WAC™ Fragment Screening Technology: Principles & Advantages

Fragment library screening in drug discovery aims to identify a molecule (small organic molecule) or a series of compounds that can bind to a drug target and modulate its activity. Several biophysical methods support fragment screening, with X-ray crystallography and NMR spectroscopy among the most common. About eight years ago, SARomics Biostructures and RG Discovery introduced weak affinity chromatography (WAC™) fragment screening services. The technology is a version of affinity chromatography that detects weak-affinity ligand binding to a target molecule (see image below). It originated from academic research by Prof. Sten Ohlson and his co-workers.

Unlike traditional affinity chromatography, where the molecule of interest is tightly bound to the immobilized target and subsequently eluted, for example, with a drastic pH shift or an ionic strength change, WAC™ offers a gentler approach. A mixture of small-molecular-weight fragments or drug-like compounds is injected into a standard high-performance liquid chromatography (HPLC) column packed with silica, onto which the protein target has been immobilized. During elution, fragments with higher affinity for the protein stay on the column longer than those with lower affinity. Although small fragments usually bind weakly, with Kd values often in the mM range, modern methods such as mass spectrometry or UV spectroscopy enable efficient detection and classification of binders. With its ability to simultaneously identify weak binders (mM) in complex mixtures, WAC™ fragment screening technology has proven to be an efficient and low-cost choice compared to other fragment screening methods, e.g., surface plasmon resonance (SPR), X-ray crystallography, NMR spectroscopy, affinity selection-mass spectrometry (AS-MS), or thermal shift assay (TSA).

The efficiency of WAC has been validated in our laboratories in over 50 projects we have run on around 20 distinct target classes over the years since its commercial implementation. Among the advantages we can highlight:

• Physiological buffer as a mobile phase
• Label-free detection by MS (enables built-in quality control)
• Straightforward detection of retention times from which Kd can be calculated
• Potential for high-throughput screening (up to 5000 fragments per week)
• Low material consumption (<5 mg protein)
• mM hits can be identified by screening fragments at low concentrations (1-5 µM)
• Immediate hit ranking and control of hit quality
• High hit rates from 1-20% (avg 6%)

WAC fragment-based drug discovery projects run by SARomics Biostructures & RG Discovery included screening for novel fragment hits as project starting points, assessing the druggability of new targets, ranking of fragment hits identified by X-ray crystallographic fragment screening (XFS), finding differentiated backups in mature projects, and as a rescuing mode for challenging targets (PPIs, etc.). Target classes included kinases, GTPases, cytokine/protein-protein interactions (PPIs), bromodomains, integrins, helicases/ATPases, transcription factors, deubiquitinases, ubiquitin ligases, and many others.

After initial hit identification, follow-up activities may include fragment hit expansion using our integrated fragment-based drug discovery platform, built on our joint capabilities in medicinal chemistry, computational chemistry, X-ray crystallography, biophysical validation, biochemical and cell-based assays, and in vitro ADME. Protein NMR spectroscopy and protein X-ray crystallography can verify compound binding and gain insights into the interactions with the target protein. In addition, the WAC™ column lasts several months and can be reused to rapidly rank acquired or synthesized analogs.

WAC Use Case Studies

In a recent case study, we used WAC fragment screening technology to study ligand binding to interleukin-23 (IL-23). The joint SARomics Biostructures & Red Glead Discovery team ran the project and reported it at the Drug Discovery Chemistry conference in San Diego (April 2024). IL-23 belongs to a class of inflammatory proteins called cytokines, which mediate signal transduction between cells. Monoclonal antibody inhibitors of IL-23 that block its action have been approved for the clinical treatment of inflammation, and an orally administered small-molecule inhibitor of the IL-23 pathway has the potential to provide significant benefits for patients with autoimmune inflammatory disorders. The reported case study used a library of 1013 fragments for the WAC™ screening. With a 2% hit rate, 19 fragments were identified, and six were nominated for co-crystallization with the target. Two complex structures at resolutions of 2.7 Å and 2.95 Å were obtained. Although the two fragments do not block biologically relevant receptor interactions, analyzing their binding modes provided valuable insights for further expanding the molecules to design efficient inhibitors.

Another case study involved screening the transcription regulator BRG1 (SMARCA4). This protein is part of the large chromatin remodeling complex SWI/SNF, which is involved in transcriptional gene activation. A ligandability screen with 250 compounds gave 20 hits. Five crystal structures of complexes with ligands were solved, and a new ligand binding site was identified (for details, see poster).

Our publications page provides many examples of our contributions to clients’ projects. Our methods and knowledge center provides an overview of structure-based drug design strategies.

WAC fragment screening offer

We are unlocking the power of WAC fragment screening technology for a broader audience with our new, more affordable package deals! We’re excited to offer more accessible options, including a new fixed-price package with a comprehensive WAC™ screen of 500 fragments, plus analog search, selection, and WAC testing. WAC™ is also ideal for ranking your weak affinity hits from XFS. Please contact us to learn more!