A team from ETH Zurich and Karolinska Institutet has developed a cross-linking MALDI mass spectrometry workflow that captures both functional response and target binding in a single assay.
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Drug screening has traditionally been split between functional assays and binding assays. However, neither tells the full story. Functional assays indicate whether a drug works, but not how, while binding assays indicate whether a drug binds, but not what effect the binding has. This information gap is one factor driving the high failure rate of drug candidates; clinical efficacy accounts for 40% to 50% of clinical failures. This is especially true for protein-protein interactions (PPIs), which lack well-defined binding sites.
How cross-linking unlocks a new capability
Conventional MALDI-MS is primarily an identification and measurement tool used to measure the masses of proteins, widely used for enzyme activity assays and quality control. However, it can’t reliably detect intact protein-protein complexes. This is because the laser ionization process used to measure the proteins is strong enough to break apart the weak, noncovalent forces that hold proteins together. The new method adds a chemical cross-linking step. An NHS-ester reagent is added to the protein-drug mixture. This creates covalent bonds between two proteins wherever they are in contact, locking the complex together. After the complex is “locked”, it is analyzed by mass spectrometry, delivering a double output of both functional response and target binding.
The researchers screened 17 FDA-approved drug candidates that target the SARS-CoV-2 spike protein’s receptor-binding domain (RBD) and ACE2, the protein the virus binds to in order to enter human cells. They found that two compounds that looked virtually identical by conventional assays, amentoflavone and dalbavancin, had critical differences. Dalbavancin binds to ACE2 with approximately 10-fold stronger affinity and shows preferential, on-target engagement, while amentoflavone has weaker and less specific binding.
A cell-based antiviral assay confirmed what MALDI showed. Dalbavancin significantly improved cell viability in SARS-CoV-2-infected cells, while amentoflavone showed no benefit, and even had mild toxicity at higher concentrations.
Implications and limitations
The richer data provided by this method could help researchers make smarter decisions about which compounds to advance and which to cut, saving valuable time and resources. The potential for this extends beyond inhibitors. The platform could also identify molecular stabilizers and allosteric activators of protein complexes.
Some limitations do exist. The binding parameters are semi-quantitative. Absolute binding affinities may be underestimated due to laser-induced dissociation during the MALDI process. While this proof-of-concept is exciting, further validation across other PPI targets is still needed.
Additionally, cross-linking requires amine-free buffers. Currently, the authors envision MALDI as best positioned for post-primary screening when candidate pools have already been narrowed.
Filed Under: Drug Discovery
Tagged With: ACE2, allosteric activators, amentoflavone, antiviral, binding affinity, binding assay, cell viability, clinical efficacy, clinical failure, covalent bonds, cross-linking, dalbavancin, drug candidates, drug discovery, Drug Screening, ETH Zürich, functional assay, high-throughput screening, Karolinska Institutet, laser-induced dissociation, MALDI-MS, mass spectrometry, molecular stabilizers, NHS-ester, noncovalent forces, post-primary screening, protein-protein interactions, RBD, receptor-binding domain, SARS-CoV-2, semi-quantitative