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).

This presentation will outline a strategy for discovering and characterizing non-orthosteric ligands of the phosphatase domain of PTPN22. We opted for a fragment screen to identify ligands of PTPN22 and utilized a multidisciplinary approach to characterize them. This included the integration of experimental data-driven molecular dynamics when co-crystallization of fragments with PTPN22 was unsuccessful. With this approach we identified and progressed fragments that bind PTPN22 at two novel non-orthosteric sites.

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.

BCL2A1, a member of the BCL-2 anti-apoptotic protein family, is overexpressed in severalhematologic malignancies and solid tumors. To identify a novel inhibitor of BCL2A1, we initiated a DEL screen. This screen identified a promising molecular scaffold featuring a unique but ultimately intractable reversible covalent bond with a buried lysine. Leveraging early covalent fragment-type mass spectrometry-based assays, we developed a new covalent lead that irreversibly engages a critical cysteine.

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.

Molecular glues modulate protein proximity, yet have resisted function-first screening.  Here, we present a direct-to-biology approach which can distinguish glues from non-glues. Using high-throughput synthesis and affinity-selection mass spectrometry, we can identify a molecular glue from within 20,000 reaction mixtures. Orthogonal assays confirm gluing behavior. Our results outline a roadmap for de novo glue discovery via kinetic profiling of unpurified small molecules against protein pairs.

By removing the synthesis bottleneck associated with traditional hit-to-lead optimization, high-throughput synthesis and direct-to-biology (D2B) screening accelerates the discovery of drug candidates with first-in-class structures and mechanisms. Building on the success of D2B implemented at Carmot Therapeutics, we present Kimia’s ATLAS platform, a next-generation D2B and direct-to-ADME (D2A) platform that integrates multiple chemistries and machine learning to guide the search for novel drugs, exemplifying the approach with several case studies.