Effect of Miltefosine on membrane restructuring events during the enzymatic generation of ceramide or diacilglierol

ZULUETA DÍAZ, YENISLEIDY DE LAS MERCEDES; FANANI, M. LAURA

Virtual

Simposio; IUPAB2021: Young Scientists Program Webinar Series.; 2021

IUPAB (International Union for Pure and Applied Biophysics)

Alkylphosphocholines such as Miltefosine (HePC), are natural phospholipids analogues. These compounds onstitutes a new class of antineoplastic agents which exerts their cytostatic effect by interacting with lipid membranes and afecting membrane-dependent processes. We hypothesize that HePC could exert its pharmacological function by affecting the action of phospholipases indirectly by modulating membrane properties. Therefore, we study the effect of HePC on changes either at the level of enzymatic activity or on the changes that the formation of products has on the physical properties of those membranes.In the present study, we used large unilamellar vesicles (LUVs) composed of DOPC (PLC substrate): DOPE or SM16 (SMase substrate): DOPE in the absence and presence of HePC. In these systems, the activity of both enzymes was determined by thin plate chromatography (TLC) analysis. Simultaneously, we studied the change in turbidity of the LUV suspensions by spectrophotometry and the changes induced in the particle size distribution by Dynamic Light Dispersion (DLS). Our results indicated that the presence of HePC did not affect the initial hydrolysis rate of DOPC or SM16 (or the production of DAG or Cer) at short times. However, in the case of PLC, the addition of 20 mol % of HePC inhibited the restructuring of membranes, characteristic of the DAG enzymatic formation process. A drastic decrease in vesicle aggregation, DAG formation and generation of smaller LUVs populations compared to the control was observed at long times (40 min). These results show for the first time that HePC is able to inhibit the enzymatic action and therefore the aggregation induced by the PLC in a remarkable way. In the case of SMase, a modest effect on vesicle restructuring was observed.In general, results obtained appear not to be related to a direct enzyme inhibition or interaction of HePC with the substrates or products of the reaction but with a non-specific geometric compensation of HePC with the membrane components. HePC has an effective cone-shaped geometry, which when inserted in bilayers can induce a positive curvature counteracting the effect caused by DAG. Therefore, these results gain relevance in the understanding of the mechanism of action by which the HePC regulates the lipid metabolism and signal transduction pathways in which these enzymes are involve.Abbreviations: DOPC: dioleoylphosphatidylcholine; DOPE: dioleoylphosphatidylethanolamine; DAG: diacyl glycerol; Cer: ceramide; Hexadecylphosphocholine: HePC.