ABSTRACT
The E2 protein is required for the replication of human papillomaviruses (HPVs), which are responsible for anogenital warts and cervical carcinomas. Using an NMR-based screen, we tested compounds for binding to the DNA-binding domain of the HPV-E2 protein. Three classes of compounds were identified which bound to two distinct sites on the protein. Biphenyl and biphenyl ether compounds containing a carboxylic acid bind to a site near the DNA recognition helix and inhibit the binding of E2 to DNA. Benzophenone-containing compounds which lack a carboxylic acid group bind to the beta-barrel formed by the dimer interface and exhibit negligible effects on the binding of E2 to DNA. Structure-activity relationships from the biphenyl and biphenyl ether compounds were combined to produce a compound [5-(3'-(3",5"-dichlorophenoxy)-phenyl)-2,4-pentadienoic acid] with an IC50 value of approximately 10 microM. This compound represents a useful lead for the development of antiviral agents that interfere with HPV replication and further illustrates the usefulness of the SAR by NMR method in the drug discovery process.
Subject(s)
Antiviral Agents/chemistry , DNA-Binding Proteins/antagonists & inhibitors , DNA/metabolism , Drug Design , Repressor Proteins/metabolism , Trans-Activators/metabolism , Viral Proteins/antagonists & inhibitors , Antiviral Agents/pharmacology , Binding Sites , Biphenyl Compounds/pharmacology , Bovine papillomavirus 1 , Crystallography, X-Ray , DNA-Binding Proteins/metabolism , Humans , Ligands , Magnetic Resonance Spectroscopy , Models, Chemical , Models, Molecular , Papillomaviridae , Protein Conformation , Structure-Activity Relationship , Viral Proteins/metabolismABSTRACT
Heterodimerization between members of the Bcl-2 family of proteins is a key event in the regulation of programmed cell death. The molecular basis for heterodimer formation was investigated by determination of the solution structure of a complex between the survival protein Bcl-xL and the death-promoting region of the Bcl-2-related protein Bak. The structure and binding affinities of mutant Bak peptides indicate that the Bak peptide adopts an amphipathic alpha helix that interacts with Bcl-xL through hydrophobic and electrostatic interactions. Mutations in full-length Bak that disrupt either type of interaction inhibit the ability of Bak to heterodimerize with Bcl-xL.
Subject(s)
Membrane Proteins/chemistry , Protein Conformation , Proto-Oncogene Proteins c-bcl-2 , Proto-Oncogene Proteins/chemistry , Amino Acid Sequence , Apoptosis , Crystallography, X-Ray , Dimerization , Magnetic Resonance Spectroscopy , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Protein Structure, Secondary , Proto-Oncogene Proteins/metabolism , Sequence Deletion , bcl-2 Homologous Antagonist-Killer Protein , bcl-X ProteinABSTRACT
A nuclear magnetic resonance (NMR)-based method is described in which small organic molecules that bind to proximal subsites of a protein are identified, optimized, and linked together to produce high-affinity ligands. The approach is called "SAR by NMR" because structure-activity relationships (SAR) are obtained from NMR. With this technique, compounds with nanomolar affinities for the FK506 binding protein were rapidly discovered by tethering two ligands with micromolar affinities. The method reduces the amount of chemical synthesis and time required for the discovery of high-affinity ligands and appears particularly useful in target-directed drug research.
Subject(s)
Carrier Proteins/metabolism , DNA-Binding Proteins/metabolism , Heat-Shock Proteins/metabolism , Ligands , Magnetic Resonance Spectroscopy , Proteins/metabolism , Tacrolimus/metabolism , Anilides/metabolism , Binding Sites , Models, Molecular , Structure-Activity Relationship , Tacrolimus Binding ProteinsABSTRACT
We present the NMR structure of the PTB domain of insulin receptor substrate-1 (IRS-1) complexed to a tyrosine-phosphorylated peptide derived from the IL-4 receptor. Despite the lack of sequence homology and different binding specificity, the overall fold of the protein is similar to that of the Shc PTB domain and closely resembles that of PH domains. However, the PTB domain of IRS-1 is smaller than that of Shc (110 versus 170 residues) and binds to phosphopeptides in a distinct manner. We explain the phosphopeptide binding specificity based on the structure of the complex and results of site-directed mutagenesis experiments.
