Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine-1-phosphate (S1P) in cellular processes. Dihydroceramide desaturase (Des1), the last enzyme in the de novo synthesis of ceramide (Cer), regulates the balance between dihydroceramides (dhCers) and Cers. Both SKs and Des1 have interest as therapeutic targets. Here we show that SKI II is a noncompetitive inhibitor (K_i = 0.3 μM) of Des1 activity with effect also in intact cells without modifying Des1 protein levels. Molecular modeling studies support that the SKI II-induced decrease in Des1 activity could result from inhibition of NADH-cytochrome b5 reductase. SKI II, but not the SK1-specific inhibitor PF-543, provoked a remarkable accumulation of dhCers and their metabolites, while both SKI II and PF-543 reduced S1P to almost undetectable levels. SKI II, but not PF543, reduced cell proliferation with accumulation of cells in the G0/G1 phase. SKI II, but not PF543, induced autophagy. These overall findings should be taken into account when using SKI II as a pharmacological tool, as some of the effects attributed to decreased S1P may actually be caused by augmented dhCers and/or their metabolites.

Amino acids are of paramount importance as chiral building blocks of life, for drug development in modern medicinal chemistry, and for the manufacture of industrial products. In this work, the stereoselective synthesis of (S)- and (R)-2-amino-4-hydroxybutanoic acid was accomplished using a systems biocatalysis approach comprising a biocatalytic one-pot cyclic cascade by coupling of an aldol reaction with an ensuing stereoselective transamination. A class II pyruvate aldolase from E. coli, expressed as a soluble fusion protein, in tandem with either an S- or R-selective, pyridoxal phosphate dependent transaminase was used as a catalyst to realize the conversion, with formaldehyde and alanine being the sole starting materials. Interestingly, the class II pyruvate aldolase was found to tolerate formaldehyde concentrations of up to 1.4 M. The cascade system was found to reach product concentrations for (S)- or (R)-2-amino-4-hydroxybutanoic acid of at least 0.4 M, rendering yields between 86% and >95%, respectively, productivities of >80 g L^–1 d^–1, and ee values of >99%.