{"id":5822,"date":"2025-12-04T11:14:47","date_gmt":"2025-12-04T11:14:47","guid":{"rendered":"https:\/\/www.sygnaturediscovery.com\/?post_type=case-study&p=5822"},"modified":"2026-02-05T16:56:50","modified_gmt":"2026-02-05T16:56:50","slug":"covalent-fragment-screening","status":"publish","type":"case-study","link":"https:\/\/www.sygnaturediscovery.com\/fr\/case-study\/covalent-fragment-screening\/","title":{"rendered":"Covalent Fragment Screening of a Challenging Protein Target Using X-ray Crystallography"},"content":{"rendered":"\n

In this article\u00a0Jijin Ayanath<\/a>\u00a0describes how we at Sygnature were able to successfully identify covalent binders to a structurally challenging target. This was achieved through the development of a robust crystal soaking system, that allowed the screening of our focused covalent fragment library.<\/h2>\n\n\n\n

Fragment-based drug discovery (FBDD) has gained prominence as an effective strategy for initiating drug discovery, in recent years. FBDD uses smaller molecules (fragments) that explore a diverse chemical space, unlike the traditional High-Throughput screening (HTS) methods, where larger more complex molecules are screened. Fragments typically exhibit weak binding affinities in the micromolar to millimolar range and have low molecular weights (100 \u2013 300 Da). With technological advancements, methods such as Surface Plasmon Resonance (SPR), Thermal shift assays, NMR and X-ray crystallography are being used to detect these weak interactions. Although crystallographic screening lacks direct evaluation of Kd, what sets it apart from the rest is the ability to visualize fragment binding to a target at atomic level. The details of the specificity and interactions between the fragment and residues in the target protein obtained through crystallography provides a compelling foundation for iterative optimization of drug candidates.<\/p>\n\n\n\n

Covalent Fragments at Sygnature Discovery<\/h3>\n\n\n\n

Covalent fragments offer unique advantages by binding irreversibly through their electrophilic war heads to nucleophilic residues such as cysteine, serine, threonine, lysine etc. on protein targets. Covalent fragment screening using crystallography can yield specific hits to proteins deemed \u2018undruggable\u2019. The hits identified may be further optimized for enhancing selectivity and binding affinity, thereby serving as promising lead candidates for drug discovery.<\/p>\n\n\n\n

Our curated covalent fragment screen library at Sygnature Discovery has approximately 400 covalent fragments which were hand-picked from multiple commercial libraries and filtered using Rule-of-3 with focus on Cysteine-specific war heads. Many of the fragments have acrylamide warheads, frequently found in approved covalent drugs. Solubility analysis of the 405 fragments was performed using Dynamic Light Scattering (DLS) instrument. For each sample, Normalized intensity of Static Light Scattering (SLS) was measured at the desired concentration in the crystal-soaking buffer, allowing us to choose the best soluble 300 fragments for soaking crystals.<\/p>\n\n\n\n

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Figure 1: A)<\/strong>\u00a0A data table showing the normalized intensity readings collected for a few samples. A visual image rating: green, yellow and red corresponding to if there is no precipitate, light precipitate or heavy precipitate in the sample is also given.\u00a0B)<\/strong>\u00a0The Normalized intensity values are plotted, and soluble fragments are selected for fragment screening based on their values and visual image rating.<\/em><\/figcaption><\/figure>\n\n\n\n

Screening covalent fragments<\/h3>\n\n\n\n

Our client\u2019s protein target posed significant challenges as it lacked deep pockets for inhibitor binding and had multiple binding partners with overlapping protein-protein interaction sites. However, the presence of numerous cysteine residues and the need for highly specific inhibition made it ideal target for covalent fragment screening. The screening process involves soaking protein crystals in high concentrations of covalent fragments and diffracting them to generate electron density maps, which reveal the presence of bound fragments. Robust, reproducible and good diffraction-quality crystals are required in large numbers to successfully carry out a fragment screening experiment. The client provided pure protein as well as the initial crystallization conditions. The crystals were reproduced at 8\u00a0o<\/sup>C in multiple conditions in 96-well plates. The crystallization set-up was optimized to generate enough crystals for conducting a fragment screen. Roughly ~ 1000 crystals are required for completing a 300-fragment screen. A soaking strategy was also developed by testing DMSO tolerance of the crystals by both visual inspection and diffraction experiments. Additional factors such as, the pH of the soaking buffer and its salt concentration were optimized. Once the strategy was in place, soaking was done, and 300 crystals were flash-frozen (one crystal\/fragment).<\/p>\n\n\n\n

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