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Acid sphingomyelinase (ASM), which regulates sphingolipid metabolism and lipid signaling, has been considered as a new potential target for the treatment of atherosclerosis. In this study, a series of benzene-heterocyclic-based ASM inhibitors were rationally designed, synthesized, and screened for the first time. As a result, some compounds showed favorable inhibitory activity against recombinant human ASM. The detailed SARs are also discussed. Compound 4i revealed good pharmacokinetic data and in vivo inhibitory activity against ASM by reducing the level of ceramide in mice plasma and liver. Pharmacodynamic studies confirmed that 4i could lessen lipid plaques in the aortic arch and aorta and reduce plasma ceramide concentration and Ox-LDL levels. Moreover, 4i was found to significantly decrease LPS-induced and Ox-LDL-induced cell inflammation by regulating the levels of ceramide and sphingomyelin. Overall, this study preliminarily demonstrates that ASM may be an effective target against atherosclerosis for the first time.

Measuring the activity of low-abundance enzymes, down to a few molecules in one living cell, is important but challenging to elucidate their biological function. Here, an electrochemical molecule trap is established at the tip of a nanopipette with an electrochemical detector, in which the diffusion of the molecules away from the electrochemical detector is prevented by electro-osmotic flow (EOF). Accordingly, a limited amount of enzymes is trapped to continuously catalyze the conversion of the substrate to generate a sufficient amount of the byproduct hydrogen peroxide for electrochemical measurements. The resistive pulse sensing of the enzymes in single liposomes validates the detection sensitivity down to 15 molecules. Using this ultrasensitive electrochemical strategy, the activity of 60 sphingomyelinase molecules inside single unstimulated living J774 cells is measured, which was hardly detected by previous methods. The established electrochemical molecule trap-based sensing approach opens the door toward single-molecule electrochemical detection in one living cell. This success will solve the long-standing problem regarding the study of the activity of low-abundance proteins in cells in their native physiological state and greatly enhance the understanding of the roles of proteins in cellular behavior.

Extracellular vesicles (EVs) can carry pathological cargo and play an active role in disease progression. Neutral sphingomyelinase-2 (nSMase2) is a critical regulator of EV biogenesis, and its inhibition has shown protective effects in multiple disease states. 2,6- D imethoxy-4-(5- p henyl-4- t hiophen-2-yl-1 H - i midazol-2-yl) p henol (DPTIP) is one of the most potent (IC 50 = 30 nM) inhibitors of nSMase2 discovered to date. However, DPTIP exhibits poor oral pharmacokinetics (PK), limiting its clinical development. To overcome DPTIP's PK limitations, we synthesized a series of prodrugs by masking its phenolic hydroxyl group. When administered orally, the best prodrug ( P18 ) with a 2',6'-diethyl-1,4'-bipiperidinyl promoiety exhibited >fourfold higher plasma (AUC 0- t = 1047 pmol·h/mL) and brain exposures (AUC 0- t = 247 pmol·h/g) versus DPTIP and a significant enhancement of DPTIP half-life (2 h vs ∼0.5 h). In a mouse model of acute brain injury, DPTIP released from P18 significantly inhibited IL-1β-induced EV release into plasma and attenuated nSMase2 activity. These studies report the discovery of a DPTIP prodrug with potential for clinical translation.

Sphingomyelinase (SMase) is closely related to diseases like Niemann-Pick disease and atherosclerosis, and the development of a simple method for the assay of SMase activity is very useful to screen new potential inhibitors or stimulators of SMase or biomarkers of disease. Fluorophore-encapsulated nanoliposomes (FENs) are emerging as a new fluorescent probe for sensing the enzymatic activity. In this work, two fluorochromes (cy7 and IR780) were encapsulated into the liposome of sphingomyelin, and therefore, a sphingomyelin-based ratiometric FEN probe for the SMase activity assay was constructed. The probe shows high selectivity and sensitivity to acid SMase with a detection limit of 4.8 × 10 -4 U/mL. Sphingomyelin is the natural substrate of SMase; therefore, the probe has native ability for all kinds of SMase activity assays. Moreover, the probe has been successfully applied to the analysis of acid SMase activity in cells and urine samples. As far as we know, this is the first example of a nanoliposome fluorescence method for assaying acid SMase, and the method is biocompatible and much simpler than the existing ones, which might provide a new strategy for developing new methods for other important esterases.

In rat sperm heads, sphingomyelin (SM) species that contain very long-chain polyunsaturated fatty acid (V-SM) become ceramides (V-Cer) after inducing in vitro the acrosomal reaction. The reason for such a specific location of this conversion, catalyzed by a sphingomyelinase (SMase), has received little investigation so far. Here, the effects of SMase were compared in unilamellar vesicles (large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs)) containing phosphatidylcholine, and either V-SM or a palmitate-rich SM (P-SM). In uniformly sized LUVs at 37 °C, more V-Cer was generated and more rapidly than P-Cer. Nephelometry and dynamic light scattering showed that LUVs tended to form large lipid particles more intensely, and Förster resonance energy transfer (FRET) increases suggested that lateral lipid mixing was more marked when V-Cer rather than P-Cer was produced. As reported by 6-dodecanoyl-2-dimethyl-aminopnaphthalene (Laurdan) and 1,6-diphenyl-1,3,5,-hexatriene (DPH), the production of V-Cer resulted in higher and faster restriction in lipid mobility than that of P-Cer, implying a stronger increase in membrane dehydration and microviscosity. Moreover, DPH anisotropy suggested a higher solubility of V-Cer than that of P-Cer in the liquid-disordered phase. At room temperature, liquid-condensed lateral domains appeared in P-SM- but not in V-SM-containing GUVs. The former maintained their size while losing their contents gradually during SMase action, whereas the latter became permeable earlier and reduced their size in few minutes until suddenly collapsing. The fast and potent generation of V-Cer may contribute to the membrane restructuring events that occur on the acrosome-reacted sperm head.

The formation of domains in supported lipid membranes has been studied extensively as a model for the 2D organization of cell membranes. The compartmentalization of biological membranes to give domains such as cholesterol-rich rafts plays an important role in many biological processes. This article summarizes experiments from the author's laboratory in which a combination of atomic force microscopy and near-field scanning optical microscopy is used to probe phase separation in supported monolayers and bilayers as models for membrane rafts. These techniques are used to study binary and ternary lipid mixtures that have gel-phase or liquid-ordered domains that vary in size from tens of nanometers to tens of micrometers, surrounded by a fluid-disordered membrane. Examples are presented in which these models are used to investigate the distribution of glycolipid membrane raft markers and the preference for peptide and protein localization in ordered versus fluid membrane phases. Finally, the enzyme-mediated restructuring of membranes containing liquid-ordered domains provides an in vitro model for the coalescence of membrane rafts to give signaling platforms. Overall, the results demonstrate the importance of using techniques that can probe the nanoscale organization of membranes and of combining techniques that yield complementary information. Furthermore, the ability of supported lipid bilayers to model some aspects of membrane compartmentalization provides an important approach to understanding natural membranes.