ABSTRACT
Human kallikrein 5 and 7 (KLK5 and KLK7) are trypsin-like and chymotrypsin-like serine proteases, respectively, and promising targets for the treatment of skin desquamation, inflammation and cancer. In an effort to develop new inhibitors for these enzymes, we carried out enzymatic inhibition assays and docking studies with three isocoumarin compounds. Some promising inhibitors were uncovered, with vioxanthin and 8,8'-paepalantine being the most potent competitive inhibitors of KLK5 (K(i)=22.9 µM) and KLK7 (K(i)=12.2 µM), respectively. Our docking studies showed a good correlation with the experimental results, and revealed a distinct binding mode for the inhibitors at the binding sites of KLK5 and KLK7. In addition, the docking results suggested that the formation of hydrogen bonds at the oxyanion hole is essential for a good inhibitor.
Subject(s)
Isocoumarins/chemistry , Isocoumarins/pharmacology , Kallikreins/antagonists & inhibitors , Serine Endopeptidases/chemistry , Serine Endopeptidases/pharmacology , Humans , Kallikreins/metabolism , Models, Molecular , Protein BindingABSTRACT
The isocoumarins (1-50 microM) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin isolated from Paepalanthus bromelioides, were assessed for antioxidant activity using isolated rat liver mitochondria and non-mitochondrial systems, and compared with the flavonoid quercetin. The paepalantine and paepalantine dimers, but not vioxanthin, were effective at scavenging both 1,1-diphenyl-2-picrylhydrazyl (DPPH(*)) and superoxide (O(2)(-)) radicals in non-mitochondrial systems, and protected mitochondria from tert-butylhydroperoxide-induced H(2)O(2) accumulation and Fe(2+)-citrate-mediated mitochondrial membrane lipid peroxidation, with almost the same potency as quercetin. These results point towards paepalantine, followed by paepalantine dimer, as being a powerful agent affording protection, apparently via O(2)(-) scavenging, from oxidative stress conditions imposed on mitochondria, the main intracellular source and target of those reactive oxygen species. This strong antioxidant action of paepalantine was reproduced in HepG2 cells exposed to oxidative stress condition induced by H(2)O(2).
Subject(s)
Antioxidants/pharmacology , Eriocaulaceae/chemistry , Isocoumarins/pharmacology , Mitochondria, Liver/drug effects , Animals , Antioxidants/chemistry , Antioxidants/isolation & purification , Cell Line, Tumor , Dimerization , Dose-Response Relationship, Drug , Humans , Hydrogen Peroxide/metabolism , Isocoumarins/chemistry , Isocoumarins/isolation & purification , Lipid Peroxidation/drug effects , Mitochondria, Liver/metabolism , Molecular Structure , Naphthols/chemistry , Naphthols/isolation & purification , Naphthols/pharmacology , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Rats , Reactive Oxygen Species/metabolismABSTRACT
Isolated mitochondria may undergo uncoupling, and in presence of Ca(2+) at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 microM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 microM Ca(2+); inhibition of these processes was assessed in non-energized organelles in the presence of 300 microM t-butyl hydroperoxide plus 500 microM Ca(2+). Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca(2+). Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.
Subject(s)
Eriocaulaceae/chemistry , Isocoumarins/isolation & purification , Isocoumarins/pharmacology , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Mitochondrial Membranes/drug effects , Animals , Biological Transport , Cytochromes c/metabolism , Dimerization , Isocoumarins/chemistry , Male , Mitochondrial Swelling/drug effects , Molecular Structure , Oxidation-Reduction , Rats , Rats, Wistar , Sulfhydryl Compounds/metabolismABSTRACT
The microdilution technique, using a microplate photometer, to determine the minimal inhibitory concentration (MIC) for a natural product was compared to the serial tube dilution method. The MIC obtained for Paepalantine against S. aureus was the same by the two methods, showing an antimicrobial effect similar to chloramphenicol.
Subject(s)
Ampicillin , In Vitro Techniques , Drug Resistance, Microbial , Enzyme-Linked Immunosorbent Assay , MethodsABSTRACT
Numerous natural compounds have a potential for therapeutic applications, but may have to be chemically modified to alter toxic side effects. We investigated structural parameters that could affect the cytotoxicity of isocoumarins similar to 9,10-dihydroxy-5,7-dimethoxy-1H-naphtho(2,3c)pyran-1-one (paepalantine 1). Paepalantine 1 has antimicrobial activity, as well as significant in vitro cytotoxic effects in the McCoy cell line. Two other natural and two semi-synthetic isocoumarins with similar structures obtained from the capitula of Paepalanthus bromelioides were tested on the same cell line by the neutral red assay. Substitution of the 9 and/or 10-OH group made these compounds less cytotoxic.
