ABSTRACT
BACKGROUND: 17-Allylamino-17-demethoxygeldanamycin (17-AAG) inhibits heat shock protein 90, promotes degradation of oncoproteins, and exhibits synergy with paclitaxel in vitro. We conducted a phase I study in patients with advanced malignancies to determine the recommended phase II dose of the combination of 17-AAG and paclitaxel. METHODS: Patients with advanced solid malignancies that were refractory to proven therapy or without any standard treatment were included. 17-AAG (80-225 mg/m2) was given on days 1, 4, 8, 11, 15, and 18 of each 4-week cycle to sequential cohorts of patients. Paclitaxel (80-100 mg/m2) was administered on days 1, 8, and 15. Pharmacokinetic studies were conducted during cycle 1. RESULTS: Twenty-five patients were accrued to five dose levels. The median number of cycles was 2. Chest pain (grade 3), myalgia (grade 3), and fatigue (grade 3) were dose-limiting toxicities at dose level 4 (225 mg/m2 17-AAG and 80 mg/m2 paclitaxel). None of the six patients treated at dose level 3 with 17-AAG (175 mg/m2) and paclitaxel (80 mg/m2) experienced dose-limiting toxicity. Disease stabilization was noted in six patients, but there were no partial or complete responses. The ratio of paclitaxel area under the concentration to time curve when given alone versus in combination with 17-AAG was 0.97 +/- 0.20. The ratio of end-of-infusion concentration of 17-AAG (alone versus in combination with paclitaxel) was 1.14 +/- 0.51. CONCLUSIONS: The recommended phase II dose of twice-weekly 17-AAG (175 mg/m2) and weekly paclitaxel (80 mg/m2/wk) was tolerated well. There was no evidence of drug-drug pharmacokinetic interactions.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Antineoplastic Combined Chemotherapy Protocols/pharmacokinetics , Neoplasms/drug therapy , Adult , Aged , Area Under Curve , Benzoquinones/administration & dosage , Benzoquinones/adverse effects , Benzoquinones/pharmacokinetics , Drug Administration Schedule , Drug Interactions , Female , Humans , Lactams, Macrocyclic/administration & dosage , Lactams, Macrocyclic/adverse effects , Lactams, Macrocyclic/pharmacokinetics , Male , Maximum Tolerated Dose , Middle Aged , Paclitaxel/administration & dosage , Paclitaxel/adverse effects , Paclitaxel/pharmacokineticsABSTRACT
Mitogen-activated protein kinase phosphatase (MKP)-1 is a dual-specificity phosphatase that negatively regulates the activity of mitogen-activated kinases and that is overexpressed in human tumors. Contemporary studies suggest that induction of MKP-1 during chemotherapy may limit the efficacy of clinically used antineoplastic agents. Thus, MKP-1 is a rational target to enhance anticancer drug activity, but suitable small-molecule inhibitors of MKP-1 are currently unavailable. Here, we have used a high-content, multiparameter fluorescence-based chemical complementation assay for MKP activity in intact mammalian cells to evaluate the cellular MKP-1 and MKP-3 inhibitory activities of four previously described, quinone-based, dual-specificity phosphatase inhibitors, that is, NSC 672121, NSC 95397, DA-3003-1 (NSC 663284), and JUN-1111. All compounds induced formation of reactive oxygen species in mammalian cells, but only one (NSC 95397) inhibited cellular MKP-1 and MKP-3 with an IC(50) of 13 mumol/L. Chemical induction of MKP-1 by dexamethasone protected cells from paclitaxel-induced apoptosis but had no effect on NSC 95397. NSC 95397 phenocopied the effects of MKP-1 small inhibitory RNA by reversing the cytoprotective effects of dexamethasone in paclitaxel-treated cells. Isobologram analysis revealed synergism between paclitaxel and NSC 95397 only in the presence of dexamethasone. The data show the power of a well-defined cellular assay for identifying cell-active inhibitors of MKPs and support the hypothesis that small-molecule inhibitors of MKP-1 may be useful as antineoplastic agents under conditions of high MKP-1 expression.
Subject(s)
Dexamethasone/antagonists & inhibitors , Dexamethasone/pharmacology , Drug Resistance, Neoplasm/drug effects , Mitogen-Activated Protein Kinase Phosphatases/antagonists & inhibitors , Naphthoquinones/pharmacology , Paclitaxel/pharmacology , Antineoplastic Combined Chemotherapy Protocols , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Survival/drug effects , Cytoprotection/drug effects , Drug Evaluation, Preclinical , Drug Synergism , Dual Specificity Phosphatase 1/antagonists & inhibitors , Dual Specificity Phosphatase 1/metabolism , Dual Specificity Phosphatase 6/antagonists & inhibitors , Dual Specificity Phosphatase 6/metabolism , HeLa Cells , Humans , Models, Biological , Naphthoquinones/administration & dosage , Paclitaxel/administration & dosage , Quinones/pharmacology , Reactive Oxygen Species/metabolism , Tumor Cells, CulturedABSTRACT
Compounds that bind to microtubules (MTs) and alter their dynamics are highly sought as a result of the clinical success of paclitaxel and docetaxel. The naturally occurring compound (-)-dictyostatin binds to MTs, causes cell cycle arrest in G(2)/M at nanomolar concentrations, and retains antiproliferative activity in paclitaxel-resistant cell lines, making dictyostatin an attractive candidate for development as an antineoplastic agent. In this study, we examined a series of dictyostatin analogs to probe biological and biochemical structure-activity relationships. We used a high-content multiparameter fluorescence-based cellular assay for MT morphology, chromatin condensation, mitotic arrest, and cellular toxicity to identify regions of dictyostatin that were essential for biological activity. Four analogs (6-epi-dictyostatin, 7-epi-dictyostatin, 16-normethyldictyostatin, and 15Z,16-normethyldictyostatin) retained low nanomolar activity in the cell-based assay and were chosen for analyses with isolated tubulin. All four compounds were potent inducers of MT assembly. Equilibrium binding constant (K(i)) determinations using [(14)C]epothilone B, which has a 3-fold higher affinity for the taxoid binding site than paclitaxel, indicated that 6-epi-dictyostatin and 7-epi-dictyostatin displaced [(14)C]epothilone B with K(i) values of 480 and 930 nM, respectively. 16-Normethyldictyostatin and 15Z,16-normethyldictyostatin had reduced affinity (K(i) values of 4.55 and 4.47 muM, respectively), consistent with previous reports showing that C16-normethyldictyostatin loses potency in paclitaxel-resistant cell lines that have a Phe270-to-Val mutation in the taxoid binding site of beta-tubulin. Finally, we developed a set of quantitative structure-activity relationship equations correlating structures with antiproliferative activity. The equations accurately predicted biological activity and will help in the design of future analogs.
Subject(s)
Macrolides/chemistry , Macrolides/pharmacology , Microtubules/drug effects , Quantitative Structure-Activity Relationship , Alkanes/metabolism , Alkanes/pharmacology , Alkanes/toxicity , Animals , Benzimidazoles/metabolism , Binding Sites , Brain Chemistry , Carbamates/metabolism , Carbamates/pharmacology , Carbamates/toxicity , Carcinoma/drug therapy , Carcinoma/pathology , Cattle , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Proliferation/drug effects , Epothilones/antagonists & inhibitors , Epothilones/pharmacology , Epothilones/toxicity , Female , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Antibody Technique, Indirect , Fluorescent Dyes/metabolism , G2 Phase/drug effects , HeLa Cells , Histones/metabolism , Humans , Kinetics , Lactones/metabolism , Lactones/pharmacology , Lactones/toxicity , Macrolides/chemical synthesis , Macrolides/metabolism , Molecular Structure , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/pathology , Paclitaxel/metabolism , Paclitaxel/pharmacology , Paclitaxel/toxicity , Phosphorylation/drug effects , Protein Binding , Pyrones/metabolism , Pyrones/pharmacology , Pyrones/toxicity , Radioligand Assay , Tubulin/biosynthesis , Tubulin Modulators/metabolism , Tubulin Modulators/pharmacology , Tubulin Modulators/toxicityABSTRACT
The structure-activity relationship of the crucial C16 region of (-)-dictyostatin was established through total synthesis of analogs followed by detailed biological characterization. A versatile synthetic strategy was used to prepare milligram quantities of 16-normethyldictyostatin, 16-epi-dictyostatin, and the C16-normethyl-C15Z isomer. Along the way, a number of other E/Z isomers and epimers were prepared, and a novel lactone ring contraction to make iso-dictyostatins with 20-membered macrolactones (instead of 22-membered macrolactones) was discovered. The synthesis of 16-normethyl-15,16-dehydrodictyostatin is the first of any dictyostatin by a maximally convergent route in which three main fragments are assembled, coupled in back-to-back steps, and then processed through refunctionalization and macrolactonization. Cell-based and biochemical evaluations showed 16-normethyl-15,16-dehydrodictyostatin and 16-normethyldictyostatin to be the most potent of the new agents, only 2- and 5-fold less active than (-)-dictyostatin itself. This data and that from previously generated dictyostatin analogs are combined to produce a picture of the structure-activity relationships in this series of anticancer agents.
Subject(s)
Antineoplastic Agents/chemical synthesis , Macrolides/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Binding, Competitive , Cattle , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Macrolides/chemistry , Macrolides/pharmacology , Microtubules/chemistry , Stereoisomerism , Structure-Activity Relationship , Tubulin/chemistryABSTRACT
Total syntheses of (-)-dictyostatin, 6,16-bis-epi-dictyostatin, 6,14,19-tris-epi-dictyostatin and a number of other isomers and analogs are reported. Three main fragments-top, middle and bottom-were first assembled and then joined by olefination or anionic addition reactions. After appending the two dienes at either end of the molecule, macrolactonization and deprotection completed the syntheses. The work proves both the relative and absolute configurations of (-)-dictyostatin. The compounds were evaluated by cell-based measurements of increased microtubule mass and antiproliferative activity, and in vitro tubulin polymerization assays as well as competitive assays with paclitaxel for its binding site on microtubules. These assays showed dictyostatin to be the most potent of the agents and further showed that the structural alterations caused from 20- to >1000-fold decreases in activity.
ABSTRACT
Structure-activity analyses of synthetic disorazole C(1) and eight of its analogs indicate that the presence of a vinyl oxirane moiety or a tetraene sequence is not necessary for potent cytotoxic and antimitotic properties. Using an automated multiparameter fluorescence-based cellular assay to simultaneously probe the effects of disorazole analogs on cellular microtubules, mitotic arrest, and cytotoxicity, we found that disorazole C(1) enhanced the mitotic index and chromatin condensation and arrested cells in the G2/M phase of the cell cycle. All structural analogs and synthesis precursors of disorazole C(1) were at least two orders of magnitude less potent than the parent compound, thus indicating that both the functional group array and the three-dimensional conformation of the parent compound are critical for interaction with the biological target. We conclude that disorazole C(1) is a potent inducer of mitotic arrest and hypothesize that this biological activity may be mediated by microtubule perturbation.
Subject(s)
Antimitotic Agents/chemistry , Biological Products/chemistry , Cell Cycle/drug effects , Oxazoles/chemistry , Antimitotic Agents/metabolism , Antimitotic Agents/pharmacology , Biological Products/metabolism , Biological Products/pharmacology , Dose-Response Relationship, Drug , Flow Cytometry , Fluorescent Antibody Technique , HeLa Cells , Humans , Macrolides , Microtubules/drug effects , Mitosis/drug effects , Oxazoles/metabolism , Oxazoles/pharmacology , Oxazoles/toxicity , Structure-Activity RelationshipABSTRACT
Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase that is overexpressed in many human tumors and can protect cells from apoptosis caused by DNA-damaging agents or cellular stress. Small molecule inhibitors of MKP-1 have not been reported, in part because of the lack of structural guidance for inhibitor design and definitive assays for MKP-1 inhibition in intact cells. Herein we have exploited a high content chemical complementation assay to analyze a diverse collection of pure natural products for cellular MKP-1 inhibition. Using two-dimensional Kolmogorov-Smirnov statistics, we identified sanguinarine, a plant alkaloid with known antibiotic and antitumor activity but no primary cellular target, as a potent and selective inhibitor of MKP-1. Sanguinarine inhibited cellular MKP-1 with an IC50 of 10 microM and showed selectivity for MKP-1 over MKP-3. Sanguinarine also inhibited MKP-1 and the MKP-1 like phosphatase, MKP-L, in vitro with IC50 values of 17.3 and 12.5 microM, respectively, and showed 5-10-fold selectivity for MKP-3 and MKP-1 over VH-1-related phosphatase, Cdc25B2, or protein-tyrosine phosphatase 1B. In a human tumor cell line with high MKP-1 levels, sanguinarine caused enhanced ERK and JNK/SAPK phosphorylation. A close congener of sanguinarine, chelerythrine, also inhibited MKP-1 in vitro and in whole cells, and activated ERK and JNK/SAPK. In contrast, sanguinarine analogs lacking the benzophenanthridine scaffold did not inhibit MKP-1 in vitro or in cells nor did they cause ERK or JNK/SAPK phosphorylation. These data illustrate the utility of a chemical complementation assay linked with multiparameter high content cellular screening.