Bioactive sphingolipids: Advancements and contributions from the laboratory of Dr. Lina M. Obeid.

Sphingolipids and their synthetic enzymes have emerged as critical mediators in numerous diseases including inflammation, aging, and cancer. One enzyme in particular, sphingosine kinase (SK) and its product sphingosine-1-phosphate (S1P), has been extensively implicated in these processes. SK catalyzes the phosphorylation of sphingosine to S1P and exists as two isoforms, SK1 and SK2. In this review, we will discuss the contributions from the laboratory of Dr. Lina M. Obeid that have defined the roles for several bioactive sphingolipids in signaling and disease with an emphasis on her work defining SK1 in cellular fates and pathobiologies including proliferation, senescence, apoptosis, and inflammation.

Molecular dynamics of conformation-specific dopamine transporter-inhibitor complexes.

The recreational psychostimulant cocaine inhibits dopamine reuptake from the synapse, resulting in excessive stimulation of postsynaptic dopamine receptors in brain areas associated with reward and addiction. Cocaine binds to and stabilizes the outward- (extracellular-) facing conformation of the dopamine transporter (DAT) protein, while the low abuse potential DAT inhibitor benztropine prefers the inward- (cytoplasmic-) facing conformation. A correlation has been previously postulated between psychostimulant abuse potential and preference for the outward-facing DAT conformation. The 3ß-aryltropane cocaine analogs LX10 and LX11, however, differ only in stereochemistry and share a preference for the outward-facing DAT, yet are reported to vary widely in abuse potential in an animal model. In search of the molecular basis for DAT conformation preference, complexes of cocaine, benztropine, LX10 or LX11 bound to each DAT conformation were subjected to 100ns of all-atom molecular dynamics simulation. Results were consistent with previous findings from cysteine accessibility assays used to assess an inhibitor's DAT conformation preference. The respective 2ß- and 2α-substituted phenyltropanes of LX10 and LX11 interacted with hydrophobic regions of the DAT S1 binding site that were inaccessible to cocaine. Solvent accessibility measurements also revealed subtle differences in inhibitor positioning within a given DAT conformation. This work serves to advance our understanding of the conformational selectivity of DAT inhibitors and suggests that MD may be useful in antipsychostimulant therapeutic design.

Contact Pin-Printing onto Porous Silicon for Creating Microarrays with High Chemical Diversity.

We explore the size and spatial microheterogeneity of contact pin-printed spots formed on porous silicon (pSi). Glycerol was contact printed at room temperature onto as-prepared, hydrogen-passivated pSi (ap-pSi) using 50 or 200 µm diameter solid pins. The pSi was then subjected to a strong oxidizing environment (gaseous O3) and washed to remove the glycerol masks. The glycerol-free regions were converted to oxidized pSi (ox-pSi); the glycerol-coated regions were protected from O3, but not entirely. The final array is described as circularly shaped "ap-pSi" regions on a field of ox-pSi. When comparing the areas outside and inside the glycerol-masked pSi spots, one finds dramatic differences in the Si-O-Si, SiHx (x = 1-3) and OySiHx (y, x = 1-3) levels with a spatially dependent continuum of compositions across the spot diameter. Experimental conditions could be adjusted to tune the final ap-pSi spot diameter and edge widths from 90 µm to 520 µm and 20 µm to 130 µm, respectively. The resulting ap-pSi spot diameter is explained by using molecular kinetic theory and time-dependent glycerol imbibement into the pSi within a one-dimensional Darcy's law model.

2-Substituted 3ß-Aryltropane Cocaine Analogs Produce Atypical Effects without Inducing Inward-Facing Dopamine Transporter Conformations.

Previous structure-activity relationship studies indicate that a series of cocaine analogs, 3ß-aryltropanes with 2ß-diarylmethoxy substituents, selectively bind to the dopamine transporter (DAT) with nanomolar affinities that are 10-fold greater than the affinities of their corresponding 2α-enantiomers. The present study compared these compounds to cocaine with respect to locomotor effects in mice, and assessed their ability to substitute for cocaine (10 mg/kg, i.p.) in rats trained to discriminate cocaine from saline. Despite nanomolar DAT affinity, only the 2ß-Ph2COCH2-3ß-4-Cl-Ph analog fully substituted for cocaine-like discriminative effects. Whereas all of the 2ß compounds increased locomotion, only the 2ß-(4-ClPh)PhCOCH2-3ß-4-Cl-Ph analog had cocaine-like efficacy. None of the 2α-substituted compounds produced either of these cocaine-like effects. To explore the molecular mechanisms of these drugs, their effects on DAT conformation were probed using a cysteine-accessibility assay. Previous reports indicate that cocaine binds with substantially higher affinity to the DAT in its outward (extracellular)- compared with inward-facing conformation, whereas atypical DAT inhibitors, such as benztropine, have greater similarity in affinity to these conformations, and this is postulated to explain their divergent behavioral effects. All of the 2ß- and 2α-substituted compounds tested altered cysteine accessibility of DAT in a manner similar to cocaine. Furthermore, molecular dynamics of in silico inhibitor-DAT complexes suggested that the 2-substituted compounds reach equilibrium in the binding pocket in a cocaine-like fashion. These behavioral, biochemical, and computational results show that aryltropane analogs can bind to the DAT and stabilize outward-facing DAT conformations like cocaine, yet produce effects that differ from those of cocaine.