ABSTRACT
A new fragment library for lead discovery has been designed and experimentally validated for use in surface plasmon resonance (SPR) biosensor-based screening. The 930 compounds in the library were selected from 4.6 million commercially available compounds using a series of physicochemical and medicinal chemistry filters. They were screened against 3 prototypical drug targets: HIV-1 protease, thrombin and carbonic anhydrase, and a nontarget: human serum albumin. Compound solubility was not a problem under the conditions used for screening. The high sensitivity of the sensor surfaces allowed the detection of interactions for 35% to 97% of the fragments, depending on the target protein. None of the fragments was promiscuous (i.e., interacted with a stoichiometry ≥5:1 with all 4 proteins), and only 2 compounds dissociated slowly from all 4 proteins. The use of several targets proved valuable since several compounds would have been disqualified from the library on the grounds of promiscuity if fewer target proteins had been used. The experimental procedure allowed an efficient evaluation and exploration of the new fragment library and confirmed that the new library is suitable for SPR biosensor-based screening.
Subject(s)
Biosensing Techniques/methods , Drug Evaluation, Preclinical/methods , Surface Plasmon Resonance/methods , Animals , Carbonic Anhydrase II/metabolism , Cattle , Enzyme Inhibitors/metabolism , HIV Protease/metabolism , Humans , Protein Binding , Serum Albumin/metabolism , Small Molecule Libraries , Thrombin/metabolismABSTRACT
The soluble acetylcholine binding protein (AChBP) is a homologue of the ligand-binding domain of the nicotinic acetylcholine receptors (nAChR). To guide future fragment-screening using surface plasmon resonance (SPR) biosensor technology as a label-free, direct binding, biophysical screening assay, a focused fragment library was generated based on deconstruction of a set of α7 nAChR selective quinuclidine containing ligands with nanomolar affinities. The interaction characteristics of the fragments and the parent compounds with AChBP were evaluated using an SPR biosensor assay. The data obtained from this direct binding assay correlated well with data from the reference radioligand displacement assay. Ligand efficiencies for different (structural) groups of fragments in the library were correlated to binding with distinct regions of the binding pocket, thereby identifying ligand efficiency hot spots (LE hot spots). These hot spots can be used to identity the most promising hit fragments in a large scale fragment library screen.
Subject(s)
Carbamates/chemistry , Carrier Proteins/chemistry , Models, Molecular , Pyridines/chemistry , Quinuclidines/chemistry , Receptors, Nicotinic/chemistry , Animals , Aplysia , Binding Sites , Binding, Competitive , Biosensing Techniques , Carbamates/chemical synthesis , Humans , Ligands , Lymnaea , Pyridines/chemical synthesis , Quinuclidines/chemical synthesis , Radioligand Assay , Structure-Activity Relationship , Surface Plasmon ResonanceABSTRACT
Small inhibitors of matrix metalloproteinase 12 (MMP-12) have been identified with a biosensor-based screening strategy and a specifically designed fragment library. The interaction between fragments and three variants of the target and a reference protein with an active-site zinc ion was measured continuously by surface plasmon resonance. The developed experimental design overcame the inherent instability of MMP-12 and allowed the identification of fragments that interacted specifically with the active-site of MMP-12 but not with the reference protein. The interaction with MMP-12 for selected compounds were analyzed for concentration dependence and saturability. Compounds interacting distinctly with the target were further evaluated by an activity-based assay, verifying MMP-12 inhibition. Two effective inhibitors were identified, and the compound with highest affinity was confirmed to be a competitive inhibitor with an IC50 of 290 nM and a ligand efficiency of 0.7 kcal/mol heavy atom. This procedure integrates selectivity and binding site identification into the screening procedure and does not require structure determination.
Subject(s)
Biosensing Techniques , Drug Design , Matrix Metalloproteinase 12/chemistry , Matrix Metalloproteinase Inhibitors , Small Molecule Libraries , Benzimidazoles/chemistry , Benzoxazines/chemistry , Binding Sites , Carbonic Anhydrase II/chemistry , Carbonic Anhydrase Inhibitors/chemistry , Cations, Divalent , Hydroxamic Acids , Indoles/chemistry , Kinetics , Ligands , Protein Binding , Quinolines/chemistry , Surface Plasmon Resonance , Thermodynamics , Thiazoles/chemistry , Zinc/chemistryABSTRACT
From a set of weakly potent lead compounds, using in silico screening and small library synthesis, a series of 2-alkyl-3-aryl-3-alkoxyisoindolinones has been identified as inhibitors of the MDM2-p53 interaction. Two of the most potent compounds, 2-benzyl-3-(4-chlorophenyl)-3-(3-hydroxypropoxy)-2,3-dihydroisoindol-1-one (76; IC(50) = 15.9 +/- 0.8 microM) and 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (79; IC(50) = 5.3 +/- 0.9 microM), induced p53-dependent gene transcription, in a dose-dependent manner, in the MDM2 amplified, SJSA human sarcoma cell line.
Subject(s)
Antineoplastic Agents/chemical synthesis , Indoles/chemical synthesis , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Suppressor Protein p53/metabolism , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Combinatorial Chemistry Techniques , Drug Screening Assays, Antitumor , Humans , Indoles/chemistry , Indoles/pharmacology , Models, Molecular , Protein Binding , Stereoisomerism , Structure-Activity Relationship , Transcription, Genetic , Tumor Suppressor Protein p53/geneticsABSTRACT
A series of 2-N-alkyl-3-aryl-3-alkoxyisoindolinones has been synthesised and evaluated as inhibitors of the MDM2-p53 interaction. The most potent compound, 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (NU8231), exhibited an IC50 of 5.3 +/- 0.9 microM in an ELISA assay, and induced p53-dependent gene transcription in a dose-dependent manner, in the SJSA human sarcoma cell line.
Subject(s)
Indoles/chemistry , Indoles/pharmacology , Nuclear Proteins/drug effects , Proto-Oncogene Proteins/drug effects , Tumor Suppressor Protein p53/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Humans , Indoles/chemical synthesis , Models, Molecular , Molecular Structure , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Protein Binding/drug effects , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Structure-Activity Relationship , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
We have investigated the efficacy of generating multiple sidechain conformations using a rotamer library in order to find the experimentally observed ligand binding site conformation of a protein in the presence of a bound ligand. We made use of a recently published algorithm that performs an exhaustive conformational search using a rotamer library to enumerate all possible sidechain conformations in a binding site. This approach was applied to a dataset of proteins whose structures were determined by X-ray and NMR methods. All chosen proteins had two or more structures, generally involving different bound ligands. By taking one of these structures as a reference, we were able in most cases to successfully reproduce the experimentally determined conformations of the other structures, as well as to suggest alternative low-energy conformations of the binding site. In those few cases where this procedure failed, we observed that the bound ligand had induced a high-energy conformation of the binding site. These results suggest that for most proteins that exhibit limited backbone motion, ligands tend to bind to low energy conformations of their binding sites. Our results also reveal that it is possible in most cases to use a rotamer search-based approach to predict alternative low-energy protein binding site conformations that can be used by different ligands. This opens the possibility of incorporating alternative binding site conformations to improve the efficacy of docking and structure-based drug design algorithms.
Subject(s)
Models, Molecular , Proteins/chemistry , Proteins/metabolism , Algorithms , Binding Sites , Crystallography, X-Ray , Ligands , Magnetic Resonance Spectroscopy , Protein BindingABSTRACT
Knowledge about how secondary-structure elements combine together to form the tertiary structure is crucial for understanding protein folding. We have examined packing solutions for alpha-helices by performing a crystal survey of the underlying backbone-backbone inter-geometry of tertiary contacts. The information content is different from, and complementary to, the results of side-chain to side-chain crystal surveys and studies of helix-helix-crossing angles. Six geometry descriptors were recorded from each tertiary contact in nonredundant data sets of globular and transmembrane proteins. The descriptors included distances, angles, and dihedral angles that together describe completely the underlying geometry of each contact. From the results it is possible to identify differences in the geometry requirements of tertiary contacts between alpha-helices in general and transmembrane alpha-helices. The differences become more apparent when the correlation between the different geometry descriptors is analyzed. The results are compared with those of other types of secondary structure. Finally, an investigation of how the geometry of tertiary contacts changes with the amino-acid types involved in the contact is performed using multivariate techniques. The results of this study provide a well-defined overview of the underlying structural framework of tertiary contacts between alpha-helices, in both globular and TM environments, that will have valuable implications for predicting protein tertiary structure.
Subject(s)
Peptides/chemistry , Amino Acid Sequence , Computer Graphics , Crystallography, X-Ray/statistics & numerical data , Multivariate Analysis , Protein Interaction Mapping/methods , Protein Interaction Mapping/statistics & numerical data , Protein Structure, TertiaryABSTRACT
Computational ligand-protein docking is routinely used for binding mode prediction. We have quantified the effect of considering multiple docking solutions on the success rate of obtaining the crystallographic binding mode. By selection of a small set of representatives, the experimentally observed binding mode can be predicted with a higher probability after a ligand-protein docking simulation. The proportion of correctly predicted complexes improved from 69% to 87% when five distinct binding modes were considered.
Subject(s)
Ligands , Proteins/chemistry , Crystallography, X-Ray , Models, Molecular , Molecular Structure , Protein BindingABSTRACT
A major difficulty in structure-based molecular design is the prediction of the structure of the protein-ligand complex because of the enormous number of degrees of freedom. Commonly, the target protein is kept rigid in a single low-energy conformation. However, this does not reflect the dynamic nature of protein structures. In this work, we investigate the influence of receptor flexibility in virtual screening of reagents on a common scaffold in the S1' pocket of human collagenase (matrix metalloproteinase-1). We compare screening using a single-crystal structure and multiple NMR structures, both apo and holo forms. We also investigate two computational methods of addressing receptor flexibility that can be used when NMR data are not available. The results from virtual screening using the experimental structures are compared to those obtained using the two computational methods. From the results, we draw conclusions about the impact of target flexibility on the identification of active and diverse reagents in a virtual screening protocol.
Subject(s)
Matrix Metalloproteinase 1/chemistry , Apoenzymes/chemistry , Binding Sites , Crystallography, X-Ray , Holoenzymes/chemistry , Humans , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Phenyl Ethers/chemistry , Pyridines/chemistry , Sulfones/chemistryABSTRACT
A novel hybrid optimization method called quantum stochastic tunneling has been recently introduced. Here, we report its implementation within a new docking program called EasyDock and a validation with the CCDC/Astex data set of ligand-protein complexes using the PLP score to represent the ligand-protein potential energy surface and ScreenScore to score the ligand-protein binding energies. When taking the top energy-ranked ligand binding mode pose, we were able to predict the correct crystallographic ligand binding mode in up to 75% of the cases. By using this novel optimization method run times for typical docking simulations are significantly shortened.
Subject(s)
Algorithms , Proteins/chemistry , Computer Simulation , Crystallography, X-Ray , Drug Design , Ligands , Protein Binding , ThermodynamicsABSTRACT
Side-chain flexibility of ligand-binding sites needs to be considered in the rational design of novel inhibitors. We have developed a method to generate conformational ensembles that efficiently sample local side-chain flexibility from a single crystal structure. The rotamer-based approach is tested here for the S1' pocket of human collagenase-1 (MMP-1), which is known to undergo conformational changes in multiple side-chains upon binding of certain inhibitors. First, a raw ensemble consisting of a large number of conformers of the S1' pocket was generated using an exhaustive search of rotamer combinations on a template crystal structure. A combination of principal component analysis and fuzzy clustering was then employed to successfully identify a core ensemble consisting of a low number of representatives from the raw ensemble. The core ensemble contained geometrically diverse conformers of stable nature, as indicated in several cases by a relative energy lower than that of the minimised template crystal structure. Through comparisons with X-ray crystallography and NMR structural data we show that the core ensemble occupied a conformational space similar to that observed under experimental conditions. The synthetic inhibitor RS-104966 is known to induce a conformational change in the side-chains of the S1' pocket of MMP-1 and could not be docked in the template crystal structure. However, the experimental binding mode was reproduced successfully using members of the core ensemble as the docking target, establishing the usefulness of the method in drug design.