Hit and lead generation by fragment-based methods has become an established approach which is now routinely applied alongside complementary methods in early drug discovery, with ¹⁹F NMR-based methods proven to be of particular utility as they offer unique advantages. In this presentation, recent developments which increase the feasibility of assessing large and chemically diverse libraries by ¹⁹F NMR as well as case studies of difficult-to-drug targets will be discussed.

Identification of fragment-binding positions on the surface of macromolecules is a key to locating druggable sites, assessing the druggability of novel targets, and developing starting points for finding new chemical entities. Computational hot-spot mapping was performed across a set of known or potential drug targets, and a novel approach for clustering was employed to select the top druggable sites, including those at active and allosteric sites. Within this context we assessed the utility of experimentally-determined vs. AlphaFold-generated models. Some of the latest techniques such as FTMove and its use with AlphaFold models (AI generated protein models) will be described.

Rapid synthon-based screening approaches like V-SYNTHES have shown practical utility in hit and lead discovery for many clinically relevant targets, however, like any structure-based method it is limited to the structurally well-defined binding pockets. Here, we explore the new approaches to incorporate experimental information obtained in classical fragment screening into the V-SYNTHES pipeline to discover potent lead-like and drug-like ligands for cryptic pockets usually considered undruggable.

A screen of 17k fragments identified 3 classes on NNMT inhibitors. One of the classes were turnover inhibitors which are substrates of the enzyme. We characterized this inhibitory mechanism in detail using a newly developed surface biosensor methodology that quantify enzymatic turnover. Systematic medicinal chemistry resulted in identification of more potent, extremely ligand efficient, inhibitors, for which we were able to verify the inhibitory mechanism in vivo.

Fragment-based drug design presents a unique opportunity to identify allosteric footholds for therapeutic targets that have resisted conventional active-site-focused drug discovery. We leveraged crystallographic fragment screening and machine learning to discover binders and inhibitors at the allosteric L16 site of PTP1B. We characterized these and existing ligands using multiple biophysical assays, including HDX-MS. The results include a variety of responses in protein dynamics, including surprisingly diverse and widespread effects on conformational dynamics. Alongside complementary work on fragment screening with room-temperature crystallography, these results emphasize the powerful effects fragments and lead compounds have on protein structure and dynamics.

The rational search for covalent drugs has typically relied on the development of potent reversible binders, followed by the appending of an electrophilic warhead. Recently, a distinct approach involving the screening of covalent fragment-sized molecules has proved to be a viable method for the discovery of hits against previously intractable targets, including PPIs. This talk describes how AstraZeneca are pioneering the use of the electrophile-first approach for covalent drug discovery.

Astex has pioneered the application of structure-based approaches in drug discovery. I present a case study where crystal engineering was used to trap a therapeutic target protein kinase in distinct conformational states and deliver novel soakable crystal systems. This allowed the characterization of binding modes and mechanism-of-action of covalent and non-covalent compounds currently in the clinic. The combined results identified strategies to dial-out off-target effects and improve ligand selectivity.

We developed the covalent alternatives of Astex's MiniFrags that allow mapping potential binding sites for covalent inhibitors. Covalent MiniFrags are 5- and 6-membered electrophilic heterocycles that covalently bond at their binding site. Screening hits can be identified by simple biochemical assay, and the binding site can be located by mass spectrometry. The utility of this methodology was demonstrated by discovering the first leadlike covalent inhibitor of HDAC8.

This study demonstrates a novel method employing data from HDX experiments to guide small molecules into cryptic pockets to crystallographic resolution with a success rate greater than 85%. A cryptic pocket is a binding site that requires the ligand in order to exist. These pockets elude successful interrogation by computational techniques, such as virtual screening and present a considerable challenge for drug discovery. The method will be described using a dynamic kinase with solved X-ray structures reflecting many binding modes before demonstrating its utility on a fragment-based drug discovery program, alongside critical aspects of the HDX experiments that enable this work.

I'll present how we program microbes to build small-molecule modulators that bind to cryptic pockets. We use these pockets to guide the discovery of novel hits in drug-like chemical space. Our pocket-finding probes are sp3-rich and largely nonpolar; our final hits are soluble, drug-like, and amenable to rapid chemical elaboration.

An important challenge for ligase-based targeted protein degradation (TPD) is identifying new ligands for existing ligases. Because ubiquitin ligases are usually part of a multi-subunit protein that contains one or more binding partners, hit-finding assays need to differentiate binding locations. To identify new chemotypes for the VHL and cereblon ligases, we used various hit finding methods including fragment screening. I will describe our results and the complexities we encountered.

A fragment-based screening strategy was employed to identify allosteric binders for tau tubulin kinase 1 (TTBK1). Several hit classes identified by leveraging biophysical, computational, and crystallographic approaches were prioritized based on the biophysical profile, potential ligandability, and potential of binding site for inhibitory selectivity. The identified allosteric pockets and corresponding fragment hits will be discussed with regard to their potential and early elaboration to provide kinome selectivity for TTBK1.