ABSTRACT
Multidrug-resistant (MDR) bacteria is a severe threat to public health. Therefore, it is urgent to establish effective screening systems for identifying novel antibacterial compounds. In this study, a highly miniaturized droplet microarray (DMA) based high-throughput screening system is established to screen over 2000 compounds for their antimicrobial properties against carbapenem-resistant Klebsiella pneumoniae and methicillin resistant Staphylococcus aureus (MRSA). The DMA consists of an array of hydrophilic spots divided by superhydrophobic borders. Due to the differences in the surface wettability between the spots and the borders, arrays of hundreds of nanoliter-sized droplets containing bacteria and different drugs can be generated for screening applications. A simple colorimetric viability readout utilizing a conventional photo scanner is developed for fast single-step detection of the inhibitory effect of the compounds on bacterial growth on the whole array. Six hit compounds, including coumarins and structurally simplified estrogen analogs are identified in the primary screening and validated with minimum inhibition concentration assay for their antibacterial effect. This study demonstrates that the DMA-based high-throughput screening system enables the identification of potential antibiotics from novel synthetic compound libraries, offering opportunities for development of new treatments against multidrug-resistant bacteria.
Subject(s)
Anti-Bacterial Agents , Methicillin-Resistant Staphylococcus aureus , Anti-Bacterial Agents/pharmacology , Drug Resistance, Multiple, Bacterial , Microbial Sensitivity Tests , BacteriaABSTRACT
The protein arginine deiminase 4 (PAD4) is a calcium-dependent enzyme, which catalyses the irreversible conversion of peptidyl-arginines into peptidyl-citrullines and plays an important role in several diseases such as in the rheumatoid arthritis, multiple sclerosis, Alzheimer's disease, Creutzfeldt-Jacob's disease and cancer. In this study, we report the inhibition profiles and computational docking toward the PAD4 enzyme of a series of 1,2,3-triazole peptidomimetic-based derivatives incorporating the ß-phenylalanine and guanidine scaffolds. Several effective, low micromolar PAD4 inhibitors are reported in this study.
Subject(s)
Enzyme Inhibitors/pharmacology , Hydrolases/antagonists & inhibitors , Peptidomimetics/pharmacology , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Hydrolases/metabolism , Molecular Structure , Peptidomimetics/chemical synthesis , Peptidomimetics/chemistry , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Structure-Activity RelationshipABSTRACT
Transcriptional activators that respond to ligands with no cellular targets are powerful tools that can confer regulated expression of a transgene in almost all biological systems. In this study, we altered the ligand-binding specificity of the human estrogen receptor alpha (hER alpha) so that it would recognize a non-steroidal synthetic compound with structural similarities to the phytoestrogen resveratrol. For this purpose, we performed iterative rounds of site-specific saturation mutagenesis of a fixed set of ligand-contacting residues and subsequent random mutagenesis of the entire ligand-binding domain. Selection of the receptor mutants and quantification of the interaction were carried out by exploiting a yeast two-hybrid system that reports the ligand-dependent interaction between hER alpha and steroid receptor coactivator-1 (SRC-1). The screen was performed with a synthetic ligand (CV3320) that promoted growth of the reporter yeast strain to half maximal levels at a concentration of 3.7 microM. The optimized receptor mutant (L384F/L387M/Y537S) showed a 67-fold increased activity to the synthetic ligand CV3320 (half maximal yeast growth at 0.055 microM) and a 10-fold decreased activity to 17beta-estradiol (E2; half maximal yeast growth at 4 nM). The novel receptor-ligand pair partially fulfills the requirements for a specific 'gene switch' as it responds to concentrations of the synthetic ligand which do not activate the wildtype receptor. Due to its residual responsiveness to E2 at concentrations (4 nM) that might occur in vivo, further improvements have to be performed to render the system applicable in organisms with endogenous E2 synthesis.
Subject(s)
Directed Molecular Evolution , Estrogen Receptor alpha/metabolism , Estradiol/metabolism , Estrogen Receptor alpha/chemistry , Estrogen Receptor alpha/genetics , Histone Acetyltransferases/metabolism , Humans , Ligands , Mutagenesis, Site-Directed , Mutation , Nuclear Receptor Coactivator 1 , Protein Engineering , Resveratrol , Stilbenes/metabolism , Transcription Factors/metabolismABSTRACT
A library of 17 novel estrogen analogues 3 and 4 containing different substituents at rings A and D (steroid nomenclature) was prepared in a five- to seven-step synthesis. The key transformation is a Sonogashira-coupling of cyclic vinyl iodides of type 7 or 8 with phenylacetylenes of type 9. Reduction of the keto function in 3 led to the estradiol analogue 5.
Subject(s)
Estradiol/analogs & derivatives , Estradiol/chemical synthesis , Estrone/analogs & derivatives , Estrone/chemical synthesis , Small Molecule Libraries/chemical synthesis , Estradiol/chemistry , Estrone/chemistry , Molecular Conformation , StereoisomerismABSTRACT
A novel enantioselective total synthesis of the oral contraceptive desogestrel (2) is described, in which the tetracyclic steroid core is formed by a sequence of two consecutive Heck reactions. Conversion of the known enantiopure diketone 7 led to the chiral bicycle 6 which was used for a diastereoselective intermolecular Heck reaction with vinyliodide 5 to give 15. In the following intramolecular Heck reaction, the tetracyclic ring system was formed to give 4, from which the synthesis of desogestrel (2) was furnished.
