Identifying the structure-activity relationship of leelamine necessary for inhibiting intracellular cholesterol transport

Abstract

Leelamine is an anticancer chemotherapeutic agent inhibiting intracellular cholesterol transport. Cell death mediated by leelamine occurs due to the lysosomotropic property of the compound, its accumulation in the lysosome, and inhibition of cholesterol transport leading to lack of availability for key processes required for functioning of cancer cells. The present study dissects the structure-activity-relationship of leelamine using synthesized derivatives of leelamine and abietic acid, a structurally similar compound, to identify the moiety responsible for anti-cancer activity. Similar to leelamine, all active derivatives had an amino group or a similar moiety that confers a lysosomotropic property to the compound enabling its accumulation in the lysosome. Active derivatives inhibited intracellular cholesterol transport and hindered xenografted melanoma tumor development without obvious systemic toxicity. In silico studies suggested that active derivatives accumulating in lysosomes bound to NPC1, a protein responsible for cholesterol export from the lysosome, to inhibit its activity that then caused accumulation, and lack of cholesterol availability for other key cellular activities. Thus, active derivatives of leelamine or abietic acid maintained lysosomotropic properties, bound to NPC1, and disrupted cellular cholesterol transport as well as availability to retard tumor development.

Keywords: AKT; abietic acid; leelamine; melanoma; structure-activity relationship.

Figure 1. Schemes used for synthesizing abietic acid (Scheme 1 & 2) and leelamine (Scheme 3) derivative compounds

Figure 2. Lysosomotropism mediated by the amino group of leelamine is needed for vacuolization in cancer cell death

(A and B) Lysosomotropism was measured by assessing the induction of cellular vacuolization by light microscopy of UACC 903 cells following treatment with active and inactive derivatives of abietic acid and leelamine alone or in combination with BafA1. (C and D) Viability of melanoma cells was assessed by MTS assay of cells treated with active derivatives 4a, 4b, 4c, 5a, 5b and 5e in the absence or presence of V-ATPase inhibitor BafA1 or β-cyclodextrin, which functions to remove cholesterol from the cells.

Figure 3. Leelamine and abietic acid derivatives containing an amino-group-like-moiety inhibited autophagic flux and cellular endocytosis

(A) Active derivatives dose dependently induced accumulation of p62 and LC3B proteins indicating inhibition of autophagic flux in a manner similar to that of control leelamine. (B) Receptor mediated uptake of Alexa Fluor 488 conjugated transferrin protein, visualized by fluorescence miscopy, was inhibited with active derivatives in a manner similar to that occurring with leelamine. Abietic acid and DMSO vehicle served as a negative control.

Figure 4. Active derivatives were capable of lysosomotropism and interacted with NPC1 in a manner similar to that of cholesterol and leelamine

(A) Visualization of the predicted docking of cholesterol, leelamine and active derivatives in the cholesterol-binding pocket of NPC1 protein (PDB ID: 3GKI). (B) Two-dimensional interaction of cholesterol, leelamine and active derivatives in the cholesterol-binding pocket of NPC1.

Figure 5. Active derivatives inhibit three key signaling pathways regulating melanoma development

(A–C) Treatment with 3 to 5 μmol/L of active derivatives for 24 hours decreased PI3K/Akt (A), STAT3 (B) and MAPK (C) pathways in the UACC 903 melanoma cell line. 30–50 μmol/L treatments with of inactive derivatives did not affect the signaling cascades. Alpha-enolase served as a control for equal protein loading.

Figure 6. Amino group containing active derivative 4a of abietic acid inhibits melanoma tumor development with negligible toxicity

(A) Active abietic acid derivative 4a inhibits melanoma tumor development by an average of 55%. Athymic nu/nu mice were s.c. injected with UACC 903 melanoma cells and six days later, treated orally daily with leelamine or compound 4a. 10% PEG was administered as vehicle control (4 mice/group; 2 tumors/mouse). Inset (A) No significant difference was observed in the body weight of mice following 24-days of treatment, indicating negligible toxicity. (B) Levels of blood biomarkers used to indicate major organ related toxicity, suggested negligible toxicity at the doses evaluated.