AD state and stage diagnosis, paving the way for the development not only for an innovative blood diagnostic assay for AD precise diagnosis, progressive clinical assessment, and screening but also for new effective treatments. 3559 GGGCIEGPCLEG(2) WVGCHGEWCGVW(1) HRGCIEGPCLDA(1) 3 Phage display (common panning) 4 PMID:32176482 Anti-F1 capsular antigen monoclonal antibody (YPF19) and human sera pool IgG of Alzheimer’s disease (AD) patients F1 capsule antigen Not determined. Not determined. pVIII-12aa-Cys phage display library (X3CX4CX3) ELISA Immunoscreening:positive spots on the immunoblots were detected using the Stable DAB chromogen system (Life Technologies, Monza, Italy). ELISA:the ELISA signal was measured at 450 nm using Labsystem Multiskan Bichromatic. Briefly, in the first round of selection, 500 μL of DYN−mAb YPF19 were incubated with 100 μL of each of the four phage libraries with a titer of e12 for 3−4 h at room temperature under mild stirring. The beads were washed 3 times in PBS−0.05% Tween 20 and separated with a magnetic device for 1−2 min to eliminate supernatants containing phage that did not bind the target present on the functionalized beads. Selected phage clones were eluted from antibodies with 500 μL of eluting buffer, 0.2 M of glycine-HCl (pH 2.2) + 0.1% BSA, neutralized immediately with 1 M Tris−HCl, pH 9.6. The enriched phage pools were amplified by infecting TG1 E. coli, purified twice by PEG precipitation, titrated, and used as the input for further panning. Biopanning affinity selection was repeated in the second round against DYN−IgG-AD, then in the third round against DYN−mAb YPF19 again, and finally a fourth selection round was carried out as the second one. 3560 CQGPTFKCDAIWREC(20)[3.4±0.2] CVPQPDTCAKIDPRC(8)[>300] CLLQQPVCGEWNPKC(7)[>300] CYAAQGGCLRDWTRC(4)[~100] CYAAQGGCLRDWTRC(3)[>300] CYAINQRCLQEWSRC(3)[~250] CFADGSNCVVEWSEC(3)[N.D.] CIVQQGLCHEWNPRC(3)[>300] CVWLQATCTRSWSGC(3)[~300] CRPQPDTCVSLSGEC(2)[>300] CVPQPDTCANMDPEC(1)[>300] CKPQPDTCLHTTGKC(1)[N.D.] CYAAKGGCQPNWTQC(1)[~100] CYQSWPVCQAWNPRC(1)[>300] CQHAGAVCHWWNPRC(1)[>300] CNQDLMVCRFWNSRC(1)[>300] CRKMAEDCGWGVLVC(1)[>300] CKKVSMSCGWGEAVC(1)[>300] CRERMAVCGWGVQVC(1)[>300] CNWQTQVCVRDWLGC(1)[~300] CSWQQGECTRTWGGC(1)[>300] 21 Phage display (common panning) 3 PMID:31251434 72 kDa type IV collagenase Not determined. Not determined. Not determined. CX6CX6C phage display library Fluorescence The inhibitory activity of bicyclic peptides was determined by measuring sidual protease activity with a fluorogenic substrate. For the FS-6 substrate, excitation was measured at 325 nm and emission at 400 nm. For the OMNIMMP® RED substrate, excitation was measured at 545 nm and emission at 576 nm. The inhibition constants (Ki) were calculated according to the equation of Cheng and Prusoff Ki = IC50/(1+([S]0/Km) wherein IC50 is the peptide concentration at which 50% of the enzymaticactivity is inhibited, [S]0 is the total substrate concentration, and KM is the Michaelis-Menten constant. Ki (μM) values were shown. Bacterial cells of a phage library glycerol stock were added to a 2 L falcon flask containing 500 ml of 2YT/chloramphenicol (30 μg/ml) medium to obtain an OD600 of 0.1. The culture was shaken (200 rpm) for 16 hr at 30°C. Bacterial cells were pelleted by centrifugation at 16,000 g and 4°C for 30 min, and the phage in the supernatant were purified by PEG-precipitation as follows. Briefly, 0.2 volumes of PEG solution (20% (w/v) polyethylene glycol 6000, 2.5 M NaCl) was added to the supernatant, mixed, incubated on ice for 30 min, and centrifuged at 2,700 g and 4°C for 30 min. PEG-purified phage, typically 1011–1012 t.u. (transducing units) were resuspended in 20 ml of 20 mM NH4HCO3, 5 mM EDTA, pH 8.0. Disulfide bridges were reduced by addition of 1 mM of TCEP and incubation at 42°C for 1 hr. The concentration of TCEP was subsequently reduced by repetitive concentration and dilution steps with reaction buffer (20 mM NH4HCO3, 5 mM EDTA, pH 8.0, degassed) in a Vivaspin-20 filter (MWCO of 10,000, Sartorius-Stedim Biotech GmbH). The volume of the phage solution was adjusted to 32 ml with reaction buffer and 8 ml of 50 μM tris-(bromomethyl)benzene (TBMB) in acetonitrile (MeCN) were added to obtain a final TBMB concentration of 10 μM. The reaction was incubated at 30°C for 1 hr before non-reacted TBMB was removed by precipitation of the phage with 0.2 volumes of 20% (w/v) polyethylene glycol 6000, 2.5 M NaCl on ice and centrifugation at 2,700 g and 4°C for 30 minutes. The phage pellet was resuspended in 3 ml of washing buffer (10 mM Tris-Cl, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 1 mM CaCl2). Biotinylated active MMP-2 (180 μg) was added to 50 μl magnetic streptavidin beads (Dynabeads M-280 from Invitrogen Dynal Biotech AS) in washing buffer and incubated on a rotating wheel for 10 min at room temperature (RT). The magnetic beads were then washed with 0.5 ml washing buffer and incubated for 30 min at RT with 0.5 ml washing buffer containing 1% (w/v) BSA and 0.1% (v/v) Tween 20. At the same time the chemically modified phage (typically e10–e11 t.u. dissolved in 3 ml of washing buffer) were blocked by addition of 1.5 ml of washing buffer containing 3% (w/v) BSA and 0.3% (v/v) Tween 20 for 30 minutes. The blocked beads/target protein mixture (0.5 ml) and phage (4.5 ml) were mixed together and incubated for 30 minutes on a rotating wheel at room temperature. The beads were washed eight times with washing buffer containing 0.1% (v/v) Tween 20 and twice with washing buffer. The phage were eluted by incubation with 100 μl of 50 mM glycine, pH 2.2 for 5 minutes (pH-dependent elution), and then transferred to 50 μl of 1 M Tris-Cl, pH 8.0 for neutralization. The eluted phage were incubated with 30 ml of TG1 cells at OD600 of 0.4 for 90 minutes at 37°C, and the cells were plated on large 2YT/chloramphenicol (30 μg/ml chloramphenicol) plates. The second and third round of panning were performed following the same procedure but using in the second round neutravidin-coated magnetic beads instead of streptavidin to prevent the enrichment of streptavidin-specific peptide binders. Neutravidin beads were prepared by reacting 0.8 mg neutravidin (Pierce) with 0.5 ml of tosyl-activated magnetic beads (2e9 beads/ml; Dynabeads M-280, Invitrogen Dynal Biotech AS) according to the supplier's instructions. The abundant peptide M21 (ACQGPTFKCDAIWRECG) inhibited MMP-2 with a Ki of 3.4±0.2 μM. An alanine scan of M21 revealed that the first four amino acids of the C-terminal ring, Asp-Ala-Ile-Trp, were most important for the binding. 3561 WIPNSEFEHERT(23/50)[2.0] 1 Phage display (subtractive panning) 3 PMID:31301216 Fab A Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Fluorescence polarization The labeled peptide was diluted in PBS to prepare a stock solution of 500 nM. Fab A was diluted in PBS to two different concentrations (2 and 20 μM). Concentrations of peptide and Fab A were measured using a NanoDrop UV Spectrophotometer (Harlow Scientific). The level of binding was evaluated using fluorescence polarization (FP) by mixing equal volumes (20 μL) of the peptide and each Fab concentration in a 384 well black plate, resulting in a final peptide concentration of 250 nM. The dissociation constants (KD, μM) were determined. Three positive selections with Fab A as target and two negative selections were performed. For the first two positive rounds, Fab A was bound to protein L functionalized beads, while the last positive round employed covalent immobilization of the Fab on NHS beads. The negative selections were included to eliminate nonspecific binding of the phages to plastic, protein L, magnetic beads, and/or BSA. The first negative selection was performed by incubating with a BSA blocked tube containing protein L magnetic beads after the first positive panning rather than before, to avoid losing promising phage candidates that may have possessed significant binding to the target. The second negative selection was performed after the second positive selection, equal to the first one. Peptide B1 (WIPNSEFEHERT) was shown to selectively recognize Fab A. B1 having the lowest dissociation constant (KD) of 2 μM to a single Fab target. 3562 WHYNWQDVSDRQ[4.1] HQNHHSTFWEIY[6.5] 2 Phage display (subtractive panning) 4 PMID:31301216 Fab A,Fab Z and Fab D Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Fluorescence polarization The labeled peptide was diluted in PBS to prepare a stock solution of 500 nM. Fab A was diluted in PBS to two different concentrations (2 and 20 μM). Concentrations of peptide and Fab A were measured using a NanoDrop UV Spectrophotometer (Harlow Scientific). The level of binding was evaluated using fluorescence polarization (FP) by mixing equal volumes (20 μL) of the peptide and each Fab concentration in a 384 well black plate, resulting in a final peptide concentration of 250 nM. The dissociation constants (KD, μM) were determined. The first round of screen was performed with Fab A as target. Amplified phage from the first positive was diluted to a concentration of e11 pfu/200 μL and incubated thrice (first negative) in: (a) BSA blocked tube, for 1 hr at RT; (b, c) protein L magnetic beads, without Fab, for 50 and 40 min, respectively, at RT. In each incubation, the supernatant of the previous step was used. The supernatant after the first set of negative rounds was collected and incubated with Fab Z immobilized on Protein L magnetic beads, for 1 hr at RT (second positive). All the amplified phage from the second positive (concentration of e11 pfu/200 μL) was used in a second negative selection round, similar to the first negative (second negative). The supernatant of the second negative was collected and incubated with Fab D immobilized in protein L magnetic beads, for 45 min at RT. The amplified phage from the third positive was diluted to a concentration of e11 pfu/200 μL and added to four different tubes containing NHS beads (fourth positive) with: (a) a mixture of the three used Fabs, (b) Fab A, (c) Fab Z, and (d) Fab D. The top two peptide binders, A5 (WHYNWQDVSDRQ) and C7 (HQNHHSTFWEIY), were determined to have KD values of 4.1 μM and 6.5 μM, respectively. 3563 HAMRAQP(8)[243.3722±14.9784] NAPDWPA(4)[87.7108±23.5104] SPSTHWK(3)[24.0053±5.9724] STSFWIT(2)[41.7329±5.8776] NESHSRT(2)[26.1857±5.8776] GFFHKTT(1)[NT] LPAGRVL(1)[NT] NGLTAWT(1)[NT] AAPDWAG(1)[NT] HAKRARA(1)[NT] TLHPAAD(1)[NT] HVQLWAT(1)[NT] SAAIGTL(1)[NT] LSNNNLR(1)[NT] HAXRA,APDW 14 3 PMID:35103339 Colon cancer cell line SW480 Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA Binding selectivity of the frequently occurring selected phage clones to colon cancer cells was analyzed by cell-based ELISA. ELISA values for binding of each phage clone to each cell type were calculated by dividing the OD of selected phage clone to the OD of control phage (no displayed peptide). Data are reproduced from Figure 4 and presented as the mean ± SD. In the first round of in vitro selection, an aliquot of the primary library was added to an empty well (depletion well). The supernatant composed of phages that do not bind to the plastic surface of six-well plates was removed and poured into a serum-treated well. The supernatant containing phages that do not interact with serum was removed and transferred to the well with blocked absorber cells. The subtracted phage supernatant was recovered, applied to the target SW480 cells. According to the results of cell binding assay and phage cell-based ELISA, one of the isolated peptides denoted as CCBP1 (with the sequence HAMRAQP) was indicated to have the highest binding efficiency, selectivity, and specificity toward colon cancer cells with no significant binding to control cells. Peptide competitive inhibition assay revealed that binding of the phage-displayed CCBP1 is competitively inhibited by the same free peptide, suggesting that CCBP1 specific binding to the target cell is independent of the phage context. Taken together, our findings provide support for the notion that CCBP1 binds specifically to colon cancer cells and might be a potential lead candidate for targeted delivery of imaging agents or therapeutic genes/drugs to colon tumors. 3564 EHHRSHL(4)[0.6674±0.0648] HGSGVHA(3)[0.4796±0.076] APGGHSS(3)[0.8127±0.0425] VGYSGRD(2)[0.9651±0.0445] YMNDRMY(2)[0.8104±0.0516] DKSHVGL(2)[0.8398±0.0155] HPIKHLR(2)[0.7032±0.0335] SAQIAPH(2)[1.0009±0.0267] TGLIGQK(1)[0.5511±0.0335] GTQFFNK(1)[0.9448±0.0267] GYWNKFD(1)[0.7054±0.067] 12 Phage display (subtractive panning) 4 PMID:34202166 Copper ion, Cu(2+) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA An ELISA assay to determine the binding affinity of the 12 selected peptides to Cu(II) was performed. Absorbance was recorded at 405 nm with an ultraviolet spectrophotometer (UV 1800 Pharma Spec, Japan). Data are reproduced from Figure 2A and presented as the mean ± SE. In order to eliminate the phages bound to the resin, the IDA resin was first subjected to reverse biopanning followed by Cu(II) affinity biopanning. In brief, 100 μL of the phage solution (~2e12 plaque forming units, PFUS) in the NEB original peptide library was dissolved in 900 μL of TBST solution and added to 100 μL of IDA resin, followed by gently shaking for 25 min at 25°C. The supernatant was extracted and further infected with E. coli ER2738 cell culture for amplification according to the manufacturer’s instructions. Then affinity screening was carried out for screening Cu(II)-binding peptide. 100 μL of the phage solution (~0.8e12 virions) from the reverse screening amplification was dissolved in 900 μL of TBST solution, and added to 100 μL of Cu(II) chelating resin, followed by gently shaking for 25 min at 25°C. The supernatant was discarded, and the resin was washed twice with 1 mL of TBST solution to remove unbound phages. Finally, 1 mL of EDTA (0.5 M, pH 8.0) was added, and the eluate was collected by shaking at 200 rpm at 25°C for 10 min. The eluate was centrifuged with a 100 KD centrifugal filter device (Millipore, U.S.) at 4°C, 5000 rpm, 15 min to remove Cu(II). The remaining phages are amplified and subjected to the next round of screening. Besides, to obtain high affinity phages, 4 rounds of biopanning were conducted. In the fourth round of biopanning, Cu(II) loaded on the resin beads was reduced by washing with citrate buffer solution (pH 4.4) to improve the biopanning affinity. The Cu(II)-binding peptide (SAQIAPH, PCu) identified from the phage display heptapeptide library was used to explore the mechanism of PCu inhibition of Cu2+-mediated Aβ aggregation and Aβ production. In vitro experiments revealed that PCu directly inhibited Cu2+-mediated Aβ aggregation and regulated copper levels to reduce biological toxicity. Furthermore, PCu reduced the production of Aβ by inhibiting Cu2+-induced BACE1 expression and improving Cu(II)-mediated cell oxidative damage. Cell culture experiments further demonstrated that PCu had relatively low toxicity. This Cu(II)-binding peptide that we have identified using phage display technology provides a potential therapeutic approach to prevent or treat Alzheimer's disease. 3565 GWRVSEF(24) GWRVSEL(1) GFHYSLH(1) IVGSQVT(1) GWRVSE 4 3 PMID:34314166 Cowpea mosaic virus, CPMV Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) ELISA The plate was incubated in the dark for 10 min, and the absorbance was measured at 370 nm by using an Infinite 200 Rx plate reader (Tecan Life Sciences) with 25 flashes in 96-well flat-bottom plate mode. The input library was preincubated overnight with blocking agent (2% (w/v) bovine serum albumin; BSA) each round to remove potential BSA binders. Biopanning and characterization of lead candidates resulted in isolation of the motif “GWRVSEF/L” as the plant virus cowpea mosaic virus (CPMV) specific motif with phenylalanine (F) at the seventh position being stronger than leucine (L). Specificity to CPMV was demonstrated, and cross-reactivity toward other plant viruses was not observed. To demonstrate cargo loading, GWRVSEF was tagged with biotin, fluorescein isothiocyanate (FITC), and a human epidermal growth factor receptor 2 (HER2)-specific targeting peptide ligand. Display of the active ingredient was confirmed, and utility of tagged and targeted CPMV in cell binding assays was demonstrated. The CPMV-binding peptides (CBP) functionalization strategy offers a new avenue for CPMV nanoparticle functionalization and should offer a versatile tool to add active ingredients that otherwise may be difficult to conjugate or display. 3566 AHWNPFWLATPF[0.409] YWVDSSAWVAHK[0.3389] NNDPLQLRSQRY[0.4388] KLDVFTKPLVFT[0.2597] 4 3 PMID:35755076 Purified serum IgG from human T-lymphotropic virus 1 (HTLV-1) infected people Human T-lymphotropic virus 1 Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA After the screening performed in the first Phage ELISA, the phage clones containing the peptides that presented a ratio between the absorbances of the positive and control samples >2 were submitted to a second Phage ELISA. Although most of the peptides could discriminate individuals with HTLV-1 infection from seronegative control individuals, four peptides (A6, A8, B6, and D7) stood out from the rest. In the phage ELISA, dilutions of 1:50, 1:100, 1:500, and 1:1000 were used, and the best results were obtained at a dilution of 1:1000. The biopanning process for selection of HTLV-1 peptides included a subtractive step, which consisted of incubating the phage library (1.0e11 phage particles of the PD library) first with IgG from HTLV-seronegative individuals for 30 min at room temperature. Then, the supernatant containing unbound phages was added to IgG of HTLV-2-seropositive individuals with incubation for 30 min at room temperature. The next step consisted of positive selection, in which the final supernatant of the subtractive step was incubated with purified IgG from HTLV-1-positive individuals for 30 min at room temperature. Three biopanning cycles were performed for the selection of HTLV-1 peptides.The phages bound to the magnetic microspheres of the positive selections were recovered by acid elution and subjected to steps of amplification, titration, supernatant production, DNA extraction, and phage DNA sequencing. Bioprospection of peptides mimicking HTLV-1 using phage display led to the identification of four clones, three related to gp46 (A6, B6, and D7) and one related to protease and Tax (A8). The analysis of accessibility, antigenicity, and hydrophilicity showed that clones A6 and B6 are potentially suitable for diagnostic use and that clone B6 has great potential for use in vaccine tests. The best reactivity, evaluated by phage ELISA, was that of clone B6, which showed good AUC, sensitivity, specificity, and LR. Thus, the successful testing of the B6 clone peptide would be relevant to diagnostic tests on platforms that allow rapid results and to the availability of more affordable testing for the overall population, and it would contribute to the prevention and control of HTLV-1 infection. 3567 CTDKASSSC[0.6487±0.0543] CHMYHNATC[0.5415±0.0545] 2 Phage display (competitive panning) 4 PMID:35032803 Ovomucoid Not determined. Not determined. Not determined. Ph.D.-C7C phage display library (CX7C) ELISA ELISA signal was measured using a microplate spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA) at 405 nm. Streptavidin-coated plates (streptavidin high binding capacity coated 96-well plates) (Thermo Scientific, USA) were used for phage display. After adding 1 μL (1.0e11 PFU/mL) of each M13 library to 100 μL (24.75 μg/mL) of biotinylated ovomucoid, the mixture was stirred at 100 rpm for 1 h at room temperature. When the agitation of the phage-protein complex was completed, the streptavidin-coated plate was washed three times repeatedly using 0.1 M PBS, pH 7.2. Thereafter, the phage-protein complex was added to the streptavidin-coated plates and allowed to react at 110 rpm for 10 min to enable the specific binding between avidin and biotin. Then, 1 μL of biotin (0.1 mM) was added to the plate and incubated for 5 min for blocking, followed by repeated washing 10 times using PBST containing 0.1 M PBS with 0.1 % Tween 20. Finally, phages specifically bound to ovomucoid were eluted in 0.2 M Glycine-HCl (pH 2.2) 1 mg/mL BSA solution, and 15 μL of Tris-HCl (pH 9.0) was added for neutralization. After the characterization of binding affinities of selected phages, whole phage particles were covalently attached to a gold electrode using crosslinking chemistry (MUA-EDC/NHS and Sulfo-LC/SPDP); the developed phage sensor was characterized using cyclic voltammetry (CV), square wave voltammetry (SWV), and electrochemical impedance spectroscopy (EIS). The cyclic peptide (CTDKASSSC) displayed phage sensor modified using EDC/NHS chemistry exhibited significantly better binding affinity (Kd = 2.36 ± 0.44 μg/mL) and limit of detection (LOD, 0.12 μg/mL) for ovomucoid than the linear phage sensor, resulting in good reproducibility and recovery, even in an actual egg and white wine samples. This approach may provide an alternative and more efficient way of sensing food allergens with desirable sensitivity, selectivity, and feasibility in food diagnostic applications. 3568 LQAYIGPKATWW[0.5068±0.0774] HHSRFSTLFNWP[0.5013±0.0917] WWQPYSSAPRWL[0.8965±0.0631] 3 Phage display (competitive panning) 4 PMID:35032803 Ovomucoid Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) ELISA ELISA measurements were performed to compare the relative binding affinities of the five M13 phage candidates as follows. First, the streptavidin-coated plate was pre-washed thrice with 200 μL 0.1 M PBS for 5 min each, following which 100 μL biotinylated ovomucoid (24.75 μg/mL) was added to the streptavidin-coated plate and stirred at room temperature for 1 h. Subsequently, the protein solution was removed and 200 μL blocking solution (5% BSA in NaHCO3, pH 8.6) was added and incubated at 4 ℃ for 1.5 h. Then, after repeated washing with 0.5% PBST six times, each phage was added at a concentration of 1012 PFU/mL, followed by stirring at 100 rpm at room temperature for 1 h. After washing six times with 0.5 % PBST, horseradish peroxidase (HRP)-conjugated anti-M13 monoclonal antibody (1:2,500 v/v) diluted in blocking solution was added and the microplate was then incubated for 1 h at 25 °C. Unbound antibody solution was removed, and the microplate was washed again with TBST. Freshly prepared HRP substrates, tetramethylbenzidine (TMB) were introduced to the microplate and the ELISA signal was measured using a microplate spectrophotometer (Multiskan FC, Thermo Scientific, Waltham, MA, USA) at 405 nm. Streptavidin-coated plates (streptavidin high binding capacity coated 96-well plates) (Thermo Scientific, USA) were used for phage display. After adding 1 μL (1.0e11 PFU/mL) of each M13 library to 100 μL (24.75 μg/mL) of biotinylated ovomucoid, the mixture was stirred at 100 rpm for 1 h at room temperature. When the agitation of the phage-protein complex was completed, the streptavidin-coated plate was washed three times repeatedly using 0.1 M PBS, pH 7.2. Thereafter, the phage-protein complex was added to the streptavidin-coated plates and allowed to react at 110 rpm for 10 min to enable the specific binding between avidin and biotin. Then, 1 μL of biotin (0.1 mM) was added to the plate and incubated for 5 min for blocking, followed by repeated washing 10 times using PBST containing 0.1 M PBS with 0.1 % Tween 20. Finally, phages specifically bound to ovomucoid were eluted in 0.2 M Glycine-HCl (pH 2.2) 1 mg/mL BSA solution, and 15 μL of Tris-HCl (pH 9.0) was added for neutralization. 3569 IPPVPTTTKASV(4)[48.1236±3.7248] WHWTWLSEYPPP(3)[34.79±5.3442] HWTSFSWLGSWN(3)[55.1952±1.9973] IPPVPRLKTSYK(2)[69.8783±7.0177] FHWSLPWLPGLP(2)[77.5438±3.3469] WHFEWWRATPSG(2)[31.767±5.0203] HWWNTPWWAWHP(2)[43.1572±7.9893] HWNFWMHNAHWN(2)[46.828±1.9973] HTWWLWPPQLPP(2)[54.4935±3.6708] WHTTELLSLPWR(1)[31.1192±13.0097] WHWNPFRVSQPL(1)[32.4148±1.0257] WHFSYSYKPALS(1)[36.7874±12.038] HWNFWMQNATRS(1)[46.828±2.3212] ILLGRRLKYSTA(1)[47.4758±3.6708] WHTTELLSLPWR(1)[48.1236±3.7248] WHWNAWALPTHG(1)[54.4935±5.3442] NHWPSYFAQWMT(1)[89.2579±0.7018] WHW, HW, WH and HWW 17 4 PMID:35068948 72 kDa type IV collagenase Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) MMP-2 (15 ng) was incubated overnight with 2e11 pfu/mL phage clones that were enriched for binding against active MMP-2. After 18 h, each mixture was run on 7.5% SDS-PAGE with 20 mg/mL gelatin. After electrophoresis, the gel was washed twice at room temperature with 2.5% Triton-X 100 for 30 min. The gel was then kept overnight in zymogram retention solution (6.06 g Tris-HCl, 1.47 g CaCl2, and 2.92 g NaCl per liter). The next day, the gel was stained with Coomassie Blue R. The density of the bands were analyzed using the Bio-Rad Multi-Analyst program (Tajhya et al., 2017). The gel was scanned using a digital scanner, and MMP-2 activity was determined by measuring the peak area of each band. The peak percentage was normalized relative to the activity of MMP-2 (positive control) and wild-type M13 phage (negative control). The inhibitory effects of the peptides on the gelatinase activity of MMP-2 was reproduced from Figure 4. For subtractive screening, 200 μL of the Ph.D.-12 library (containing 4e10 phages) was first added to wells coated with active matrix metalloproteinase-9 (MMP-9) ligand incubated for 1 h at room temperature. Unbound phages were recovered and transferred to wells coated with active-MMP-2. MMP-2 inhibition by selected peptides was evaluated through zymogram analyses, which revealed that four peptides (WHFEWWRATPSG, WHWNPFRVSOPL, WHTTELLSLPWR and WHWTWLSEYPPP) inhibited MMP-2 activity by at least 65%. These four peptides were synthesized and used for in vitro wound healing using human umbilical vein endothelial cells, and two peptides, AOMP12 (WHTTELLSLPWR) and AOMP29 (WHWTWLSEYPPP), were found to inhibit wound healing by 40%. These peptides are, thus, potential candidates for MMP- 2 inhibition for cancer treatment. Furthermore, our findings suggest that our substractive biopanning screening method is a suitable strategy for identifying peptides that selectively inhibit MMP-2. 3570 SLNTTWVSPMMK(19) YNTHGVRDNAWL(6) IESRYLTKEAVH(3) 3 Phage display (common panning) 2-4 PMID:37044252 Microcentrifuge tube (polypropylene, PP) Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) 3571 TPPSSNSYDWLV(17) METRPVAPHEFR(8) ALKIGPETTIYM(4) 3 Phage display (common panning) 2-4 PMID:37044252 Boiled Daphnia magna asexual egg Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) 3572 LYALPLSHLKSH(13)[95.55%] SPSSAYPRHGPD(12)[87.50%] DPLLFPGTSRQM(6)[47.00%] 3 Phage display (common panning) 2-4 PMID:37044252 Daphnia magna asexual egg Not determined. Not determined. Not determined. Ph.D.-12 phage display library (X12) Binding assay Ten microliters of each phage was added to 200 μL of ER2738 culture grown to an OD600 of 0.7 and incubated at RT for 5 min. The cells with infected phages were transferred to a conical tube containing 5 mL of Top Agar at around 40 ◦C and poured directly onto an LB/IPTG/Xgal plate containing 1 mL of IPTG/Xgal stock in 25 mL of DMF. After standing for 1 h to solidify, the plates were incubated at 37 ◦C overnight. After counting the number of blue plaques on each plate, we calculated the pfu/mL value while considering the dilution factor of each phage. Attached % was calculated as: ((Input phage-Output phage)/Input phage) × 100. We identified a peptide, DEP1 (LYALPLSHLKSH), with the highest binding affinity to D. magna eggs. DEP1 did not affect zebrafish eggs, but it inhibited normal hatching and reproductive ability in D. magna eggs, and hindered growth in neonates before their first ecdysis. Morphological analysis revealed that DEP1 caused intestinal damage and tissue abnormalities. Our findings demonstrate that the whole cell-based phage display technique is successful in presenting antigens in their natural form, and that the DEP1 peptide can be applied to regulate the growth cycle of D. magna. These results have implications for the use of phage display in environmental research and the potential use of DEP1 for hazardous organisms in aquatic systems. 3573 CTEWDYLTVC 1 Phage display (common panning) 4 PMID:33508757 Prostate specific antigen (PSA(-/lo)) human prostate cancer cell line LNCaP Not determined. Not determined. Not determined. fUSE55-based CX8C phage display library (CX8C) Prostate cancer (PCa) contains both differentiated (PSA(+)) and undifferentiated (PSA(−/lo)) tumor cells. PSA was short for prostate-specific antigen. PSA(+) and PSA(-/lo) cells were mixed in equal proportions and incubated with CX8C phage display library. A measure of 1.0e9 of the phage display peptide library in 5 ml of 1 ×phosphate buffered saline (PBS) was added to the confluent cell monolayer and incubated for 2 h on a rocker platform at 4 °C. The cells were washed four times with PBS containing 0.2% Tween 20. Cells were subjected to fluorescence-activated cell sorting (FACS) for GFP(+) and GFP(−) cells. Sorted cells were lysed to release phage particles that were used to infect K91 bacteria. Individual bacterial colonies that emerged on the Tet/Kanamycin plates were isolated, amplified, and phage particles reisolated and used in 2–3 more similar cycles. PSA(−/lo) PCa cell-specific targeting peptide (TAP1, CTEWDYLTVC) suppressed PCa cell growth both in vitro and in vivo and improved the drug sensitivities of anti-androgens and chemotherapeutic agents at least through shortening the length of telomere and reducing the expression of HOXB9 and TGF-β2. 3574 STTGTQY(4)[30.7011±2.0436] DLFVSSL(1)[NT] VNLNLLP(1)[NT] SIWQSLN(1)[NT] AKLHILR(1)[NT] KHINPSI(1)[NT] YWPGYSM(1)[NT] SPLLIPQ(1)[NT] NQVLARH(1)[NT] TWQVLRP(1)[NT] YDRENHP(1)[NT] LEVTPWW(1)[NT] YLSPVPM(1)[NT] 13 Phage display (subtractive panning) 3 PMID:33706101 Human embryonic kidney cells 293 (HEK293 cells) transfected with free fatty acid receptor 1 (FFAR1) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) HEK293 cells were seeded in a 24-well plate at a density of 1.0e5 cells/well and incubated overnight. Expression vectors (pCAGGS/FFAR1 and pCAGGS/APTGF- a) were kindly provided by Dr. Inoue (Tohoku University) and used for transfection. A mixture of 125 ng pCAGGS/AP-TGF-α and 50 ng pCAGGS/FFAR1 per well in 24-well plates was transfected using PEI (49553-93-7, Polysciences, Warrington, PA, USA) and incubated overnight. The following day, HEK293 cells were harvested by trypsinization and re-seeded in 96-well plates at a volume of 80 mL/well. After incubation for 1 h, 20 mL of palmitic acid (PA) and peptide samples in HBSS containing 5 mM HEPES (pH 7.4) were added and incubated for 1 h. Eighty microliters of supernatant containing released AP-TGF-α was transferred into an empty 96- well plate. A substrate for alkaline phosphatase, paranitrophenylphosphate (p-NPP, 34045, Thermo Fisher Scientific) was added to the transferred medium, and absorbance at 405 nm of the plates was measured using a microplate reader before and after 30 min incubation at 37 ◦C. We calculated AP-TGF-α release as a TGF-α shedding response. First, 1.0e7 HEK293 cells in 1 mL phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) were incubated with 1.5e11 phages on a shaking machine (120 rpm) for 2 h at 4 °C. This mixture was centrifuged at 1500 rpm for 1 min, and the supernatant containing phages that could not bind HEK293 cells was incubated with other HEK293 cells. This selection (negative selection) was repeated twice to remove phage-binding HEK293 cells. Next, the supernatant was incubated with 5.0e6 HEK293/FFAR1 cells on a shaking machine (120 rpm) for 2 h at 4 °C. The HEK293/FFAR1 cells were washed five times in cold PBS containing 1% BSA and 0.1% Tween-20. HEK293/FFAR1 cells binding phages were eluted with Glycine-HCl (pH 9.1) containing 1 mg/mL BSA on ice for 10 min. The 10 μL phages were titered according to the manufacturer’s instructions, and the amplified phage using E. coli ER2738 was used in the next round of selection. STTGTQY determined by phage display promoted glucose-stimulated insulin secretion in pancreatic MIN6 cells. 3575 DLFVSSL(18)[24.3461±1.1215] AETVESC(2)[NT] STTGTQY(1)[NT] WSLDPSS(1)[NT] SLVVERL(1)[NT] 5 Phage display (subtractive panning) 4 PMID:33706101 Human embryonic kidney cells 293 (HEK293 cells) transfected with free fatty acid receptor 1 (FFAR1) Not determined. Not determined. Not determined. Ph.D.-7 phage display library (X7) HEK293 cells were seeded in a 24-well plate at a density of 1.0e5 cells/well and incubated overnight. Expression vectors (pCAGGS/FFAR1 and pCAGGS/APTGF- a) were kindly provided by Dr. Inoue (Tohoku University) and used for transfection. A mixture of 125 ng pCAGGS/AP-TGF-α and 50 ng pCAGGS/FFAR1 per well in 24-well plates was transfected using PEI (49553-93-7, Polysciences, Warrington, PA, USA) and incubated overnight. The following day, HEK293 cells were harvested by trypsinization and re-seeded in 96-well plates at a volume of 80 mL/well. After incubation for 1 h, 20 mL of palmitic acid (PA) and peptide samples in HBSS containing 5 mM HEPES (pH 7.4) were added and incubated for 1 h. Eighty microliters of supernatant containing released AP-TGF-α was transferred into an empty 96- well plate. A substrate for alkaline phosphatase, paranitrophenylphosphate (p-NPP, 34045, Thermo Fisher Scientific) was added to the transferred medium, and absorbance at 405 nm of the plates was measured using a microplate reader before and after 30 min incubation at 37 ◦C. We calculated AP-TGF-α release as a TGF-α shedding response. First, 1.0e7 HEK293 cells in 1 mL phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA) were incubated with 1.5e11 phages on a shaking machine (120 rpm) for 2 h at 4 °C. This mixture was centrifuged at 1500 rpm for 1 min, and the supernatant containing phages that could not bind HEK293 cells was incubated with other HEK293 cells. This selection (negative selection) was repeated twice to remove phage-binding HEK293 cells. Next, the supernatant was incubated with 5.0e6 HEK293/FFAR1 cells on a shaking machine (120 rpm) for 2 h at 4 °C. The HEK293/FFAR1 cells were washed five times in cold PBS containing 1% BSA and 0.1% Tween-20. HEK293/FFAR1 cells binding phages were eluted with Glycine-HCl (pH 9.1) containing 1 mg/mL BSA on ice for 10 min. The 10 μL phages were titered according to the manufacturer’s instructions, and the amplified phage using E. coli ER2738 was used in the next round of selection. DLFVSSL showed free fatty acid receptor 1 (FFAR1) agonist activity, although it was lower than that of STTGTQY.