ABSTRACT
Diverse pathways drive resistance to BRAF/MEK inhibitors in BRAF-mutant melanoma, suggesting that durable control of resistance will be a challenge. By combining statistical modeling of genomic data from matched pre-treatment and post-relapse patient tumors with functional interrogation of >20 in vitro and in vivo resistance models, we discovered that major pathways of resistance converge to activate the transcription factor, c-MYC (MYC). MYC expression and pathway gene signatures were suppressed following drug treatment, and then rebounded during progression. Critically, MYC activation was necessary and sufficient for resistance, and suppression of MYC activity using genetic approaches or BET bromodomain inhibition was sufficient to resensitize cells and delay BRAFi resistance. Finally, MYC-driven, BRAFi-resistant cells are hypersensitive to the inhibition of MYC synthetic lethal partners, including SRC family and c-KIT tyrosine kinases, as well as glucose, glutamine, and serine metabolic pathways. These insights enable the design of combination therapies that select against resistance evolution.
Subject(s)
Melanoma/drug therapy , Proto-Oncogene Proteins c-myc/metabolism , Antineoplastic Agents, Hormonal/therapeutic use , Benzimidazoles/pharmacology , Cell Line, Tumor , Estradiol/analogs & derivatives , Estradiol/therapeutic use , Evolution, Molecular , Female , Fulvestrant , Humans , Imidazoles/pharmacology , Imidazoles/therapeutic use , Indoles/pharmacology , Male , Oximes/therapeutic use , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Proto-Oncogene Proteins B-raf/metabolism , Pyridones/therapeutic use , Pyrimidinones/therapeutic use , Quinolines/pharmacology , Signal Transduction/drug effects , Sulfonamides/pharmacologyABSTRACT
Shape is an inherent trait of a molecule that dictates how it interacts with other molecules, either in binding events or intermolecular reactions. Large-ring macrocyclic compounds in particular leverage their shape when they are selectively bound by biomolecules and also when they exhibit macrocyclic diastereoselectivity. Nonetheless, rules that link structural parameters to the conformation of a macrocycle are still rudimentary. Here we use a structural investigation of a family of [13]-macrodilactones as a case study to develop rules that can be applied generally to macrocycles of different sizes and with a variety of functionality. A characteristic "ribbon" shape is adopted by the [13]-macrodilactones in the absence of stereogenic centres, which exhibits planar chirality. When one stereogenic centre at key positions on the backbone is incorporated into the structure, the planar chirality is dictated by the configuration of the centre. In cases where two stereogenic centres are present, their relationships can either reinforce the characteristic ribbon shape or induce alternative shapes to be adopted. The rules established in the case study are then applied to the analysis of a structure of the natural product migrastatin. They lay the groundwork for the development of models to understand macrocycle-biomolecule interactions and for the preparation of macrocycles with designed properties and activities.
ABSTRACT
The synthesis and characterization of new [13]-macrodilactones substituted at stereogenic centers α- to the carbonyl are reported. When one center is substituted, it directs the topology of the macrocycle; when two centers are substituted, both the shape and the topology are influenced. The findings indicate that the number and configuration of α-centers fine-tune macrocyclic structure.
ABSTRACT
The cardiac glycosides ouabain and digitoxin, established Na(+)/K(+) ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na(+)/K(+) ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure-activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na(+)/K(+) ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein-small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na(+)/K(+) transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1-M6 correlates with the Na(+) pump activity and cell migration. Other Na(+)/K(+) ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na(+)/K(+) ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.
Subject(s)
Breast Neoplasms/metabolism , Cardiac Glycosides/pharmacology , Ouabain/analogs & derivatives , Ouabain/pharmacology , Sodium-Potassium-Exchanging ATPase/metabolism , Cardiac Glycosides/chemistry , Cell Line, Tumor , Cell Movement/drug effects , Female , Humans , Ion Transport , Ouabain/chemistry , Structure-Activity RelationshipABSTRACT
Natural product-like macrocycles were designed as potential antibacterial compounds. The macrocycles featured a D-glucose unit fused into a 12- or 13-member macrolactone. The rings are connected via the C6' and anomeric (C1') positions of the monosaccharide. The new macrocycles/macrolides were characterized by X-ray crystallography. Their structures showed that, in addition to the ester and alkene units, the dihedral angle about the glycosidic linkage (exo-anomeric effect) influenced the overall shape of the molecules. Glycosylation of an available hydroxy group on the macrocycle gave a hybrid macrolide with features common to erythromycin and sophorlipid macrolactone. Weak antibiotic activity (MICs <100 µg/mL) was observed for several of the compounds.
ABSTRACT
Accumulating evidence suggests that Raf kinase inhibitor protein (RKIP), which negatively regulates multiple signaling cascades including the Raf and nuclear factor-κB (NF-κB) pathways, functions as a metastasis suppressor. However, the basis for this activity is not clear. We investigated this question in a panel of breast cancer, colon cancer and melanoma cell lines. We found that RKIP negatively regulated the invasion of the different cancer cells through three-dimensional extracellular matrix barriers by controlling the expression of matrix metalloproteinases (MMPs), particularly, MMP-1 and MMP-2. Silencing of RKIP expression resulted in a highly invasive phenotype and dramatically increased levels of MMP-1 and MMP-2 expression, while overexpression of RKIP decreased cancer cell invasion in vitro and metastasis in vivo of murine tumor allografts. Knockdown of MMP-1 or MMP-2 in RKIP-knockdown cells reverted their invasiveness to normal. In contrast, when examining migration of the different cancer cells in a two-dimensional, barrier-less environment, we found that RKIP had either a positive regulatory activity or no activity, but in no case a negative one (as would be expected if RKIP suppressed metastasis at the level of cell migration itself). Therefore, RKIP's function as a metastasis suppressor appears to arise from its ability to negatively regulate expression of specific MMPs, and thus invasion through barriers, and not from a direct effect on the raw capacity of cells to move. The NF-κB pathway, but not the Raf pathway, appeared to positively control the invasion of breast cancer cells. A regulatory loop involving an opposing relationship between RKIP and the NF-κB pathway may control the level of MMP expression and cell invasion.
Subject(s)
Matrix Metalloproteinases/metabolism , NF-kappa B/metabolism , Neoplasms/metabolism , Phosphatidylethanolamine Binding Protein/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Cell Movement , Cell Proliferation , Female , Gene Expression Regulation, Neoplastic , Humans , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Matrix Metalloproteinase 1/genetics , Matrix Metalloproteinase 1/metabolism , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinases/genetics , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness , Neoplasms/genetics , Neoplasms/pathology , Phosphatidylethanolamine Binding Protein/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Signal TransductionABSTRACT
The biological activities of a family of novel, lipid-linked 13-membered-ring macro-dilactones are reported. These [13]-macro-dilactones were synthesized by diacylation of functionalized diols, followed by ring-closing metathesis under conditions we had previously reported. Antimigratory, cytostatic and cytotoxic activities of the compounds against cancer cells were evaluated. Compound 13 was the most potent in the series, while compound 10 had the broadest concentration range of subtoxic antiproliferative activity. These compounds share common structural components, namely the [13]-macro-dilactone templated by an octyl alpha-glucoside 4,6-diol.