Eaton, S. A. | Search bioRxiv

bioRxiv2026

A family portrait of lanmodulin selectivity for enhanced rare-earth separations

Proteins offer a molecular design space to create bespoke ligands for the separation of critical metals, like rare earth elements (REs). However, data-intensive approaches to fine-tune metalloprotein selectivity are bottlenecked by the low-throughput nature of existing characterization methods. Here we invented an assay called SpyTag-Catcher Immobilization of Lanmodulin for Assaying Metal-Binding Selectivity (SpyCI-LAMBS) to measure metalloprotein selectivity en masse. This 96-format workflow was used to study the selectivity of 621 lanmodulin (LanM) orthologs for 15 REs, revealing eight distinct selectivity profiles based on sequence-to-function analyses. We discovered >200 LanMs with dampened selectivity for low-value LaIII relative to the prototypical LanM. This includes a LanM that can perform a challenging one-stage separation of PrIII from LaIII with up to >99.9 mol% purity and 83% yield. SpyCI-LAMBS is a powerful tool that can rapidly collect high-fidelity selectivity data to inform metal ion separations and machine learning-assisted metalloprotein design.

bioengineering↗

bioRxiv2025

Covalent Binding of Boronic Acid-Based Inhibitor GSK4394835A to Phosphodiesterase 3B, a Drug Target for Cardiovascular Disease

Boronic acids have become increasingly prominent in the pharmacopoeia ever since the US Food and Drug Administration approved Bortezomib (Velcade) for the treatment of multiple myeloma more than 20 years ago. Since then, four additional boronic acid-based drugs have been approved for clinical use in the treatment of cancer, fungal and bacterial infections, and eczema. The boron atom is inherently electrophilic due to being electron deficient, so the mode of inhibition by a boronic acid very often involves addition of a nucleophile, such as an enzyme-bound residue or a water molecule, to yield a tetrahedral boronate anion. Recently, a new class of boronic acid-based inhibitors of human phosphodiesterase 3B (PDE3B) have been reported, along with the 2.7 [A]-resolution crystal structure of PDE3B complexed with one of these inhibitors, GSK4394835A [Rowley et al. (2024) Discovery and SAR study of boronic acid-based selective PDE3B inhibitors from a novel DNA-encoded library. J. Med. Chem. 67, 2049-2065]. Surprisingly, the boronic acid moiety of the inhibitor was reported to bind as an intact, unreacted boronic acid. Additional discrepancies were evident in the reported refinement statistics, so we downloaded structure factor amplitudes from the Protein Data Bank (accession code 8SYC) and re-solved the structure. Our initial electron density maps clearly indicated nucleophilic addition of H737 to the boronic acid moiety of the inhibitor, generating a tetrahedral boronate anion in the active site. We re-refined the structure with excellent refinement statistics, concluding that GSK4394835A is a reversible covalent inhibitor of PDE3B.

biochemistry↗