Virtual Screening Strategy and In Vitro Tests to Identify New Inhibitors of the Immunoproteasome

Area of Science:

• Medicinal Chemistry
• Biochemistry
• Pharmacology

Background:

• Immunoproteasome inhibition is a key strategy for treating hematological malignancies, autoimmune diseases, and inflammatory conditions.
• Non-covalent inhibitors present a promising alternative to existing covalent drugs, potentially reducing off-target toxicity.

Purpose of the Study:

• To biologically evaluate a collection of compounds identified through virtual screening for immunoproteasome inhibition.
• To identify novel non-covalent inhibitors of the immunoproteasome β1i and/or β5i catalytic subunits.

Main Methods:

• Virtual screening incorporating dynamic pharmacophore modeling and docking approaches.
• In vitro enzymatic assays to assess inhibition of β1i and β5i subunits.
• Tian continuous assays to determine dissociation constants (Ki) and binding affinities.

Main Results:

• Seven out of thirty-four screened compounds demonstrated inhibition of β1i and/or β5i subunits.
• Compound 3 showed potent inhibition of the β1i subunit (Ki = 11.84 ± 1.63 µM).
• Compound 17 exhibited significant inhibition of the β5i subunit (Ki = 12.50 ± 0.77 µM).
• Compound 2 displayed dual inhibitory activity against both β1i (Ki = 12.53 ± 0.18 µM) and β5i (Ki = 31.95 ± 0.81 µM) subunits.

Conclusions:

• The identified compounds represent promising starting points for developing non-covalent immunoproteasome inhibitors.
• Compound 2, as a dual inhibitor, is particularly interesting for further optimization towards new therapeutic agents.