Kehrein, J. | Search bioRxiv

bioRxiv2026

Exploring lipid nanoparticle design spaces using self-regulating microfluidic machines and multiplexed in vivo biodistribution

Delivering therapeutic mRNA relies on lipid nanoparticles (LNPs). Finding optimal process parameters for new lipid combinations in LNP formulations remains a challenge. In our work, we used an automated, self-regulating microfluidic platform that actively changes process parameters to tune LNP formulations for preset, desired quality standards. We tested new LNPs by swapping poly(ethylene glycol) (PEG) lipopolymers for alternatives based on poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx). For each lipopolymer variant, the platform independently identified optimal production conditions in four or fewer iterative cycles, yielding particles of the preset size and high mRNA encapsulation. Small-angle X-ray scattering revealed that smaller LNPs modified with PEtOx had more structural surface variety and higher mRNA loading efficiency. When multiplexing these formulations in mice, the PEtOx-containing LNPs accumulated more in bone marrow compared to those with PEG, indicating trends that the chemistry of the lipopolymer affects the biodistribution of the resulting LNPs. By combining automated formulation and in vivo multiplexed testing, our approach provides a practical way to rapidly plan, formulate, and evaluate large pharmaceutical design spaces, to select excipients and process parameters yielding optimal biological performance of LNPs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/729611v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1a08b68org.highwire.dtl.DTLVardef@120724corg.highwire.dtl.DTLVardef@12fed98org.highwire.dtl.DTLVardef@197aa41_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2022

Fragment screening using biolayer interferometry reveals ligands targeting the SHP-motif binding site of the AAA+ ATPase p97

Biosensor techniques have become increasingly important for fragment-based drug discovery during the last years. Here, we describe a biolayer interferometry-based fragment screen targeting the AAA+ ATPase p97, an essential protein with key roles in protein homeostasis and a possible target for cancer chemotherapy. Currently available p97 inhibitors target its ATPase activity and globally impair p97-mediated processes. In contrast, inhibition of cofactor binding to the N-domain by a protein-protein-interaction inhibitor would enable the selective targeting of specific p97 functions. We demonstrate that a region known as SHP-motif binding site can be targeted with small molecules. Guided by molecular dynamics simulations, the binding sites of selected screening hits were postulated and experimentally validated using protein- and ligand-based NMR techniques, as well as X-ray crystallography, ultimately resulting in the first structure of a small molecule in complex with the N-domain of p97. The identified fragments provide insights into how this region could be targeted and present first chemical starting points for the development of a protein-protein interaction inhibitor preventing the binding of selected cofactors to p97.

biochemistry↗