ABSTRACT
Members of the BET family of bromodomain containing proteins have been identified as potential targets for blocking proliferation in a variety of cancer cell lines. A two-dimensional NMR fragment screen for binders to the bromodomains of BRD4 identified a phenylpyridazinone fragment with a weak binding affinity (1, Ki = 160 µM). SAR investigation of fragment 1, aided by X-ray structure-based design, enabled the synthesis of potent pyridone and macrocyclic pyridone inhibitors exhibiting single digit nanomolar potency in both biochemical and cell based assays. Advanced analogs in these series exhibited high oral exposures in rodent PK studies and demonstrated significant tumor growth inhibition efficacy in mouse flank xenograft models.
Subject(s)
Macrocyclic Compounds/chemistry , Macrocyclic Compounds/pharmacology , Pyridones/chemistry , Pyridones/pharmacology , Animals , Crystallography, X-Ray , Drug Discovery , Macrocyclic Compounds/pharmacokinetics , Molecular Structure , Pyridones/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.
Subject(s)
Antineoplastic Agents/chemistry , Nuclear Proteins/antagonists & inhibitors , Pyrroles/chemistry , Transcription Factors/antagonists & inhibitors , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/therapeutic use , Binding Sites , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , Drug Design , Half-Life , Humans , Mice , Molecular Dynamics Simulation , Multiple Myeloma/drug therapy , Nuclear Proteins/metabolism , Pyrroles/chemical synthesis , Pyrroles/pharmacokinetics , Pyrroles/therapeutic use , Structure-Activity Relationship , Transcription Factors/metabolism , Transplantation, HeterologousABSTRACT
Successful siRNA therapeutics requires the optimal integration of multiple components, including an efficient delivery system, a disease indication that is appropriate for siRNA-based therapy, and a potent and nontoxic siRNA against a robust therapeutic target. Although all currently available delivery systems have limitations, it is important to recognize that a careful selection of the disease indication, therapeutic target, and siRNA molecule could partially compensate for deficiencies associated with the delivery system and makes it possible to advance a therapeutic siRNA regimen. In this study, we present the development of siRNA therapeutics for hepatocellular carcinoma using an integrated approach, including the development of an efficient lipid nanoparticle delivery system, the identification of a robust therapeutic target that does not trigger liver toxicity upon target knockdown, and the selection of potent and nonimmunogenic siRNA molecules against the target. The resulting siRNA-containing lipid nanoparticles produced significant antitumor efficacy in orthotopic hepatocellular carcinoma models, and, thus, represent a promising starting point for the development of siRNA therapeutics for hepatocellular carcinoma.
Subject(s)
Carcinoma, Hepatocellular/drug therapy , Drug Delivery Systems/methods , Genetic Therapy/methods , Liver Neoplasms/drug therapy , Nanoparticles , RNA, Small Interfering/administration & dosage , Animals , Carcinoma, Hepatocellular/genetics , Cell Line, Tumor , Female , Hep G2 Cells , Humans , Liver Neoplasms/genetics , Liver Neoplasms, Experimental , Mice , Mice, SCID , RNA, Small Interfering/genetics , RNA, Small Interfering/toxicityABSTRACT
Delivering small interfering RNA (siRNA) to tumors is the major technical hurdle that prevents the advancement of siRNA-based cancer therapy. One of the difficulties associated with the development of clinically relevant delivery systems is the lack of reliable tools for monitoring siRNA delivery to tumors in vivo. We describe here a novel, positive-readout system where siRNA-mediated target knockdown elicits a rapid and robust increase of reporter activity. Using the positive-readout system, we created (1) ß-galactosidase-based tumor models that allow the detection of target knockdown in 1%-2% of tumor cells and can distinguish between tumor areas where effective target knockdown occurs versus tumor areas that are not accessible to delivery, and (2) luciferase-based tumor models that allow the quantitative assessment of a large number of delivery systems. Using these positive-readout models, we screened a number of literature-described siRNA delivery systems and identified lipid nanoparticles as a promising delivery platform for siRNA-based cancer therapy.
Subject(s)
Gene Knockdown Techniques , Monitoring, Physiologic/methods , Neoplasms/therapy , RNA, Small Interfering/administration & dosage , Animals , Base Sequence , Cell Line, Tumor , Female , Genes, Reporter , Genetic Vectors , Liposomes , Mice , Mice, SCID , Molecular Sequence Data , Nanoparticles/administration & dosage , RNA, Small Interfering/therapeutic use , Xenograft Model Antitumor Assays , beta-Galactosidase/geneticsABSTRACT
This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.
Subject(s)
Biomimetic Materials/chemistry , Biomimetic Materials/metabolism , Gene Transfer Techniques , Integrin alphaVbeta3/metabolism , Oligopeptides/chemistry , Polyethylene Glycols/chemistry , Polyethyleneimine/chemistry , Biocompatible Materials/chemical synthesis , Biocompatible Materials/chemistry , Biocompatible Materials/metabolism , Biomimetic Materials/chemical synthesis , Cell Line, Tumor , DNA/metabolism , Gene Expression Regulation , Humans , Ligands , Maleimides/chemistry , Propionates/chemistry , Sulfhydryl Compounds/chemistry , TransfectionABSTRACT
Matrix metalloproteinases (MMPs) have been implicated in several pathologies. At Abbott Laboratories, the matrix metalloproteinases inhibitor drug discovery program has focused on the discovery of a potent, selective, orally bioavailable MMP inhibitor for the treatment of cancer. The program evolved from early succinate-based inhibitors to utilizing in-house technology such as SAR by NMR to develop a novel class of biaryl hydroxamate MMP inhibitors. The metabolic instability of the biaryl hydroxamates led to the discovery of a new class of N-formylhydroxylamine (retrohydroxamate) biaryl ethers, exemplified by ABT-770 (16). Toxicity issues with this pre-clinical candidate led to the discovery of another novel class of retrohydroxamate MMP inhibitors, the phenoxyphenyl sulfones such as ABT-518 (19j). ABT-518 is a potent, orally bioavailable, selective inhibitor of MMP-2 and 9 over MMP-1 that has been evaluated in Phase I clinical trials in cancer patients.
Subject(s)
Drug Industry , Matrix Metalloproteinase Inhibitors , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Animals , Drug Design , Humans , Magnetic Resonance Spectroscopy , Structure-Activity RelationshipABSTRACT
Several heterocyclic ketones were investigated as potential inhibitors of histone deacetylase. Nanomolar inhibitors such as 22 and 25 were obtained, the anti-proliferative activity of which were shown to be mediated by HDAC inhibition.
Subject(s)
Enzyme Inhibitors/pharmacology , Heterocyclic Compounds/pharmacology , Histone Deacetylase Inhibitors , Ketones/pharmacology , Enzyme Inhibitors/chemistry , Ketones/chemistry , Kinetics , Structure-Activity RelationshipABSTRACT
Alpha-keto ester and amides were found to be potent inhibitors of histone deacetylase. Nanomolar inhibitors against the isolated enzyme and sub-micromolar inhibitors of cellular proliferation were obtained. The alpha-keto amide 30 also exhibited significant anti-tumor effects in an in vivo tumor model.
Subject(s)
Amides/chemistry , Amides/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Animals , Cell Line, Tumor , Histone Deacetylases/metabolism , Humans , Mice , Xenograft Model Antitumor Assays/methodsABSTRACT
Trifluoromethyl ketones were found to be inhibitors of histone deacetylases (HDACs). Optimization of this series led to the identification of submicromolar inhibitors such as 20 that demonstrated antiproliferative effects against the HT1080 and MDA 435 cell lines.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Histone Deacetylase Inhibitors , Ketones/chemistry , Ketones/pharmacology , Acetylation , Blotting, Western , Breast Neoplasms/metabolism , Cell Cycle/drug effects , Fibrosarcoma/metabolism , Histones/metabolism , Humans , Hydrocarbons, Fluorinated/chemistry , Hydrocarbons, Fluorinated/pharmacology , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
A novel series of sulfone N-formylhydroxylamines (retrohydroxamates) have been investigated as matrix metalloproteinases (MMP) inhibitors. The substitution of the ether linkage of ABT-770 (5) with a sulfone group 13a led to a substantial increase in activity against MMP-9 but was accompanied by a loss of selectivity for inhibition of MMP-2 and -9 over MMP-1 and diminished oral exposure. Replacement of the biphenyl P1' substituent with a phenoxyphenyl group provided compounds that are highly selective for inhibition of MMP-2 and -9 over MMP-1. Optimization of the substituent adjacent to the retrohydroxamate center in this series led to the clinical candidate ABT-518 (6), a highly potent, selective, orally bioavailable MMP inhibitor that has been shown to significantly inhibit tumor growth in animal cancer models.
