The Immunosuppressive Properties of Cyclo-[D-Pro-Pro-β3-HoPhe-Phe-] Tetrapeptide Selected from Stereochemical Variants of Cyclo-[Pro-Pro-β3-HoPhe-Phe-] Peptide
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Isolation of Human Peripheral Blood Mononuclear Cells (PBMCs)
2.3. Proliferative Response of PBMC to PHA
2.4. The Toxicity Test
2.5. Determination of TNF-α Activity Using a Bioassay
2.6. Colorimetric MTT Assay for Cell Growth and Death
2.7. Mice
2.8. Isolation of Thymocytes
2.9. Analysis of the Peripheral Blood Picture
2.10. Contact Sensitivity to Oxazolone
2.11. Carrageenan-Induced Inflammation of Air Pouches
2.12. Dextran Sulfate-Induced Colitis
2.13. Statistics
2.14. In Silico Studies
2.15. UHPLC-MS Analysis
2.16. NMR Spectroscopy
2.17. Quantum-Chemical Calculations
3. Results
3.1. Estimation of the Purity of the D- and L-Stereoisomers
3.2. Evaluation of Activities of the Peptides In Vitro
3.3. Contact Sensitivity to Oxazolone
3.4. Carrageenan-Induced Inflammation in Air Pouches
3.5. Effect of P03 on Selected Parameters in Dextran Sulfate-Induced Colitis
3.6. In Silico Activity and Target Prediction Analysis
3.6.1. ADME-T Predictions and Beyond
3.6.2. Toxicity Potential Assessment
3.6.3. Prediction of Peptide Activity
3.6.4. Bioactivity Score Prediction
3.6.5. PASS Analysis
3.6.6. CLC-Pred: Cell-Line Cytotoxicity Prediction
3.6.7. Target Protein Identification
3.7. NMR Studies
3.8. Results of Quantum-Chemical Calculations
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Peptide | Sequence | |
---|---|---|
P00 (4B8M) | cyclo[Pro-Pro-β3-HoPhe-Phe-] | |
P01 | cyclo[Pro-Pro-β3-HoPhe-D-Phe-] | |
P02 | cyclo- [D-Pro-D-Pro-β3-HoPhe-D-Phe-] | |
P03 | cyclo-[D-Pro-Pro-β3-HoPhe-Phe-] | |
P04 | cyclo-[Pro-Pro-D-β3-HoPhe-Phe-] | |
P05 | cyclo-[Pro-D-Pro-β3-HoPhe-Phe-] | |
P06 | cyclo-[D-Pro-D-Pro-D-β3-HoPhe-D-Phe-] | |
P07 | cyclo-[D-Pro-D-Pro-β3-HoPhe-Phe-] |
Compound Concentration (µg/mL) | TNF-α (pg/mL) | % of Inhibition vs. DMSO Control | Compound Concentration (µg/mL) |
---|---|---|---|
(-) | - | 92 | - |
(LPS) | - | 6668 | - |
DMSO | 1 | 8167 | - |
10 | 8006 | - | |
P00 (4B8M) | 1 | 7364 | 9.8 |
10 | 7176 | 10.4 | |
P01 | 1 | 7016 | 14.1 |
10 | 5757 | 28.1 | |
P02 | 1 | 6614 | 19.0 |
10 | 6346 | 20.7 | |
P03 | 1 | 6052 | 25.9 |
10 | 5222 | 34.8 | |
P04 | 1 | 5463 | 33.1 |
10 | 5302 | 33.8 | |
P05 | 1 | 7123 | 12.8 |
10 | 7203 | 10.0 | |
P06 | 1 | 8033 | 1.6 |
10 | 7658 | 4.4 | |
P07 | 1 | 6453 | 21.0 |
10 | 7284 | 9.0 |
Name | Structure | Suppressive Effect on Cell Proliferation | Suppressive Effect on TNF-α Production |
---|---|---|---|
P01 | cyclo(Pro-Pro-β3-HoPhe-D-Phe-) | NS | marginal |
P02 | cyclo(D-Pro-D-Pro-β3-HoPhe-D-Phe-) | NS | small |
P03 | cyclo(D-Pro-Pro-β3-HoPhe-Phe-) | significant at 100 µg/mL | strong |
P04 | cyclo(Pro-Pro-D-β3-HoPhe-Phe-) | NS | strong |
P05 | cyclo(Pro-D-Pro-β3-HoPhe-Phe-) | NS | moderate |
P06 | cyclo(D-Pro-D-Pro-D-β3-HoPhe-D-Phe-) | NS | absent |
P07 | cyclo(D-Pro-D-Pro-β3-HoPhe-Phe-) | NS | small |
P00 | P01 | P02 | P03 | P04 | P05 | P06 | P07 | |
---|---|---|---|---|---|---|---|---|
PHYSICOCHEMICAL Physicochemical | ||||||||
Molecular weight [Daltons] | 502.62 | 502.62 | 502.62 | 502.62 | 502.62 | 502.62 | 502.62 | 502.62 |
Log P | 1.83 | 1.83 | 1.83 | 1.83 | 1.83 | 1.83 | 1.83 | 1.83 |
H-bond acceptors | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
H-bond donors | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
Lipinski rule of 5 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 |
Quantitative estimate of drug-likeness | 0.67 | 0.67 | 0.67 | 0.67 | 0.67 | 0.67 | 0.67 | 0.67 |
Stereo centers | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 |
Topological polar surface area (TPSA) [Å2] | 98.82 | 98.82 | 98.82 | 98.82 | 98.82 | 98.82 | 98.82 | 98.82 |
Absorption | ||||||||
Human intestinal absorption | 0.58 | 0.62 | 0.62 | 0.52 | 0.54 | 0.59 | 0.61 | 0.56 |
Oral bioavailability | 0.64 | 0.63 | 0.63 | 0.66 | 0.65 | 0.62 | 0.63 | 0.64 |
Aqueous solubility [log mol/L] | −3.80 | −3.89 | −3.91 | −3.79 | −3.75 | −3.84 | −3.87 | −3.82 |
lipophilicity | 1.88 | 1.97 | 1.97 | 1.85 | 1.83 | 1.90 | 1.93 | 1.88 |
Hydration free energy [kcal/mol] | −10.89 | −10.82 | −10.80 | −10.91 | −10.92 | −10.85 | −10.82 | −10.87 |
Cell effective permeability [cm/s] | −5.41 | −5.39 | −5.39 | −5.41 | −5.41 | −5.41 | −5.40 | −5.41 |
PAMPA permeability | 0.50 | 0.52 | 0.52 | 0.50 | 0.51 | 0.51 | 0.52 | 0.50 |
P-glycoprotein inhibition | 0.39 | 0.41 | 0.40 | 0.38 | 0.38 | 0.39 | 0.40 | 0.38 |
Distribution | ||||||||
Blood–brain barrier penetration | 0.16 | 0.16 | 0.16 | 0.16 | 0.15 | 0.15 | 0.16 | 0.16 |
Plasma protein binding rate [%] | 82.16 | 83.23 | 83.34 | 81.84 | 81.55 | 82.57 | 82.77 | 82.28 |
Volume of distribution at steady state [L/kg] | −4.36 | −4.25 | −4.26 | −4.40 | −4.39 | −4.32 | −4.28 | −4.35 |
Metabolism | ||||||||
CYP1A2 inhibition | 1.93 × 103 | 2.31 × 103 | 2.30 × 103 | 1.69 × 103 | 1.90 × 103 | 2.17 × 103 | 2.26 × 103 | 1.92 × 103 |
CYP2C19 inhibition | 0.06 | 0.08 | 0.08 | 0.06 | 0.06 | 0.07 | 0.07 | 0.06 |
CYP2C9 substrate | 0.14 | 0.14 | 0.14 | 0.14 | 0.13 | 0.14 | 0.14 | 0.14 |
CYP2C9 inhibition | 0.03 | 0.04 | 0.03 | 0.02 | 0.02 | 0.03 | 0.03 | 0.03 |
CYP2D6 substrate | 0.08 | 0.08 | 0.07 | 0.08 | 0.08 | 0.07 | 0.08 | 0.08 |
CYP2D6 inhibition | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
CYP3A4 substrate | 0.77 | 0.78 | 0.78 | 0.77 | 0.78 | 0.77 | 0.79 | 0.77 |
CYP3A4 inhibition | 0.37 | 0.43 | 0.42 | 0.33 | 0.36 | 0.40 | 0.42 | 0.36 |
Excretion | ||||||||
Half-life [hr] | 74.20 | 74.69 | 74.44 | 73.33 | 73.75 | 74.75 | 73.95 | 73.91 |
Drug clearance (hepatocyte) [μ L·min−1 (106 cells)−1 | 29.25 | 30.80 | 31.02 | 29.01 | 28.38 | 29.64 | 30.18 | 29.53 |
Drug clearance (microsome) [mL·min−1·g−1] | 51.72 | 54.14 | 54.42 | 51.25 | 50.71 | 52.47 | 53.48 | 52.09 |
Toxicity | ||||||||
hERG blocking | 0.29 | 0.31 | 0.31 | 0.28 | 0.29 | 0.30 | 0.32 | 0.29 |
Clinical toxicity | 0.14 | 0.14 | 0.14 | 0.15 | 0.15 | 0.14 | 0.15 | 0.14 |
Mutagenicity | 0.14 | 0.15 | 0.15 | 0.14 | 0.15 | 0.15 | 0.16 | 0.14 |
Drug-induced liver injury | 0.65 | 0.67 | 0.67 | 0.64 | 0.64 | 0.66 | 0.66 | 0.65 |
Carcinogenicity | 0.19 | 0.19 | 0.19 | 0.20 | 0.18 | 0.18 | 0.19 | 0.19 |
Acute toxicity LD50 [log(1/mol/kg)] | 3.86 | 3.87 | 3.87 | 3.85 | 3.87 | 3.87 | 3.87 | 3.85 |
Skin reaction | 0.210.12 | 0.21 | 0.21 | 0.20 | 0.21 | 0.21 | 0.22 | 0.21 |
Androgen receptor (full length) | 0.03 | 0.12 | 0.12 | 0.11 | 0.12 | 0.12 | 0.12 | 0.12 |
Androgen receptor (ligand-binding domain) | 0.01 | 0.03 | 0.04 | 0.03 | 0.04 | 0.04 | 0.04 | 0.03 |
Aryl hydrocarbon receptor | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Aromatase | 0.15 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 | 0.01 |
Estrogen receptor (full length) | 0.03 | 0.15 | 0.15 | 0.15 | 0.15 | 0.16 | 0.16 | 0.15 |
Estrogen receptor (ligand-binding domain) | 0.03 | 0.03 | 0.03 | 0.02 | 0.03 | 0.03 | 0.03 | 0.03 |
Peroxisome proliferator-activated receptor gamma | 0.03 | 0.04 | 0.04 | 0.03 | 0.03 | 0.04 | 0.04 | 0.03 |
Nuclear factor (erythroid-derived 2)-like 2/antioxidant responsive element | 0.25 | 0.28 | 0.28 | 0.23 | 0.26 | 0.27 | 0.28 | 0.25 |
ATPase family AAA domain-containing protein 5 (ATAD5) | 0.02 | 0.03 | 0.03 | 0.02 | 0.02 | 0.03 | 0.03 | 0.02 |
Heat shock factor response element | 0.03 | 0.04 | 0.04 | 0.03 | 0.03 | 0.04 | 0.04 | 0.03 |
Mitochondrial membrane potential | 0.04 | 0.05 | 0.04 | 0.03 | 0.04 | 0.04 | 0.04 | 0.04 |
Tumor protein p53 | 0.09 | 0.11 | 0.11 | 0.08 | 0.09 | 0.11 | 0.11 | 0.09 |
GPCR | Ion Channel Modulator | Kinase Inhibitor | Nuclear Receptor Ligand | Protease Inhibitor | Enzyme Inhibitor |
---|---|---|---|---|---|
0.33 | 0.02 | −0.02 | 0.00 | 0.52 | 0.12 |
Pa | Pi | Activity |
---|---|---|
0.715 | 0.037 | Nootropic |
0.710 | 0.03 | Uterine relaxant |
0.702 | 0.026 | Nicotinic alpha2beta2 receptor antagonist |
0.702 | 0.057 | CYP2C12 substrate |
0.684 | 0.006 | Chitinase inhibitor |
0.678 | 0.004 | Antibiotic glycopeptide-like |
0.657 | 0.048 | Nicotinic alpha6beta3beta4alpha5 receptor antagonist |
0.610 | 0.005 | Interleukin 2 agonist |
0.585 | 0.017 | Antineoplastic (non-Hodgin’s lymphoma) |
0.577 | 0.028 | Membrane integrity antagonist |
0.555 | 0.056 | Nicotinic alpha4beta4 receptor agonist |
0.543 | 0.017 | Na+ transporting two-sector ATPase inhibitor |
0.509 | 0.003 | Somatostatin 2 agonist |
Pa | Pi | Cell Line | Description | Tissue/Organ | Type | IAP * |
---|---|---|---|---|---|---|
0.582 | 0.005 | PANC-1 | Pancreatic carcinoma | Pancreas | Carcinoma | 0.933 |
0.568 | 0.004 | RS4-11 | Adult B acute lymphoblastic leukemia | Bone marrow | Leukemia | 0.835 |
0.496 | 0.015 | RPMI-8226 | Multiple myeloma | Hematopoietic and lymphoid tissue | Myeloma | 0.883 |
0.486 | 0.044 | HeLa | Cervical adenocarcinoma | Cervix | Adenocarcinoma | 0.885 |
0.482 | 0.073 | OCI-AML2 | Adult acute myeloid leukemia | Blood | Leukemia | 0.803 |
0.449 | 0.006 | MKN-74 | Gastric tubular adenocarcinoma | Stomach | Adenocarcinoma | 0.856 |
0.426 | 0.032 | SK-MEL-28 | Melanoma | Skin | Melanoma | 0.879 |
0.418 | 0.029 | CCRF-CEM | Childhood T acute lymphoblastic leukemia | Blood | Leukemia | 0.913 |
0.411 | 0.162 | TMD8 | Diffuse large B-cell lymphoma activated B-cell type | Lymphocytes | Lymphoma | 0.811 |
0.406 | 0.027 | CA46 | Burkitts lymphoma | Blood | Lymphoma | 0.810 |
0.403 | 0.212 | A2780cisR | Cisplatin-resistant ovarian carcinoma | ovarium | carcinoma | 0.838 |
Atoms | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Amino acids | Cα | Cβ | Cβ’ | Cγ | Cδ | Cε | Cζ | Hα | Hβ1 Hβ2 | Hβ1’ Hβ2’ | Hγ1 Hγ2 | Hδ1 Hδ2 | Hε1 Hε2 | Hζ1 Hζ2 | NH | N |
1Pro | 63.36 | 30.97 | - | 21.56 | 47.39 | - | - | 4.12 | 1.97 1.77 | - | 1.74 1.58 | 3.60 3.29 | - | - | - | - |
2Pro | 60.64 | 31.38 | - | 20.46 | 47.63 | - | - | 4.06 | 2.06 1.71 | - | 1.70 1.56 | 3.49 3.38 | - | - | - | - |
Phe | 54.23 | 37.97 | - | 139.32 | 129.79 | 128.29 | 129.18 | 4.56 | 3.12 2.70 | - | - | 7.16 | 7.19 | 7.07 | 7.89 | 119.93 |
HoPhe | 54.08 | 38.48 | 41.23 | 138.64 | 129.86 | 128.47 | 128.87 | 4.33 | 3.10 2.74 | 2.32 2.08 | - | 7.13 | 7.23 | 7.11 | 8.02 | 119.07 |
Atoms | ||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Amino acids | Cα | Cβ | Cβ’ | Cγ | Cδ | Cε | Cζ | Hα | Hβ1 Hβ2 | Hβ1’ Hβ2’ | Hγ1 Hγ2 | Hδ1 Hδ2 | Hε1 Hε2 | Hζ1 Hζ2 | NH | N |
Pro | 56.85 | 27.43 | - | 25.23 | 46.33 | - | - | 4.08 | 1.88 1.83 | - | 2.13 1.61 | 3.52 3.14 | - | - | - | - |
D-Pro | 61.97 | 29.57 | - | 24.74 | 47.35 | - | - | 4.65 | 2.13 1.61 | - | 1.82 1.72 | 4.21 3.56 | - | - | - | - |
Phe | 54.72 | 35.67 | - | 137.84 | 130.16 | 128.70 | 127.02 | 4.52 | 2.87 2.75 | - | - | 7.19 | 7.26 | 7.11 | 8.10 | 124.59 |
HoPhe | 50.25 | 39.01 | 38.78 | 138.56 | 129.67 | 128.59 | 126.71 | 3.93 | 2.91 2.89 | 2.32 2.16 | - | 7.10 | 7.28 | 7.20 | 5.88 | 108.76 |
Compound | P00 | P03 |
---|---|---|
G298 [Hartree] | −1645.48329 | −1645.47100 |
ΔG298 [kJ/mol] | 0.00 | 32.27 |
[α]D [deg] | 177.0 | −30.3 |
Dipole moment [Debye] | 7.04 | 12.09 |
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© 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
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Kaczmarek, K.; Artym, J.; Bojarska, J.; Pacholczyk-Sienicka, B.; Waśko, J.; Jelemenska, I.; Wolf, W.M.; Breza, M.; Zimecki, M. The Immunosuppressive Properties of Cyclo-[D-Pro-Pro-β3-HoPhe-Phe-] Tetrapeptide Selected from Stereochemical Variants of Cyclo-[Pro-Pro-β3-HoPhe-Phe-] Peptide. Pharmaceutics 2024, 16, 1106. https://doi.org/10.3390/pharmaceutics16081106
Kaczmarek K, Artym J, Bojarska J, Pacholczyk-Sienicka B, Waśko J, Jelemenska I, Wolf WM, Breza M, Zimecki M. The Immunosuppressive Properties of Cyclo-[D-Pro-Pro-β3-HoPhe-Phe-] Tetrapeptide Selected from Stereochemical Variants of Cyclo-[Pro-Pro-β3-HoPhe-Phe-] Peptide. Pharmaceutics. 2024; 16(8):1106. https://doi.org/10.3390/pharmaceutics16081106
Chicago/Turabian StyleKaczmarek, Krzysztof, Jolanta Artym, Joanna Bojarska, Barbara Pacholczyk-Sienicka, Joanna Waśko, Ingrid Jelemenska, Wojciech M. Wolf, Martin Breza, and Michał Zimecki. 2024. "The Immunosuppressive Properties of Cyclo-[D-Pro-Pro-β3-HoPhe-Phe-] Tetrapeptide Selected from Stereochemical Variants of Cyclo-[Pro-Pro-β3-HoPhe-Phe-] Peptide" Pharmaceutics 16, no. 8: 1106. https://doi.org/10.3390/pharmaceutics16081106