Fragment-Based Drug Discovery

Small molecules are vital for studying and therapeutically targeting membrane proteins, but conventional screening often requires labeled ligands or purified proteins and misses weak binders. We demonstrate a high-resolution magic angle spinning (HRMAS) NMR approach using unpurified membranes containing the A2A adenosine receptor. Using STD-NMR, we rapidly detected and characterized novel fragment binders, revealing distinct binding poses and establishing HRMAS NMR as a useful tool for fragment-based drug discovery.

The development of “binding-first” strategies to identify chemical matter for G protein–coupled receptors (GPCRs) is increasingly important in drug discovery, particularly for orphan GPCRs where functional activity remains unknown. Advances in solubilization techniques have mitigated some of the challenges associated with working on detergent-solubilized targets. Leveraging ^19F-NMR and surface plasmon resonance (SPR), we successfully identified and optimized fragment binders for an orphan GPCR.

Directly targeting the translation initiation factor eIF4F complex has long been considered a promising anticancer strategy but it has remained ‘undruggable.' The eIF4F complex is made up of the eukaryotic initiation factors 4E (eIF4E), 4G (eIF4G) and 4A (eIF4A). Astex applied NMR and X-ray crystallographic fragment screening to discover a novel binding site on eIF4E. Subsequent structure-guided design paired with targeted protein degradation and genetic rescue approaches, enabled the development and the functional characterisation of potent small molecule inhibitors of the eIF4E:eIF4G protein-protein interaction (PPI).

I will explain the journey we underwent at FoRx towards the discovery and optimization of new inhibitors of a DNA repair target considered as undruggable. We  performed an X-ray based fragment screen leading to the discovery of new chemical starting point binding to the sub-optimal DNA binding pocket of the protein. After optimization, we ended up with the first covalent specific inhibitor of this target active in cells.

Binding sites available at protein-protein interfaces were mapped by a screening concept that combines evolutionary optimized fragment pharmacophores with the use of photoaffinty handles that enables high hit rates by LC-MS detection. Screening our library against challenging targets such as the small GTPase KRASG12D, the transcription factor STAT5B and the E3ligase FBXW7 we have discovered tractable binding sites that were characterized by MS-based peptide mapping, structural studies and modeling. These results revealed the our protocol outperforms screening traditional PPI-targeted libraries in better exploration of available binding sites and higher hit rates observed for even difficult targets.

The fragment-based drug discovery (FBDD) process begins with the identification of low affinity, low molecular weight protein-binding fragments. Subsequently, these fragments are elaborated and/or joined (if they recognize adjacent pockets on the target) to derive higher affinity leads. In this lecture, we describe a multi-step, FBDD-like workflow for the discovery of protein-binding macrocycles using bead-displayed libraries and a novel function-based screening platform. In this scheme, each position of the macrocycle is optimized for protein binding independently while the others are held invariant. This provides an alternative to creating very large libraries of macrocycles in which each position is varied simultaneously.

DNA-encoded libraries, in the setup of Encoded Self-Assembing Chemical (ESAC) libraries, featuring the stable self-assembly of two DNA-encoded sub-libraries of fragments, allow for the identification of pairs of simultaneously binding fragments. In a second step, the most efficient linkage of individual fragment pairs will be obtained by selections of a second, DNA-encoded library of linkers (Linker-DEL), displaying the respective fragment pair.

A case study shown here is screening with ASC protein, a critical adapter protein in inflammasome activation, making it a promising target for autoimmune disorders. With its PYD and CARD domains providing potential PPI interfaces for drug design, our strategy targets ASC inhibition by disrupting poly-filament formation through covalent binding at Cys173 on its CARD domain. The comprehensive efforts from primary screening, hit validation to fragment-to-lead expansion will be presented.

Covalent drug discovery benefits from early access to quantitative kinetic information, yet existing approaches often limit throughput and complicate interpretation. New methodologies integrating fragment-based discovery with kinetic analysis are described. A mass spectrometry–based diagonal dose–response time-course (dDRTC) enables practical measurement of kinact/KI for covalent fragments and leads. Surface plasmon resonance (SPR) supports later-stage discovery by providing high-resolution characterization of covalent engagement to guide lead optimization.

DNA encoded focused library is a powerful technology for the hit identification and hit to lead optimization of the specific therapeutic target which could effectively improve the success rate of the DEL campaigns. The integration between FBDD and DNA Encoded Libraries could provide an efficient way to design the focused DEL based on the privileged fragments of corresponding target identified by diverse screening methods. This lecture will disclose the detailed examples to integrate DEL with FBDD for reversible and covalent inhibitors discovery. The future direction for the FBDD and DEL integration will also be discussed.