Accelerating ionizable lipid discovery for mRNA delivery using machine learning and combinatorial chemistry.

To unlock the full promise of messenger (mRNA) therapies, expanding the toolkit of lipid nanoparticles is paramount. However, a pivotal component of lipid nanoparticle development that remains a bottleneck is identifying new ionizable lipids. Here we describe an accelerated approach to discovering effective ionizable lipids for mRNA delivery that combines machine learning with advanced combinatorial chemistry tools. Starting from a simple four-component reaction platform, we create a chemically diverse library of 584 ionizable lipids. We screen the mRNA transfection potencies of lipid nanoparticles containing those lipids and use the data as a foundational dataset for training various machine learning models. We choose the best-performing model to probe an expansive virtual library of 40,000 lipids, synthesizing and experimentally evaluating the top 16 lipids flagged. We identify lipid 119-23, which outperforms established benchmark lipids in transfecting muscle and immune cells in several tissues. This approach facilitates the creation and evaluation of versatile ionizable lipid libraries, advancing the formulation of lipid nanoparticles for precise mRNA delivery.

Combinatorial development of nebulized mRNA delivery formulations for the lungs.

Inhaled delivery of mRNA has the potential to treat a wide variety of diseases. However, nebulized mRNA lipid nanoparticles (LNPs) face several unique challenges including stability during nebulization and penetration through both cellular and extracellular barriers. Here we develop a combinatorial approach addressing these barriers. First, we observe that LNP formulations can be stabilized to resist nebulization-induced aggregation by altering the nebulization buffer to increase the LNP charge during nebulization, and by the addition of a branched polymeric excipient. Next, we synthesize a combinatorial library of ionizable, degradable lipids using reductive amination, and evaluate their delivery potential using fully differentiated air-liquid interface cultured primary lung epithelial cells. The final combination of ionizable lipid, charge-stabilized formulation and stability-enhancing excipient yields a significant improvement in lung mRNA delivery over current state-of-the-art LNPs and polymeric nanoparticles.

Oleic acid is an endogenous ligand of TLX/NR2E1 that triggers hippocampal neurogenesis.

Neural stem cells, the source of newborn neurons in the adult hippocampus, are intimately involved in learning and memory, mood, and stress response. Despite considerable progress in understanding the biology of neural stem cells and neurogenesis, regulating the neural stem cell population precisely has remained elusive because we have lacked the specific targets to stimulate their proliferation and neurogenesis. The orphan nuclear receptor TLX/NR2E1 governs neural stem and progenitor cell self-renewal and proliferation, but the precise mechanism by which it accomplishes this is not well understood because its endogenous ligand is not known. Here, we identify oleic acid (18:1ω9 monounsaturated fatty acid) as such a ligand. We first show that oleic acid is critical for neural stem cell survival. Next, we demonstrate that it binds to TLX to convert it from a transcriptional repressor to a transcriptional activator of cell-cycle and neurogenesis genes, which in turn increases neural stem cell mitotic activity and drives hippocampal neurogenesis in mice. Interestingly, oleic acid-activated TLX strongly up-regulates cell cycle genes while only modestly up-regulating neurogenic genes. We propose a model in which sufficient quantities of this endogenous ligand must bind to TLX to trigger the switch to proliferation and drive the progeny toward neuronal lineage. Oleic acid thus serves as a metabolic regulator of TLX activity that can be used to selectively target neural stem cells, paving the way for future therapeutic manipulations to counteract pathogenic impairments of neurogenesis.

Discovery of small molecules targeting the tandem tudor domain of the epigenetic factor UHRF1 using fragment-based ligand discovery.

Despite the established roles of the epigenetic factor UHRF1 in oncogenesis, no UHRF1-targeting therapeutics have been reported to date. In this study, we use fragment-based ligand discovery to identify novel scaffolds for targeting the isolated UHRF1 tandem Tudor domain (TTD), which recognizes the heterochromatin-associated histone mark H3K9me3 and supports intramolecular contacts with other regions of UHRF1. Using both binding-based and function-based screens of a ~ 2300-fragment library in parallel, we identified 2,4-lutidine as a hit for follow-up NMR and X-ray crystallography studies. Unlike previous reported ligands, 2,4-lutidine binds to two binding pockets that are in close proximity on TTD and so has the potential to be evolved into more potent inhibitors using a fragment-linking strategy. Our study provides a useful starting point for developing potent chemical probes against UHRF1.

Publisher Correction: Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Synthesis of Enantiomerically Pure 5-Substituted Piperazine-2-Acetic Acid Esters as Intermediates for Library Production.

The piperazine heterocycle is housed within a large number of FDA-approved drugs and biological probe compounds. Structurally, however, these compounds are mostly confined to substitutions on the two ring nitrogen atoms, rationalizing the expansion of piperazine chemical diversity through carbon substitutions. On the basis of the concept of systematic chemical diversity, a divergent six-step synthesis was developed in which chiral amino acids were transformed, with high diastereoselectivity, into either cis or trans 5-substituted piperazine-2-acetic acid esters that could be chromatographically rendered diastereomerically homogeneous. Starting from six commercially available amino acids or their respective amino alcohols (both antipodes), we obtained a complete set of 24 protected chiral 2,5-disubstituted piperazines, as single stereoisomers in multigram quantities. These diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as starting materials for parallel library synthesis and as intermediates for the targeted production of more complex C-substituted piperazine compounds.

Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic ß-cells.

ß-cell proliferation induction is a promising therapeutic strategy to restore ß-cell mass. By screening small molecules in a transgenic zebrafish model of type 1 diabetes, we identified inhibitors of non-canonical IκB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε), as enhancers of ß-cell regeneration. The most potent ß-cell regeneration enhancer was a cinnamic acid derivative (E)-3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA), which, acting through the cAMP-dependent protein kinase A (PKA), stimulated ß-cell-specific proliferation by increasing cyclic AMP (cAMP) levels and mechanistic target of rapamycin (mTOR) activity. A combination of PIAA and cilostamide, an inhibitor of ß-cell-enriched cAMP hydrolyzing enzyme phosphodiesterase (PDE) 3, enhanced ß-cell proliferation, whereas overexpression of PDE3 blunted the mitogenic effect of PIAA in zebrafish. PIAA augmented proliferation of INS-1ß-cells and ß-cells in mammalian islets including human islets with elevation in cAMP levels and insulin secretion. PIAA improved glycemic control in streptozotocin (STZ)-induced diabetic mice with increases in ß-cell proliferation, ß-cell area, and insulin content in the pancreas. Collectively, these data reveal an evolutionarily conserved and critical role of TBK1/IKKε suppression in expanding functional ß-cell mass.

Synthesis of Enantiomerically Pure 3-Substituted Piperazine-2-acetic Acid Esters as Intermediates for Library Production.

The piperazine heterocycle is broadly exploited in FDA-approved drugs and biologically active compounds, but its chemical diversity is usually limited to ring nitrogen substitutions, leaving the four carbon atoms underutilized. Using an efficient six-step synthesis, chiral amino acids were transformed into 3-substituted piperazine-2-acetic acid esters as diastereomeric mixtures whose cis and trans products (dr 0.56 → 2.2:1, respectively) could be chromatographically separated. From five amino acids (both antipodes) was obtained a complete matrix of 20 monoprotected chiral 2,3-disubstituted piperazines, each as a single absolute stereoisomer, all but one in multigram quantities. In keeping with our overall purpose of constructing more Csp3-enriched compound libraries for drug discovery, these diverse and versatile piperazines can be functionalized on either nitrogen atom, allowing them to be used as scaffolds for parallel library synthesis and as intermediates for the production of novel piperazine compounds.