Peptide Information

General Information of This Peptide
Peptide ID
PEP00115
Peptide Name
P1GCGT1
Structure
Sequence
NPNWGRSWYNQRFKGCGRIKPRKGYTR
Peptide Type
Linear
Receptor Name
Receptor tyrosine-protein kinase erbB-2 (ERBB2)
  Receptor Info 
PDC Transmembrane Types Cell targeting peptides (CTPs)
Formula
C148H225N51O36S
Isosmiles
[H]NCCCC[C@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CCC/N=C(/N)N[H])NC(=O)[C@H](CCC(=O)N[H])NC(=O)[C@H](CC(=O)N[H])NC(=O)[C@H](Cc1ccc(O[H])cc1)NC(=O)[C@H](Cc1cn([H])c2ccccc12)NC(=O)[C@H](CO[H])NC(=O)[C@H](CCC/N=C(/N)N[H])NC(=O)CNC(=O)[C@H](Cc1cn([H])c2ccccc12)NC(=O)[C@H](CC(=O)N[H])NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)N[H])N[H])C(=O)NCC(=O)N[C@@H](CS[H])C(=O)NCC(=O)N[C@@H](CCC/N=C(/N)N[H])C(=O)N[C@]([H])(C(=O)N[C@@H](CCCCN[H])C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC/N=C(/N)N[H])C(=O)N[C@@H](CCCCN[H])C(=O)NCC(=O)N[C@@H](Cc1ccc(O[H])cc1)C(=O)N[C@]([H])(C(=O)N[C@@H](CCC/N=C(/N)N[H])C(=O)O)[C@@H](C)O[H])[C@@H](C)CC
InChI
InChI=1S/C148H225N51O36S/c1-4-77(2)119(139(230)187-98(33-14-17-53-151)142(233)199-60-24-40-110(199)137(228)186-96(37-21-57-170-147(163)164)126(217)182-91(31-12-15-51-149)121(212)174-73-117(210)180-100(62-80-41-45-84(202)46-42-80)135(226)197-120(78(3)201)140(231)188-99(143(234)235)38-22-58-171-148(165)166)196-129(220)94(35-19-55-168-145(159)160)179-116(209)72-177-124(215)108(76-236)181-118(211)74-175-122(213)92(32-13-16-52-150)183-130(221)101(61-79-25-6-5-7-26-79)189-127(218)95(36-20-56-169-146(161)162)184-128(219)97(49-50-111(153)204)185-133(224)105(67-113(155)206)193-131(222)102(63-81-43-47-85(203)48-44-81)190-132(223)104(65-83-70-173-90-30-11-9-28-87(83)90)192-136(227)107(75-200)195-125(216)93(34-18-54-167-144(157)158)178-115(208)71-176-123(214)103(64-82-69-172-89-29-10-8-27-86(82)89)191-134(225)106(68-114(156)207)194-138(229)109-39-23-59-198(109)141(232)88(152)66-112(154)205/h5-11,25-30,41-48,69-70,77-78,88,91-110,119-120,172-173,200-203,236H,4,12-24,31-40,49-68,71-76,149-152H2,1-3H3,(H2,153,204)(H2,154,205)(H2,155,206)(H2,156,207)(H,174,212)(H,175,213)(H,176,214)(H,177,215)(H,178,208)(H,179,209)(H,180,210)(H,181,211)(H,182,217)(H,183,221)(H,184,219)(H,185,224)(H,186,228)(H,187,230)(H,188,231)(H,189,218)(H,190,223)(H,191,225)(H,192,227)(H,193,222)(H,194,229)(H,195,216)(H,196,220)(H,197,226)(H,234,235)(H4,157,158,167)(H4,159,160,168)(H4,161,162,169)(H4,163,164,170)(H4,165,166,171)/t77-,78+,88-,91-,92-,93-,94-,95-,96-,97-,98-,99-,100-,101-,102-,103-,104-,105-,106-,107-,108-,109-,110-,119-,120-/m0/s1
InChIKey
FSOBPZYHHFLALA-DIFMCXABSA-N
Pharmaceutical Properties
Molecule Weight
3326.816
Polar area
1487.26
Complexity
3324.706404
xlogp Value
-16.9759
Heavy Count
236
Rot Bonds
116
Hbond acc
45
Hbond Donor
50
The Activity Data of This Peptide
Peptide Activity Information 1 [1]
Van der Waals energy -1257.58 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-IV)
Peptide Activity Information 2 [1]
Van der Waals energy -805.29 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-II)
Peptide Activity Information 3 [1]
SASA energy -68.33 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-IV)
Peptide Activity Information 4 [1]
SASA energy -57.47 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-II)
Peptide Activity Information 5 [1]
Polar solvation energy 963.88 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-II)
Peptide Activity Information 6 [1]
Polar solvation energy 1418 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-IV)
Peptide Activity Information 7 [1]
KD 151 nM
Binding Affinity Assay
The HER2 recombinant protein was initially coupled to Chip CM5 by amide formation reaction using EDC/NHS. Different peptide concentrations (50, 25, 12.5, 6.25, and 3.125 μM) dissolved in PBST buffer flowed across the surface of the protein-coupled chip, and real-time binding signals were recorded and analyzed. The Biacore evaluation software was used to calculate the dissociation constant (KD) according to association-dissociation curves at different peptide concentrations.

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Experimental Condition Chip CM5 in the vitro
Peptide Activity Information 8 [1]
Electrostatic energy -301.04 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-IV)
Peptide Activity Information 9 [1]
Electrostatic energy -263.26 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-II)
Peptide Activity Information 10 [1]
Binding energy -208.96 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-IV)
Peptide Activity Information 11 [1]
Binding energy -162.14 kJ/mol
Binding Affinity Assay
To further study the interaction between peptides and HER2 protein, the binding free energy was calculated for each complex using the MMPBSA method.
Experimental Condition MMPBSA method (HER2-II)
Each Peptide-drug Conjugate Related to This Peptide
Full Information of The Activity Data of The PDC(s) Related to This Peptide
I-1 (NPNWGRSWYNQRFKGC=(-SS-O-COO-CPT)GRIKPRKGYTR) [Investigative]
 PDC Info 
Revealed Based on the Cell Line Data
Click To Hide/Show 5 Activity Data Related to This Level
Experiment 1 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
1.47 ± 0.54 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

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Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Breast adenocarcinoma SK-BR-3 cell CVCL_0033
Experiment 2 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
3.29 ± 0.67 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Gastric tubular adenocarcinoma NCI-N87 cell CVCL_1603
Experiment 3 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
4.29 ± 1.11 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Ovarian serous cystadenocarcinoma SK-OV-3 cell CVCL_0532
Experiment 4 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
7.60 ± 1.23 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Breast adenocarcinoma MDA-MB-231 cell CVCL_0062
Experiment 5 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
23.90 ± 1.58 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Normal MCF-10A cell CVCL_0598
I-2 (NPNWGRSWYNQRFKGC=(-SS-COO-CPT)GRIKPRKGYTR) [Investigative]
 PDC Info 
Revealed Based on the Cell Line Data
Click To Hide/Show 5 Activity Data Related to This Level
Experiment 1 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
5.76 ± 0.79 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Gastric tubular adenocarcinoma NCI-N87 cell CVCL_1603
Experiment 2 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
6.25 ± 0.96 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Ovarian serous cystadenocarcinoma SK-OV-3 cell CVCL_0532
Experiment 3 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
6.42 ± 1.21 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

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Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Breast adenocarcinoma SK-BR-3 cell CVCL_0033
Experiment 4 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
10.93 ± 1.14 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

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Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Breast adenocarcinoma MDA-MB-231 cell CVCL_0062
Experiment 5 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
20.41 ± 1.04 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

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Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Normal MCF-10A cell CVCL_0598
I-3 (NPNWGRSWYNQRFKGC=(-S-Maleimide-CPT)GRIKPRKGYTR) [Investigative]
 PDC Info 
Revealed Based on the Cell Line Data
Click To Hide/Show 5 Activity Data Related to This Level
Experiment 1 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
15.32 ± 2.25 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

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Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Gastric tubular adenocarcinoma NCI-N87 cell CVCL_1603
Experiment 2 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
23.09 ± 1.77 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

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Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Breast adenocarcinoma MDA-MB-231 cell CVCL_0062
Experiment 3 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
24.39 ± 1.45 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Ovarian serous cystadenocarcinoma SK-OV-3 cell CVCL_0532
Experiment 4 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
25.45 ± 0.64 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Breast adenocarcinoma SK-BR-3 cell CVCL_0033
Experiment 5 Reporting the Activity Data of This PDC [1]
Indication Breast cancer
Efficacy Data Half Maximal Inhibitory Concentration (IC50)
35.20 ± 2.64 µM
Administration Time 48 h
Evaluation Method MTT assay
MOA of PDC
To enhance the affinity of the human epidermal growth receptor 2 (HER2) targeted peptide developed previously, bispecific fusion peptidesP1GCGT1andP1GCGCGT1were designed using anin silicoapproach. Molecular dynamic simulation showed that both peptides strongly interacted with HER2 domains II and IV. Compared with peptides targeting each single domain,P1GCGT1andP1GCGCGT1could bind to HER2 more significantly and targeted HER2-positive cells specifically. Additionally, both peptides were used to generate peptide-drug conjugates with camptothecin (CPT), among whichI-1andI-4were screened for enhanced cellular activity and selectivity. Biological evaluation demonstrated thatI-1andI-4induced cell apoptosis, promoted cell cycle arrestin S-phase, and inhibited Topo I activity. The binding affinity assay and confocal analysis revealed thatI-1andI-4were effective at targeting HER2. Moreover,I-1andI-4showed better stability than single targeting peptide and presented enhanced antitumor activity and safety than CPT in tumor-bearing mice.

   Click to Show/Hide
Description
The cytotoxicity of conjugates was evaluated on cell lines with different expression levels of HER2. The results showed that among all conjugates with a single CPT molecule (I-1 to I-3), I-1 was the most cytotoxic against three HER2-positive cell lines (SK-BR-3, NCI-N87, and SK-OV-3 cells), and the calculated IC50 values for the three cells were 1.47 ± 0.54, 3.29 ± 0.67, and 4.29 ± 1.11 uM, respectively. Similarly, among all conjugates with two CPT molecules (I-4 to I-6), I-4 showed the highest cytotoxicity toward HER2-positive cells, and the IC50 values for SK-BR-3, NCI-N87, and SK-OV-3 cells were 0.12 ± 0.07, 1.78 ± 0.47, and 1.27 ± 0.38 uM, respectively. The activity of I-4 against SK-BR-3 cells was also slightly better than that of the positive control CPT (IC50 = 0.21 ± 0.04 uM). The cytotoxicity of I-1 and I-4 against HER2-negative MDA-MB-231 cells was comparatively lower (calculated IC50s were 7.60 ± 1.23 and 7.72 ± 0.92 uM, respectively). In contrast, CPT itself showed higher cytotoxicity against MDA-MB-231 (IC50 = 1.06 ± 0.42 uM), indicating that conjugates showed higher specificity in cell targeting. Furthermore, the cytotoxicity of I-1 and I-4 against normal cells MCF-10A (IC50 = 23.90 ± 1.58 and 17.14 ± 2.42 uM, respectively) was significantly lower than that of CPT alone (2.58 ± 0.77 uM). Based on these results, I-1 and I-4 were selected for subsequent biological evaluation.

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In Vitro Model Normal MCF-10A cell CVCL_0598
References
Ref 1 In Silico Exploration and Biological Evaluation of Bispecific Peptides Derived from Anti-HER2 Antibodies and Peptide-Camptothecin Conjugates for HER2-Positive Breast Cancer. J Med Chem. 2022 Nov 24;65(22):15123-15139. doi: 10.1021/acs.jmedchem.2c00968. Epub 2022 Nov 9.