ABSTRACT
We demonstrate a fragment-based lead discovery method that combines site-directed ligand discovery with dynamic combinatorial chemistry. Our technique targets dynamic combinatorial screening to a specified region of a protein by using reversible disulfide chemistry. We have used this technology to rapidly identify inhibitors of the drug target Aurora A that span the purine-binding site and the adaptive pocket of the kinase. The binding mode of a noncovalent inhibitor has been further characterized through crystallography.
Subject(s)
Chemistry, Pharmaceutical/methods , Combinatorial Chemistry Techniques/methods , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/chemical synthesis , Protein Serine-Threonine Kinases/antagonists & inhibitors , Aurora Kinases , Binding Sites/drug effects , Crystallography, X-Ray , Drug Design , Ligands , Mass Spectrometry/methods , Models, Chemical , Molecular Structure , Purines/chemistry , Structure-Activity RelationshipABSTRACT
We have identified a small-molecule inhibitor of tumor necrosis factor alpha (TNF-alpha) that promotes subunit disassembly of this trimeric cytokine family member. The compound inhibits TNF-alpha activity in biochemical and cell-based assays with median inhibitory concentrations of 22 and 4.6 micromolar, respectively. Formation of an intermediate complex between the compound and the intact trimer results in a 600-fold accelerated subunit dissociation rate that leads to trimer dissociation. A structure solved by x-ray crystallography reveals that a single compound molecule displaces a subunit of the trimer to form a complex with a dimer of TNF-alpha subunits.
Subject(s)
Indoles/chemistry , Indoles/pharmacology , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/chemistry , Biotinylation , Chemical Phenomena , Chemistry, Physical , Crystallography, X-Ray , Dimerization , Fluorescence , Hydrogen/chemistry , Hydrophobic and Hydrophilic Interactions , Indoles/chemical synthesis , Kinetics , Mass Spectrometry , Models, Chemical , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Conformation , Protein Subunits/chemistry , Receptors, Tumor Necrosis Factor, Type I/metabolism , Tumor Necrosis Factor-alpha/metabolismABSTRACT
In general, alpha-helical conformations in proteins depend in large part on the amino acid residues within the helix and their proximal interactions. For example, an alanine residue has a high propensity to adopt an alpha-helical conformation, whereas that of a glycine residue is low. The sequence preferences for beta-sheet formation are less obvious. To identify the factors that influence beta-sheet conformation, a series of scanning polyalanine mutations were made within the strands and associated turns of the beta-sheet region in T4 lysozyme. For each construct the stability of the folded protein was reduced substantially, consistent with removal of native packing interactions. However, the crystal structures showed that each of the mutants retained the beta-sheet conformation. These results suggest that the structure of the beta-sheet region of T4 lysozyme is maintained to a substantial extent by tertiary interactions with the surrounding parts of the protein. Such tertiary interactions may be important in determining the structures of beta-sheets in general.
Subject(s)
Alanine/genetics , Muramidase/chemistry , Muramidase/genetics , Amino Acid Sequence , Bacteriophage T4/enzymology , Crystallography, X-Ray , Models, Molecular , Molecular Sequence Data , Mutagenesis/genetics , Mutation/genetics , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , ThermodynamicsABSTRACT
Protein tyrosine phosphatases play important roles in many signaling cascades involved in human disease. The identification of druglike inhibitors for these targets is a major challenge, and the discovery of suitable phosphotyrosine (pY) mimetics remains one of the key difficulties. Here we describe an extension of tethering technology, "breakaway tethering", which is ideally suited for discovering such new chemical entities. The approach involves first irreversibly modifying a protein with an extender that contains both a masked thiol and a known pY mimetic. The extender is then cleaved to release the pY mimetic, unmasking the thiol. The resulting protein is screened against a library of disulfide-containing small molecule fragments; any molecules with inherent affinity for the pY binding site will preferentially form disulfides with the extender, allowing for their identification by mass spectrometry. The ability to start from a known substrate mimimizes perturbation of protein structure and increases the opportunity to probe the active site using tethering. We applied this approach to the anti-diabetic protein PTP1B to discover a pY mimetic which belongs to a new molecular class and which binds in a novel fashion.
