ABSTRACT
Uncialamycin analogs were evaluated as potential cytotoxic agents in an antibody-drug conjugate (ADC) approach to treating human cancer. These analogs were synthesized using Hauser annulations of substituted phthalides as a key step. A highly potent uncialamycin analog 3c with a valine-citrulline dipeptide linker was conjugated to an anti-mesothelin monoclonal antibody (mAb) through lysines to generate a meso-13 conjugate. This conjugate demonstrated subnanomolar potency (IC50â¯=â¯0.88â¯nM, H226 cell line) in in vitro cytotoxicity experiments with good immunological specificity to mesothelin-positive lung cancer cell lines. The potency and mechanism of action of this uncialamycin class of enediyne antitumor antibiotics make them attractive payloads in ADC-based cancer therapy.
Subject(s)
Anthraquinones/pharmacology , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Immunoconjugates/pharmacology , Lung Neoplasms/drug therapy , Anthraquinones/chemistry , Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Immunoconjugates/chemistry , Lung Neoplasms/pathology , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
BACKGROUND: MDX-1203 is an antibody-drug conjugate (ADC) currently in clinical trials for the treatment of renal carcinoma. The active ingredient of MDX-1203 is a DNA minor groove-binding cytotoxic drug that forms a covalently linked adduct with an adenine base. Formation of this adenine adduct prevents DNA replication, thus triggering cell death. RESULTS: A method has been developed to successfully isolate, identify and quantitate the adenine adduct using LC-MS/MS. The method is highly useful to validate the mode of action of this class of ADCs. Additionally, we have demonstrated that this method could potentially be utilized to assess the efficacy of the ADC in in vitro studies by measuring the amount of adenine adduct in various cells expressing the antigen. CONCLUSION: Upon validation, this method could serve as an invaluable tool to evaluate compounds in preclinical in vivo models and in utilizing the DNA adduct as a potential biomarker.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Biomarkers, Tumor/analysis , Carcinoma, Renal Cell/drug therapy , DNA Adducts/analysis , Immunoconjugates/pharmacology , Indoles/pharmacology , Kidney Neoplasms/drug therapy , Alkylation , Animals , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/immunology , Antineoplastic Agents/chemistry , Antineoplastic Agents/immunology , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/metabolism , Chromatography, Liquid , DNA Adducts/drug effects , DNA Adducts/metabolism , Humans , Immunoconjugates/chemistry , Immunoconjugates/immunology , Indoles/chemistry , Tandem Mass SpectrometryABSTRACT
Twenty analogues of the natural antitumor agent dolastatin 11, including majusculamide C, were synthesized and tested for cytotoxicity against human cancer cells and stimulation of actin polymerization. Only analogues containing the 30-membered ring were active. Molecular modeling and NMR evidence showed the low-energy conformations. The amide bonds are all trans except for the one between the Tyr and Val units, which is cis. Since an analogue restricted to negative 2-3-4-5 angles stimulated actin polymerization but was inactive in cells, the binding conformation (most likely the lowest-energy conformation in water) has a negative 2-3-4-5 angle, whereas a conformation with a positive 2-3-4-5 angle (most likely the lowest energy conformation in chloroform) goes through cell walls. The highly active R alcohol from borohydride reduction of dolastatin 11 is a candidate for conversion to prodrugs.
Subject(s)
Actins/metabolism , Cell Survival/drug effects , Depsipeptides , Leukemia P388/drug therapy , Models, Molecular , Molecular Conformation , Animals , Depsipeptides/chemical synthesis , Depsipeptides/chemistry , Depsipeptides/pharmacology , Drug Screening Assays, Antitumor , Humans , Leukemia P388/pathology , Mice , Molecular Structure , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
Dolastatin 11, a drug isolated from the Indian Ocean sea hare Dolabella auricularia, arrests cytokinesis in vivo and increases the amount of F-actin to stabilize F-actin in vitro, like phalloidin and jasplakinolide. However, according to the previous biochemical study, the binding of dolastatin 11 to F-actin does not compete with that of phalloidin, suggesting that the binding sites are different. To understand the mechanism of F-actin stabilization by dolastatin 11, we determined the position of bound dolastatin 11 in F-actin using the X-ray fiber diffraction from oriented filament sols. Our analysis shows that the position of dolastatin 11 is clearly different from that of phalloidin. However, these bound drugs are present in the gap between the two long-pitch F-actin strands in a similar way. The result suggests that the connection between the two long-pitch F-actin strands might be a key for the control of F-actin stabilization.
Subject(s)
Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Actins/drug effects , Actins/metabolism , Depsipeptides , Oligopeptides/metabolism , Oligopeptides/pharmacology , Peptides, Cyclic/metabolism , Peptides, Cyclic/pharmacology , Actins/chemistry , Animals , Binding Sites , Drug Stability , In Vitro Techniques , Models, Molecular , Oligopeptides/chemistry , Peptides, Cyclic/chemistry , Phalloidine/metabolism , Phalloidine/pharmacology , Protein Binding , Protein Conformation , Rabbits , Static Electricity , X-Ray DiffractionABSTRACT
The synthesis of a lyngbyastatin 1-Ibu-epilyngbyastatin 1 mixture combined with NMR and molecular modeling studies proved that natural lyngbyastatin 1 was only one Ibu epimer rather than a mixture of both and that the configuration of this epimer in the Ibu unit was R. The substance isolated with lyngbyastatin 1 was Ibu-epidolastatin 12. The extreme broadness in the proton NMR spectra of lyngbyastatin 1 and Ibu-epidolastatin 12 was exchange broadening due to rotation about the Ibu-Ala amide bond. It was a consequence of (1) a small energy difference between the cis and trans forms of this bond, (2) a substantial difference in conformation between these forms, and (3) a lowered barrier between them compared to most amide bonds (due to steric hindrance). The synthetic lyngbyastatin 1-Ibu-epilyngbyastatin 1 mixture had significant activities against cancer cells and in stimulating actin polymerization, but was less active than dolastatin 11 in all assays.