A method for the trifluoromethylation of arylsilanes is reported. The reaction proceeds with [(phen)CuCF3 ] as the CF3  source under mild, oxidative conditions with high functional-group compatibility. This transformation complements prior trifluoromethylation of arenes in several ways. Most important, this method converts arylsilanes formed by the silylation of aryl C-H bonds to trifluoromethylarenes, thereby allowing the conversion of arenes to trifluoromethylarenes. The unique capabilities of the reported method are demonstrated by the conversion of a C-H bond into a C-CF3 bond in active pharmaceutical ingredients which do not undergo this overall transformation by alternative functionalization processes, including a combination of borylation and trifluoromethylation.

A method for the oxidative coupling of arylsilanes with nitrogen nucleophiles is reported. This method occurs with a broad range of heptamethyltrisiloxylarenes and nitrogen nucleophiles, proceeds with the arylsilane as limiting reagent, and does not require a fluoride activator with electron-poor arylsilanes. The combination of this method with C-H silylation generates arylamines from unactivated arenes with site selectivity controlled by steric effects. This combination of steps gives direct access to many compounds that cannot be accessed via alternative C-H functionalization methods, including direct C-H amination or the combination of C-H borylation and amination.

KRAS mutations are one of the most common oncogenic drivers in human cancer. While small molecule inhibitors for the G12C mutant have been successfully developed, allele-specific inhibition for other KRAS hotspot mutants remains challenging. Here we report the discovery of covalent chemical ligands for the common oncogenic mutant K-Ras(G12R). These ligands bind in the Switch II pocket and irreversibly react with the mutant arginine residue. An X-ray crystal structure reveals an imidazolium condensation product formed between the α,β-diketoamide ligand and the ε- and η-nitrogens of arginine 12. Our results show that arginine residues can be selectively targeted with small molecule electrophiles despite their weak nucleophilicity and provide the basis for the development of mutant-specific therapies for K-Ras(G12R)-driven cancer.

We report on photolipid doping of giant unilamellar vesicles (GUVs) via vesicle fusion with small unilamellar photolipid vesicles (pSUVs), which enables retroactive optical control of the membrane properties. We observe that vesicle fusion is light-dependent, if the phospholipids are neutral. Charge-mediated fusion involving anionic and cationic lipid molecules augments the overall fusion performance and doping efficiency, even in the absence of light exposure. Using phosphatidylcholine analogs with one or two azobenzene photoswitches (azo-PC and dazo-PC) affects domain formation, bending stiffness, and shape of the resulting vesicles in response to irradiation. Moreover, we show that optical membrane control can be extended to long wavelengths using red-absorbing photolipids (red-azo-PC). Combined, our findings present an attractive and practical method for the precise delivery of photolipids, which offers new prospects for the optical control of membrane function.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Protein-membrane interactions (PMIs) are ubiquitous in cellular signaling. Initial steps of signal transduction cascades often rely on transient and dynamic interactions with the inner plasma membrane leaflet to populate and regulate signaling hotspots. Methods to target and modulate these interactions could yield attractive tool compounds and drug candidates. Here, we demonstrate that the conjugation of a medium-chain lipid tail to the covalent K-Ras(G12C) binder MRTX849 at a solvent-exposed site enables such direct modulation of PMIs. The conjugated lipid tail interacts with the tethered membrane and changes the relative membrane orientation and conformation of K-Ras(G12C), as shown by molecular dynamics (MD) simulation-supported NMR studies. In cells, this PMI modulation restricts the lateral mobility of K-Ras(G12C) and disrupts nanoclusters. The described strategy could be broadly applicable to selectively modulate transient PMIs.

Dopaminergic neurons innervate extensive areas of the brain and release dopamine (DA) onto a wide range of target neurons. However, DA release is also precisely regulated. In Drosophila melanogaster brain explant preparations, DA is released specifically onto α3/α'3 compartments of mushroom body (MB) neurons that have been coincidentally activated by cholinergic and glutamatergic inputs. The mechanism for this precise release has been unclear. Here we found that coincidentally activated MB neurons generate carbon monoxide (CO), which functions as a retrograde signal evoking local DA release from presynaptic terminals. CO production depends on activity of heme oxygenase in postsynaptic MB neurons, and CO-evoked DA release requires Ca²⁺ efflux through ryanodine receptors in DA terminals. CO is only produced in MB areas receiving coincident activation, and removal of CO using scavengers blocks DA release. We propose that DA neurons use two distinct modes of transmission to produce global and local DA signaling.SIGNIFICANCE STATEMENT Dopamine (DA) is needed for various higher brain functions, including memory formation. However, DA neurons form extensive synaptic connections, while memory formation requires highly specific and localized DA release. Here we identify a mechanism through which DA release from presynaptic terminals is controlled by postsynaptic activity. Postsynaptic neurons activated by cholinergic and glutamatergic inputs generate carbon monoxide, which acts as a retrograde messenger inducing presynaptic DA release. Released DA is required for memory-associated plasticity. Our work identifies a novel mechanism that restricts DA release to the specific postsynaptic sites that require DA during memory formation.

Sphingosine-1-phosphate (S1P) plays important roles as a signaling lipid in a variety of physiological and pathophysiological processes. S1P signals via a family of G-protein-coupled receptors (GPCRs) (S1P_1-5) and intracellular targets. Here, we report on photoswitchable analogs of S1P and its precursor sphingosine, respectively termed PhotoS1P and PhotoSph. PhotoS1P enables optical control of S1P_1-3, shown through electrophysiology and Ca²⁺ mobilization assays. We evaluated PhotoS1P in vivo, where it reversibly controlled S1P₃-dependent pain hypersensitivity in mice. The hypersensitivity induced by PhotoS1P is comparable to that induced by S1P. PhotoS1P is uniquely suited for the study of S1P biology in cultured cells and in vivo because it exhibits prolonged metabolic stability compared to the rapidly metabolized S1P. Using lipid mass spectrometry analysis, we constructed a metabolic map of PhotoS1P and PhotoSph. The formation of these photoswitchable lipids was found to be light dependent, providing a novel approach to optically probe sphingolipid biology.

Lysophosphatidic acid (LPA) is a phospholipid that acts as an extracellular signaling molecule and activates the family of lysophosphatidic acid receptors (LPA_1-6). These G protein-coupled receptors (GPCRs) are broadly expressed and are particularly important in development as well as in the nervous, cardiovascular, reproductive, gastrointestinal, and pulmonary systems. Here, we report on a photoswitchable analogue of LPA, termed AzoLPA, which contains an azobenzene photoswitch embedded in the acyl chain. AzoLPA enables optical control of LPA receptor activation, shown through its ability to rapidly control LPA-evoked increases in intracellular Ca²⁺ levels. AzoLPA shows greater activation of LPA receptors in its light-induced cis-form than its dark-adapted (or 460 nm light-induced) trans-form. AzoLPA enabled the optical control of neurite retraction through its activation of the LPA₂ receptor.

The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family.