ABSTRACT
Prostate cancer (PCa) therapy typically involves administration of "classical" antiandrogens, competitive inhibitors of androgen receptor (AR) ligands, dihydrotestosterone (DHT) and testosterone (tes), for the ligand-binding pocket (LBP) in the ligand-binding domain (LBD) of AR. Prolonged LBP-targeting leads to resistance, and alternative therapies are urgently required. We report the identification and characterization of a novel series of diarylhydrazides as selective disruptors of AR interaction with coactivators through application of structure and ligand-based virtual screening. Compounds demonstrate full ("true") antagonism in AR with low micromolar potency, selectivity over estrogen receptors α and ß and glucocorticoid receptor, and partial antagonism of the progesterone receptor. MDG506 (5) demonstrates low cellular toxicity in PCa models and dose responsive reduction of classical antiandrogen-induced prostate specific antigen expression. These data provide compelling evidence for such non-LBP intervention as an alternative approach or in combination with classical PCa therapy.
Subject(s)
Androgen Antagonists/chemical synthesis , Antineoplastic Agents/chemical synthesis , Hydrazines/chemical synthesis , Nuclear Receptor Coactivators/metabolism , Prostatic Neoplasms/drug therapy , Receptors, Androgen/metabolism , Androgen Antagonists/chemistry , Androgen Antagonists/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Databases, Factual , Drug Screening Assays, Antitumor , Estrogen Receptor alpha/metabolism , Estrogen Receptor beta/metabolism , Fluorescence Polarization , Fluorescence Resonance Energy Transfer , Humans , Hydrazines/chemistry , Hydrazines/pharmacology , Male , Models, Molecular , Prostate-Specific Antigen/metabolism , Receptors, Glucocorticoid/metabolism , Receptors, Progesterone/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
In this work, we describe the first application of ligand-based drug design (LBDD) to the derivation of a predictive pharmacophore for the human glucocorticoid receptor (hGR). Creation of a four feature pharmacophore in Catalyst was subsequently validated through a virtual screen of 264000 commercially available compounds. From a selected hit list of 11 diverse compounds, two nonsteroidal molecules demonstrated low micromolar activity against hGR as validated through fluorescence polarization competitive assay. Additionally, these compounds were tested for their trans-repression potential by their ability to inhibit IL-1 induced, IL-6 expression in the human A549 lung epithelial cell line. Co-treatment of A549 with 21 (MDG169) (10 microM) in combination with dexamethasone showed an improved inhibitory effect when compared to dexamethasone alone with the cooperative effect being dependent on the dexamethasone dose. Putative binding orientations in the hGR ligand binding domain crystal structure are presented. These compounds represent novel nonsteroidal hGR modulating scaffolds, rationally identified through ligand-focused computational modeling.
Subject(s)
Databases, Factual , Dibenzazepines/chemistry , Drug Design , Heterocyclic Compounds, 3-Ring/chemistry , Models, Molecular , Quantitative Structure-Activity Relationship , Receptors, Glucocorticoid/chemistry , Binding Sites , Binding, Competitive , Cell Line , Computer Simulation , Dexamethasone/pharmacology , Dibenzazepines/pharmacology , Drug Synergism , Fluorescence Polarization , Heterocyclic Compounds, 3-Ring/pharmacology , Humans , Interleukin-1/pharmacology , Interleukin-6/biosynthesis , Ligands , Molecular Conformation , Receptors, Glucocorticoid/antagonists & inhibitors , Receptors, Glucocorticoid/metabolismABSTRACT
The small GTPase Rab11 regulates the recycling of endosomes to the plasma membrane via interactions with the Rab11 family of interacting proteins (FIPs). FIPs contain a highly conserved Rab binding domain (RBD) at their C termini whose structure is unknown. Here, we have determined the crystal structure of the RBD of FIP2 in complex with Rab11(GTP) by single wavelength anomalous diffraction methods. The overall structure is a heterotetramer with dyad symmetry, arranged as a Rab11-(FIP2)2-Rab11 complex. FIP2 forms a central alpha-helical coiled coil, with both helices contributing to the Rab11 binding patch on equivalent and opposite sides of the homodimer. Switch 1 of Rab11 is embedded between the two helices, while switch 2 remains flexible and is peripherally associated with the effector. The complex reveals the structural basis for Rab11 recognition by FIPs and suggests the molecular mechanisms underlying endocytic recycling pathways.
Subject(s)
Carrier Proteins/chemistry , Membrane Proteins/chemistry , Models, Molecular , rab GTP-Binding Proteins/chemistry , Amino Acid Sequence , Carrier Proteins/genetics , Carrier Proteins/metabolism , Crystallography , DNA Primers , Dimerization , Membrane Proteins/genetics , Membrane Proteins/metabolism , Microscopy, Fluorescence , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation/genetics , Sequence Alignment , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolismABSTRACT
The small GTPase Rab11 regulates the recycling of endosomes back to the plasma membrane. In its active GTP-bound form, Rab11 binds a novel set of effectors termed the Rab11 family of interacting proteins (Rab11-FIPs) which contain a conserved C-terminal Rab-binding domain (RBD) of unknown structure. Here, a complex of Rab11 with the RBD of Rab11-FIP2 has been purified and crystallized in the trigonal space group P3(1)21, with unit-cell parameters a = 64.99, b = 64.99, c = 112.59 angstroms. Static light-scattering analyses of the molecular weight of the complex in solution are consistent with two copies of Rab11 and two copies of Rab11-FIP2 in the complex.
