A three-stage biophysical screening cascade for fragment-based drug discovery.
Nat Protoc
; 8(11): 2309-24, 2013 Nov.
Article
in En
| MEDLINE
| ID: mdl-24157549
ABSTRACT
This protocol describes the screening of a library of low-molecular-weight compounds (fragments) using a series of biophysical ligand-binding assays. Fragment-based drug discovery (FBDD) has emerged as a successful method to design high-affinity ligands for biomacromolecules of therapeutic interest. It involves detecting relatively weak interactions between the fragments and a target macromolecule using sensitive biophysical techniques. These weak binders provide a starting point for the development of inhibitors with submicromolar affinity. Here we describe an efficient fragment screening cascade that can identify binding fragments (hits) within weeks. It is divided into three stages (i) preliminary screening using differential scanning fluorimetry (DSF), (ii) validation by NMR spectroscopy and (iii) characterization of binding fragments by isothermal titration calorimetry (ITC) and X-ray crystallography. Although this protocol is readily applicable in academic settings because of its emphasis on low cost and medium-throughput early-stage screening technologies, the core principle of orthogonal validation makes it robust enough to meet the quality standards of an industrial laboratory.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Drug Discovery
Type of study:
Diagnostic_studies
/
Guideline
/
Screening_studies
Language:
En
Journal:
Nat Protoc
Year:
2013
Document type:
Article
Affiliation country:
United States