In recent years methodological and technical advancements have enabled NMR-based fragment screening to be performed in full automation and with significantly reduced consumption of fragments and unlabeled target protein. Ligand observed NMR can not only be used as a primary screening tool but also to confirm hits from other screening methods such as Thermal Shift, which results in high quality hits. Furthermore, all experiments can be performed as competition experiments in which a known binder is displaced and the Kd is determined by titration. Here we present practical aspects on how to setup ligand observed NMR screening experiments in an automated fashion for the three basic experiments:
This thesis will first discuss the method of FBLD and compare it to more traditional drug discovery approaches, such as high-throughput screening HTS.
After establishing the concepts behind FBLD, the applications of utilizing a FBLD approach towards the development of inhibitors for select metalloenzymes is discussed. A study into the use of a chelator fragment library CFL to identify new metal-binding groups MBG for metalloprotein inhibitors is discussed.
The results of screening this library against a number of metalloenzymes identified important trends between classes of chelators in addition to the identification of numerous metalloenzyme specific MBGs. Furthermore this study provides evidence that the CFL can be used as useful tool in metalloenzyme inhibitor design.
A second probes the types of key interactions a metal-binding inhibitor must posses to effectively inhibit the dinuclear metalloenzyme HIV-1 integrase HIV-1 IN. A small library of compounds varying only in composition of their MBG was prepared.
Screening this library of compounds led to the identification of a potentially unique and more potent scaffold hydroxypyrone -based compounds for HIV-1 IN inhibitors. Additionally, a small sublibrary of compounds was developed to explore the effects of manipulating the pKa of the chelator.
Lastly, the discovery of two novel metal-binding scaffolds for the development Pseudomonas aeruginosa elastase LasB inhibitors is discussed. Further use of the above mentioned CFL, helped to identify the first nonpeptidic small molecule inhibitors of LasB.
These compounds, in addition to displaying selective antagonism for LasB against a panel of similar metalloenzymes, also confirmed the role the enzyme plays in the swarming behavior of this organism Main Content.Fragment screening: an introduction A. R. Leach, M. M. Hann, J. N. Burrows diagrams etc.
contained in this article in third party publications or in a thesis or dissertation provided that the correct acknowledgement is given with the reproduced material. Weak interactions between RhoGDI2 and fragment screening hits were delineated using an integrated NMR approach.
General Interests Binders to RhoGDI2 as a potential anti -cancer target have been first reported, and their weak interactions were depicted using NMR spectroscopy. reports the construction of plasmids for qualitative screening assay for 35S promoter and NOS terminator as GMO elements, and relative quantification assays in Maize suggestions and comments during the study and my thesis defense.
Restriction Enzyme Digestion of PCR Amplified Target Fragments.. 27 Conditions for. Fragment-based drug discovery is a validated approach for the discovery of drug candidates. However, the weak affinity of fragment compounds requires highly sensitive biophysical techniques, such as nuclear magnetic resonance (NMR) or X-ray crystallography, to identify hits.
Thus the advantages of screening small fragment libraries are partly offset by the high cost of biophysical analyses. Search for new antiviral compounds using fragment screening methodology Search for new antiviral compounds using fragment screening methodology Thesis submitted by Zuzanna Kaczmarska, enrolled in the Biotechnology program at the University of .
The optimized in silico screening approach was successfully applied to identify a chemically diverse set of novel fragment-like (≤ 22 heavy atoms) H1R ligands with an exceptionally high hit rate of 73%.