Covalent fragment screening enables rapid discovery of next-generation therapeutics by unlocking targets once considered undruggable. Covalent drugs form stable covalent bonds with nucleophilic residues on the target proteins, delivering potent and long-lasting therapeutic activity. 

The FDA has recently approved several covalent drugs, such as tyrosine kinase inhibitors (TKIs) for cancer, sparking growing interest in this modality^1-3.

Covalent fragment screening makes this possible by targeting 98% of disease-modifying proteins once considered "undruggable"⁴. These small molecules (under 300 Da) access shallow, cryptic, and allosteric binding sites of challenging proteins that traditional drug discovery approaches cannot reach.

Electrophilic reactive groups, called ‘warheads’, are added to fragments to promote covalent interactions⁵. It is important for warheads to be tailored to each fragment to optimise activity and selectivity of the fragment with the target protein. 

When the fragments are ready, they are incubated with the target and screened to test ligand-target interactions. These interactions can be characterized by mass spectrometry (MS) or activity-based protein profiling (ABPP). 

The emergence of activity-based protein profiling (ABPP) is enhancing the accuracy, sensitivity, and translatability of covalent fragment screening. This method uses a library of residue-based chemical probes (RBPs) labeled with fluorescent or affinity tags. Stable isotope labeling by amino acids in cell culture (SILAC) is also commonly used in ABPP workflows. In this step, nucleophilic residues in human cells are labeled with non-radioactive isotopes, allowing for the precise detection of covalent interactions.

After incubating isotope-labeled cells with fragment libraries, data-independent acquisition (DIA)-MS can be used to analyze target-probe binding. This technique allows covalent fragments to be screened against the entire proteome, providing a comprehensive map of target reactive sites and occupancy (Figure 1). This data serves as a crucial starting point to assess probe selectivity and target druggability.

ABPP offers three key advantages that are important for accelerated covalent fragment screening (Figure 2). The three advantages are:

1. No protein purification required:

In ABPP, fragments are screened against targets in their native cellular environment, meaning protein purification is not required. Membrane proteins, protein complexes, and intrinsically disordered proteins, the "undruggable" targets that stump traditional methods, are suddenly accessible. Workflows become faster, simpler, and more biologically relevant.

1. Sensitivity that catches weak binders

High accuracy and sensitivity of DIA-MS detect low-abundance peptides and transient interactions. ABPP ensures promising hits don't slip through the cracks.

1. Real cellular context:

Isotope labelling allowed probe binding to be screened in native cellular contexts. This means fragment binding is tested in the environment where drugs need to work, providing a more holistic picture of target binding. 

We aim to expand the druggable proteome using our high-throughput ABPP (HT-ABPP) platform for covalent fragment screening. Our approach turns challenging targets into drug discovery opportunities, combining proteome-wide screening with functional validation. 

We partner with pharmaceutical and biotechnology teams to expand the druggable proteome through collaborative covalent fragment programs.

Our advanced DIA-MS analysis maps over 87,000 reactive cysteine sites across 15,800+ unique proteins and 12,000+ lysine sites on 3,500+ proteins. 

With the capacity to process up to 60 samples per day in a wide range of human cell lines and primary cells, we accelerate your fragment screening timelines. Furthermore, our sensitive DIA-MS analysis catches weak binders that traditional methods miss. Hits are validated through dose-dependent competition assays that quantify reactivity, selectivity, and ligandability, providing the confidence you need to advance fragments. 

We work alongside your team to run parallel phenotypic screens and binding assays to identify functional covalent ligands and their corresponding targets. Testing in live cells means hits are validated in biologically relevant conditions, improving clinical translatability and reducing downstream risk. 

With 25+ years of HTS expertise and 750 completed campaigns, Evotec accelerates covalent fragment programs from hit identification to lead optimization, even for targets previously considered "undruggable."

Targeting the "undruggable"? Discover how Evotec's covalent fragment screening services can identify novel starting points for your challenging targets. 

1. Cameron F, Sanford M. Ibrutinib: First Global Approval. Drugs. 2014;74(2):263-271. doi:10.1007/s40265-014-0178-8
2. Koch AL, Vellanki PJ, Drezner N, et al. FDA Approval Summary: Osimertinib for Adjuvant Treatment of Surgically Resected Non-Small Cell Lung Cancer, a Collaborative Project Orbis Review. Clin Cancer Res. 2021;27(24):6638-6643. doi:10.1158/1078-0432.CCR-21-1034
3. Nakajima EC, Drezner N, Li X, et al. FDA Approval Summary: Sotorasib for KRAS G12C-Mutated Metastatic NSCLC. Clin Cancer Res. 2022;28(8):1482-1486. doi:10.1158/1078-0432.CCR-21-3074
4. Coleman N, Rodon J. Taking Aim at the Undruggable. Am Soc Clin Oncol Educ Book. 2021;41:e145-e152. doi:10.1200/EDBK_325885
5. Keeley A, Petri L, Ábrányi-Balogh P, Keserű GM. Covalent Fragment Libraries in Drug Discovery. Drug Discov Today. 2020;25(6):983-996. doi:10.1016/j.drudis.2020.03.016