Discovery of deoxyceramide analogs as highly selective ACER3 inhibitors in live cells

Area of Science:

• Biochemistry
• Medicinal Chemistry
• Oncology

Background:

• Ceramidases, including acid (AC), neutral (NC), and alkaline ceramidase 3 (ACER3), are crucial enzymes with therapeutic potential in cancer treatment.
• Existing ceramidase inhibitors primarily target AC and NC, leaving a gap in well-characterized ACER3 inhibitors.
• Developing specific inhibitors for ACER3 is important for combination cancer therapies.

Purpose of the Study:

• To synthesize and screen novel 1-deoxy(dihydro)ceramide analogs for inhibitory activity against AC, NC, and ACER3.
• To evaluate the potency and selectivity of these analogs in various experimental settings, including cell lysates and intact cells.
• To identify selective ACER3 inhibitors for potential therapeutic applications.

Main Methods:

• Synthesis of two series of 1-deoxy(dihydro)ceramide analogs.
• Enzyme activity assays using fluorogenic substrates with recombinant human NC (rhNC) and cell lysates/intact cells enriched in AC, NC, and ACER3.
• Determination of inhibition constants (K_i) and half-maximal inhibitory concentrations (IC₅₀).

Main Results:

• No significant AC inhibitory activity was observed for any analogs.
• Several analogs showed non-competitive inhibition of rhNC with K_i values between 1-5 μM, but lost potency in cell lysates and intact cells.
• Series 2 compounds demonstrated dose-dependent inhibition of ACER3 in both cell lysates and intact cells (IC₅₀ ≈ 20 μM).
• Active Series 2 compounds increased ceramide levels in live cells, confirming ACER3 inhibition.

Conclusions:

• The study identified selective ACER3 inhibitors effective in intact cells.
• These findings provide a foundation for medicinal chemistry efforts to develop more potent and specific ACER3 inhibitors.
• The identified compounds represent promising leads for novel cancer therapeutic strategies targeting ACER3.