ABSTRACT
Fragment screening is one approach to hit identification for early stage drug discovery projects. Like any screening library, diversity is needed in fragment libraries. This includes coverage of shape and electrostatic space, as well as chemotype diversity. A new, easily interpretable shape-based fingerprint is described and its utility in probing fragment library content is demonstrated using a Rule of Three library from Maybridge. This method explicitly considers size as a component of shape. It allows interrogation of shape space on both a per conformer and a per molecule level, and includes a measure of flexibility. This allows for the identification of highly flexible compounds and their exclusion from the analysis. A comparison with two literature methods, the triangle plot approach of Sauer and Schwarz and the plane of best fit method of Firth et al., is also included.
Subject(s)
Drug Discovery , High-Throughput Screening AssaysABSTRACT
Inhibition of class IIa histone deacetylase (HDAC) enzymes have been suggested as a therapeutic strategy for a number of diseases, including Huntington's disease. Catalytic-site small molecule inhibitors of the class IIa HDAC4, -5, -7, and -9 were developed. These trisubstituted diarylcyclopropanehydroxamic acids were designed to exploit a lower pocket that is characteristic for the class IIa HDACs, not present in other HDAC classes. Selected inhibitors were cocrystallized with the catalytic domain of human HDAC4. We describe the first HDAC4 catalytic domain crystal structure in a "closed-loop" form, which in our view represents the biologically relevant conformation. We have demonstrated that these molecules can differentiate class IIa HDACs from class I and class IIb subtypes. They exhibited pharmacokinetic properties that should enable the assessment of their therapeutic benefit in both peripheral and CNS disorders. These selective inhibitors provide a means for evaluating potential efficacy in preclinical models in vivo.
Subject(s)
Drug Design , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/metabolism , Huntington Disease/drug therapy , Animals , Dose-Response Relationship, Drug , Histone Deacetylase Inhibitors/chemical synthesis , Histone Deacetylase Inhibitors/chemistry , Histone Deacetylase Inhibitors/pharmacokinetics , Histone Deacetylases/classification , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Models, Molecular , Molecular Structure , Structure-Activity RelationshipABSTRACT
Virtual screening was performed against experimentally enabled homology models of the adenosine A(2A) receptor, identifying a diverse range of ligand efficient antagonists (hit rate 9%). By use of ligand docking and Biophysical Mapping (BPM), hits 1 and 5 were optimized to potent and selective lead molecules (11-13 from 5, pK(I) = 7.5-8.5, 13- to >100-fold selective versus adenosine A(1); 14-16 from 1, pK(I) = 7.9-9.0, 19- to 59-fold selective).
Subject(s)
Adenosine A2 Receptor Antagonists/chemistry , Databases, Factual , Models, Molecular , Receptor, Adenosine A2A/chemistry , Adenosine A2 Receptor Antagonists/chemical synthesis , Adenosine A2 Receptor Antagonists/pharmacology , Animals , Binding Sites , CHO Cells , Chromones/chemical synthesis , Chromones/chemistry , Chromones/pharmacology , Cricetinae , Cricetulus , HEK293 Cells , Humans , Piperazines/chemical synthesis , Piperazines/chemistry , Piperazines/pharmacology , Radioligand Assay , Receptor, Adenosine A2A/metabolism , Structure-Activity Relationship , Triazines/chemical synthesis , Triazines/chemistry , Triazines/pharmacology , TurkeysABSTRACT
The stress-activated kinase p38α was used to evaluate a fragment-based drug discovery approach using the BioFocus fragment library. Compounds were screened by surface plasmon resonance (SPR) on a Biacore(™) T100 against p38α and two selectivity targets. A sub-set of our library was the focus of detailed follow-up analyses that included hit confirmation, affinity determination on 24 confirmed, selective hits and competition assays of these hits with respect to a known ATP binding site inhibitor. In addition, functional activity against p38α was assessed in a biochemical assay using a mobility shift platform (LC3000, Caliper LifeSciences). A selection of fragments was also evaluated using fluorescence lifetime (FLEXYTE(™)) and microscale thermophoresis (Nanotemper) technologies. A good correlation between the data for the different assays was found. Crystal structures were solved for four of the small molecules complexed to p38α. Interestingly, as determined both by X-ray analysis and SPR competition experiments, three of the complexes involved the fragment at the ATP binding site, while the fourth compound bound in a distal site that may offer potential as a novel drug target site. A first round of optimization around the remotely bound fragment has led to the identification of a series of triazole-containing compounds. This approach could form the basis for developing novel and active p38α inhibitors. More broadly, it illustrates the power of combining a range of biophysical and biochemical techniques to the discovery of fragments that facilitate the development of novel modulators of kinase and other drug targets.
Subject(s)
Drug Discovery/methods , Mitogen-Activated Protein Kinase 14/chemistry , Small Molecule Libraries/chemistry , Triazoles/chemistry , Binding Sites , Bridged Bicyclo Compounds/chemistry , Drug Evaluation, Preclinical/methods , Humans , Ligands , Molecular Conformation , Peptide Fragments/chemistry , Protein Binding , Surface Plasmon Resonance/methods , X-Ray DiffractionABSTRACT
Pin1 is an emerging oncology target strongly implicated in Ras and ErbB2-mediated tumourigenesis. Pin1 isomerizes bonds linking phospho-serine/threonine moieties to proline enabling it to play a key role in proline-directed kinase signalling. Here we report a novel series of Pin1 inhibitors based on a phenyl imidazole acid core that contains sub-µM inhibitors. Compounds have been identified that block prostate cancer cell growth under conditions where Pin1 is essential.
Subject(s)
Imidazoles/pharmacology , Peptidylprolyl Isomerase/antagonists & inhibitors , Caco-2 Cells , Drug Discovery , Humans , Imidazoles/chemistry , Models, Molecular , Molecular Structure , NIMA-Interacting Peptidylprolyl IsomeraseABSTRACT
The peptidyl prolyl cis/trans isomerase Pin1 is a promising molecular target for anti-cancer therapeutics. Here we report the structure-guided evolution of an indole 2-carboxylic acid fragment hit into a series of alpha-benzimidazolyl-substituted amino acids. Examples inhibited Pin1 activity with IC(50) <100nM, but were inactive on cells. Replacement of the benzimidazole ring with a naphthyl group resulted in a 10-50-fold loss in ligand potency, but these examples downregulated biomarkers of Pin1 activity and blocked proliferation of PC3 cells.
Subject(s)
Amino Acids/chemistry , Antineoplastic Agents/chemistry , Enzyme Inhibitors/chemistry , Peptidylprolyl Isomerase/antagonists & inhibitors , Amino Acids/chemical synthesis , Amino Acids/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Benzimidazoles/chemistry , Binding Sites , Cell Line, Tumor , Crystallography, X-Ray , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Hydrogen Bonding , Indoles/chemistry , NIMA-Interacting Peptidylprolyl Isomerase , Peptidylprolyl Isomerase/metabolism , Structure-Activity RelationshipABSTRACT
Virtual screening against a pCDK2/cyclin A crystal structure led to the identification of a potent and novel CDK2 inhibitor, which exhibited an unusual mode of interaction with the kinase binding motif. With the aid of X-ray crystallography and modelling, a medicinal chemistry strategy was implemented to probe the interactions seen in the crystal structure and to establish SAR. A fragment-based approach was also considered but a different, more conventional, binding mode was observed. Compound selectivity against GSK-3beta was improved using a rational design strategy, with crystallographic verification of the CDK2 binding mode.
Subject(s)
Cyclin-Dependent Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Crystallography, X-Ray , Drug Design , Protein Kinase Inhibitors/chemistryABSTRACT
Database searching led to the identification of potent A(2A) antagonists which do not contain the privileged furan moiety and which show selectivity over A(1) receptors. Simple substructure searching on a proprietary database identified compounds with activities in the low nM range. A targeted approach to the identification of non-furan containing compounds resulted in the identification of two novel series, with potency, selectivity and directional SAR from screening 113 compounds.
Subject(s)
Adenosine A2 Receptor Antagonists , Furans/chemistry , Furans/pharmacology , Amination , Fluorine/chemistry , Furans/chemical synthesis , Furans/metabolism , Models, Molecular , Molecular Structure , Protein Binding , Pyrimidines/chemistry , Pyrimidines/metabolism , Receptor, Adenosine A2A/metabolism , Structure-Activity RelationshipABSTRACT
Crystallographic and modelling data, in conjunction with a medicinal chemistry template-hopping approach, led to the identification of a series of novel and potent inhibitors of human cyclin-dependent kinase 2 (CDK2), with selectivity over glycogen synthase kinase-3beta (GSK-3beta). One example had a CDK2 IC(50) of 120 nM and showed selectivity over GSK-3beta of 167-fold.
Subject(s)
Cyclin-Dependent Kinase 2/antagonists & inhibitors , Drug Design , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Triazoles/chemistry , Cell Line, Tumor , Cyclin-Dependent Kinase 2/metabolism , Humans , Inhibitory Concentration 50 , Molecular Structure , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemical synthesis , Structure-Activity RelationshipABSTRACT
The protein structure guided design of a series of pyrazolo[1,5-a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3beta and other kinases.
Subject(s)
CDC2-CDC28 Kinases/antagonists & inhibitors , Pyrimidines/chemistry , Pyrimidines/pharmacology , CDC2 Protein Kinase/antagonists & inhibitors , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin-Dependent Kinase 2 , Drug Design , Humans , Inhibitory Concentration 50 , Models, Molecular , Structure-Activity RelationshipABSTRACT
Type II topoisomerases bind to DNA at the catalytic domain across the DNA gate. DNA gyrases also bind to DNA at the non-homologous C-terminal domain of the GyrA subunit, which causes the wrapping of DNA about itself. This unique mode of DNA binding allows gyrases to introduce the negative supercoils into DNA molecules. We have investigated the biochemical characteristics of Staphylococcus aureus (S. aureus) gyrase. S. aureus gyrase is known to require high concentrations of potassium glutamate (K-Glu) for its supercoiling activity. However, high concentrations of K-Glu are not required for its relaxation and decatenation activities. This is due to the requirement of high concentrations of K-Glu for S. aureus gyrase-mediated wrapping of DNA. These results suggest that S. aureus gyrase can bind to DNA at the catalytic domain independent of K-Glu concentration, but high concentrations of K-Glu are required for the binding of the C-terminal domain of GyrA to DNA and the wrapping of DNA. Thus, salt modulates the DNA binding mode and the catalytic activity of S. aureus gyrase. Quinolone drugs can stimulate the formation of covalent S. aureus gyrase-DNA complexes, but high concentrations of K-Glu inhibit the formation of S. aureus gyrase-quinolone-DNA ternary complexes. In the absence of K-Glu, ternary complexes formed with S. aureus gyrase cannot arrest replication fork progression in vitro, demonstrating that the formation of a wrapped ternary complex is required for replication fork arrest by a S. aureus gyrase-quinolone-DNA ternary complex.
Subject(s)
DNA Gyrase/metabolism , DNA Replication , DNA, Bacterial/metabolism , Glutamates/pharmacology , Quinolones/metabolism , Staphylococcus aureus/metabolism , Catalysis , Protein Binding , Staphylococcus aureus/drug effects , Staphylococcus aureus/enzymology , Staphylococcus aureus/geneticsABSTRACT
The antimicrobial natural product chuangxinmycin has been found to be a potent and selective inhibitor of bacterial tryptophanyl tRNA synthetase (WRS). A number of analogues have been synthesised. The interaction with WRS appears to be highly constrained, as only sterically smaller analogues afforded significant inhibition. The only analogue to show inhibition comparable to chuangxinmycin also had antibacterial activity. WRS inhibition may contribute to the antibacterial action of chuangxinmycin.