Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives
Abstract
:1. Introduction
2. Results and Discussion
2.1. Screening of Quinoxaline 1,4-di-N-Oxide Derivatives as FhCL1 and FhCL3 Inhibitors
2.2. Screening of Cathepsin L Inhibitors against NEJ
2.3. Analysis of Hit Compounds
2.4. Cathepsin Inhibitors Are Slow-Binding and Interact at the Active Site Cleft
2.5. Selectivity Assessment against Human Cathepsin L
2.6. Cytotoxicity Evaluation in Bovine Sperm and a Human Cell Line
3. Conclusions
4. Materials and Methods
4.1. Production of Recombinant FhCL1 and FhCL3
4.2. FhCL1 and FhCL3 Inhibition Screening
4.3. In Vitro Culture of NEJ and Treatment with the Best Inhibitors
4.4. Characterization of the Best Inhibitors
4.4.1. IC50, Time-Dependence of Inhibition, and HsCL Testing
4.4.2. Ligand-Protein Molecular Docking to Predict the Binding Site into FhCL1 and FhCL3
Preparation of Protein Structures
Preparation of Ligand Structures
Ligand-Protein Molecular Docking
4.4.3. Ligand-Protein Molecular Dynamics MD Simulations
4.5. Cytotoxicity Assay on the HepG2 Cell Line and Bovine Spermatozoa
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are not available from the authors. |
IC50 (µM) | C7 | C17 | C18 | C19 | C23 | C24 |
---|---|---|---|---|---|---|
FhCL1 | 3.5 | 1.7 | 9.0 | 5.0 | 3.0 | 1.4 |
FhCL3 | 5.2 | 4.9 | 8.0 | >50 | 11 | 0.9 |
Comp. (10 μM) | HsCL inhib.% | FhCL1 inhib.% | FhCL3 inhib.% |
---|---|---|---|
C7 | 41 | 90 | 61 |
C17 | 37 | 98 | 86 |
C18 | 25 | 55 | 61 |
C19 | 0 | 54 | 35 |
C23 | 57 | 97 | 79 |
C24 | ND | 93 | 81 |
Quinoxalines General Structures | ||||||
---|---|---|---|---|---|---|
#Comp. | R1 | R2 | R3 | R4 | %Inh FhCL1 | %Inh FhCL3 |
C1 | -CH3 | -H | -H | -H | 1 | 8 |
C2 | -CHO | -H | -H | -H | 21 | 0 |
C3 | -CN | -NH2 | -H | -H | 30 | 0 |
C4 | -CN | -NH2 | -H | 0 | 27 | |
C5 | -CN | -NH2 | -H | 20 | 39 | |
C6 | -CN | -NH2 | -H | 59 | 27 | |
C7 | -CN | -NH2 | -H | 90 | 61 | |
C8 | -CN | -NH2 | -H | 24 | 45 | |
C9 | -CN | -NH2 | -Cl | -Cl | 15 | 0 |
C10 | -CH3 | -CH3 | -H | 13 | 3 | |
C11 | -CH3 | -CH3 | -H | 56 | 32 | |
C12 | -CH3 | -CH3 | -H | 0 | 22 | |
C13 | -CH3 | -CH3 | -H | 34 | 2 | |
C14 | -CH3 | -H | -H | 28 | 5 | |
C15 | -CH3 | -H | 38 | 58 | ||
C16 | -CH3 | -Cl | -Cl | 35 | 22 | |
C17 | -Ph | -H | 98 | 86 | ||
C18 | -Ph | -H | 55 | 61 | ||
C19 | -Ph | -Cl | -Cl | 54 | 35 | |
C20 | -CH3 | -H | 72 | 0 | ||
C21 | -CH3 | -H | 47 | 54 | ||
C22 | -CH3 | -H | 23 | 49 | ||
C23 | -Ph | -H | 97 | 79 | ||
C24 | -Ph | -H | 93 | 81 | ||
C25 | -CH3 | -H | -H | 47 | 40 | |
C26 | -CH3 | -F | -F | 41 | 17 | |
C27 | -CF3 | -H | -H | 38 | 22 | |
C28 | -CF3 | -F | -F | 0 | 38 |
Enzyme | S3 | S2/S3 | S2 | |||||||
---|---|---|---|---|---|---|---|---|---|---|
Papain# | 61 | 64 | 66 | 67 | 137 | 142 | 143 | 157 | 160 | 205 |
FhCL1 | Asn64 | Gly67 | Gly69 | Leu70 | Val139 | Met144 | Met145 | Val161 | Ala164 | Leu210 |
FhCL3 | His64 | Gly67 | Gly69 | Trp70 | Ala139 | Tyr144 | Met145 | Thr161 | Ala164 | Val210 |
HsCL | Glu63 | Asn66 | Gly68 | Leu69 | Gly139 | Leu144 | Phe145 | Met161 | Gly164 | Ala214 |
Comp# | HepG2 IC50 (µM) | Sperm Motility RTC * (50 µM) |
---|---|---|
C17 | 48 ± 0.3 | 81 ± 3 |
C18 | <6.2 | 82 ± 2 |
C23 | 12 ± 0.2 | 82 ± 1 |
C24 | 16 ± 0.1 | 82 ± 1 |
TCBZ | 32 ± 0.2 | 88 ± 1 |
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Ferraro, F.; Merlino, A.; Gil, J.; Cerecetto, H.; Corvo, I.; Cabrera, M. Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives. Molecules 2019, 24, 2348. https://doi.org/10.3390/molecules24132348
Ferraro F, Merlino A, Gil J, Cerecetto H, Corvo I, Cabrera M. Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives. Molecules. 2019; 24(13):2348. https://doi.org/10.3390/molecules24132348
Chicago/Turabian StyleFerraro, Florencia, Alicia Merlino, Jorge Gil, Hugo Cerecetto, Ileana Corvo, and Mauricio Cabrera. 2019. "Cathepsin L Inhibitors with Activity against the Liver Fluke Identified From a Focus Library of Quinoxaline 1,4-di-N-Oxide Derivatives" Molecules 24, no. 13: 2348. https://doi.org/10.3390/molecules24132348