New inhibitors of glucosylceramide synthase and their effect on cell fate

Glucosylceramide (GlcCer) is an essential glycosylated lipid found in organisms ranging from fungi to mammals. It is composed of a hydrophilic β-linked glucose and a hydrophobic ceramide, with a predominant content of sphingosine in mammals (d18:1). GlcCer is the precursor of a large scale of different glycosphingolipids. This cerebrozide is synthesized from uridine diphosphate-glucose and ceramide by a GlcCer synthase (UDP-glucose:ceramide glucosyltransferase; UGCG, EC 2.4.1.80). GlcCer-based sphingolipids have been identified as important mediators of a variety of cellular functions and their disequilibrium leads to pathological process development and may induce several diseases progression. Therefore, design of UGCG inhibitor represents an important topic for pharmaceutical research. In this paper, we aimed to study effects of newly synthesized derivatives of (±)-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, known UGCG inhibitor) on: i) activity of UGCG in vitro; ii) thymocytes viability; iii) calcium transport through plasma membrane of thymocytes; iv) induction of apoptosis and autophagy in thymocytes. Thymocytes were isolated from thymus of three to seven weeks old mice (ICR strain). The key factors influencing the effect of PPMP analogues were their concentration, chemical structure and incubation time. Derivatives were able to change Ca²⁺ transport already after 15 min of cultivation, but their effects on cell viability were manifested at least after 12 h of cultivation. Four from fifteen studied compounds affected UGCG activity after four hour lasting cultivation, - but without correlation with data relating to effects on calcium transport and/or cell viability. Most potent UGCG inhibitor was chosen and applied for induction of apoptosis and autophagy in thymocytes. This inhibitor induced typical DNA fragmentation and upregulation of LC3B protein as autophagy marker, after 2 h and 4 h cultivation, respectively.