ABSTRACT
Inhibition of mutant B-Raf signaling, through either direct inhibition of the enzyme or inhibition of MEK, the direct substrate of Raf, has been demonstrated preclinically to inhibit tumor growth. Very recently, treatment of B-Raf mutant melanoma patients with a selective B-Raf inhibitor has resulted in promising preliminary evidence of antitumor activity. This article describes the design and optimization of tetrahydronaphthalene-derived compounds as potent inhibitors of the Raf pathway in vitro and in vivo. These compounds possess good pharmacokinetic properties in rodents and inhibit B-Raf mutant tumor growth in mouse xenograft models.
Subject(s)
Antineoplastic Agents/chemical synthesis , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Tetrahydronaphthalenes/chemical synthesis , Administration, Oral , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Availability , Crystallography, X-Ray , Drug Design , Melanoma, Experimental/drug therapy , Melanoma, Experimental/enzymology , Melanoma, Experimental/pathology , Mice , Mice, Nude , Models, Molecular , Mutation , Proto-Oncogene Proteins B-raf/genetics , Stereoisomerism , Structure-Activity Relationship , Tetrahydronaphthalenes/chemistry , Tetrahydronaphthalenes/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
Starting from a tripeptide screening hit, a series of dipeptide inhibitors of the proteasome with Thr as the P3 residue has been optimized with the aid of crystal structures in complex with the ß-5/6 active site of y20S. Derivative 25, (ß5 IC(50)=7.4 nM) inhibits only the chymotryptic activity of the proteasome, shows cellular activity against targets in the UPS, and inhibits proliferation.
Subject(s)
Chymotrypsin/antagonists & inhibitors , Dipeptides/chemistry , Proteasome Endopeptidase Complex/metabolism , Threonine/chemistry , Humans , Models, MolecularABSTRACT
The discovery of novel pyrazoline derivatives as B-Raf (V600E) inhibitors is described in this report. Chemical modification of the pyrazoline scaffold led to the development of SAR and identified potent and selective inhibitors of B-Raf (V600E). Determination of the pharmacokinetic properties of selected inhibitors is also reported.
Subject(s)
Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Pyrazoles/chemistry , Amino Acid Substitution , Binding Sites , Computer Simulation , Drug Evaluation, Preclinical , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Pyrazoles/chemical synthesis , Pyrazoles/pharmacokinetics , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity RelationshipABSTRACT
The mammalian 26S proteasome is a 2500 kDa multi-catalytic complex involved in intracellular protein degradation. We describe the synthesis and properties of a novel series of non-covalent di-peptide inhibitors of the proteasome based [corrected] on a capped tri-peptide that was first identified by high-throughput screening of a library of approx. 350000 compounds for inhibitors of the ubiquitin-proteasome system in cells. We show that these compounds are entirely selective for the beta5 (chymotrypsin-like) site over the beta1 (caspase-like) and beta2 (trypsin-like) sites of the 20S core particle of the proteasome, and over a panel of less closely related proteases. Compound optimization, guided by X-ray crystallography of the liganded 20S core particle, confirmed their non-covalent binding mode and provided a structural basis for their enhanced in vitro and cellular potencies. We demonstrate that such compounds show low nanomolar IC50 values for the human 20S beta5 site in vitro, and that pharmacological inhibition of this site in cells is sufficient to potently inhibit the degradation of a tetra-ubiquitin-luciferase reporter, activation of NFkappaB (nuclear factor kappaB) in response to TNF-alpha (tumour necrosis factor-alpha) and the proliferation of cancer cells. Finally, we identified capped di-peptides that show differential selectivity for the beta5 site of the constitutively expressed proteasome and immunoproteasome in vitro and in B-cell lymphomas. Collectively, these studies describe the synthesis, activity and binding mode of a new series of non-covalent proteasome inhibitors with unprecedented potency and selectivity for the beta5 site, and which can discriminate between the constitutive proteasome and immunoproteasome in vitro and in cells.
Subject(s)
Oligopeptides/pharmacology , Protease Inhibitors/pharmacology , Proteasome Inhibitors , Amino Acid Sequence , Binding Sites , Boronic Acids/pharmacology , Bortezomib , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Crystallography, X-Ray , HCT116 Cells , HT29 Cells , Humans , Kinetics , Luciferases/genetics , Luciferases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , NF-kappa B/genetics , NF-kappa B/metabolism , Oligopeptides/chemistry , Oligopeptides/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/metabolism , Protein Binding , Protein Structure, Tertiary , Protein Subunits/antagonists & inhibitors , Protein Subunits/genetics , Protein Subunits/metabolism , Pyrazines/pharmacology , RNA Interference , Sequence Homology, Amino Acid , Ubiquitin/genetics , Ubiquitin/metabolismABSTRACT
A high throughput screen identified N-aroylpyrazoline 1 as a selective inhibitor of the V600E mutant of B-Raf kinase. Parallel synthesis of acyl, aroyl, and sulfonyl derivatives led to the identification of several potent inhibitors in both enzymatic and cellular (pERK) assays such as compound 42.