Discovery and Development of a Novel mPGES-1/5-LOX Dual Inhibitor LFA-9 for Prevention and Treatment of Chronic Inflammatory Diseases

Abstract

Background: Non-steroidal anti-inflammatory drugs, cyclooxygenase (COX)-2 selective inhibitors, have been explored for prevention and treatment of several inflammatory chronic conditions including arthritis, and cancer. However, the long-term use of these drugs is associated with gastrointestinal, renal, and cardiovascular side effects. Later, COX/5-lipoxygenase (5-LOX) dual inhibitors (eg, licofelone) have been developed but did not enter into the market from the clinical trails due to COX-1/2 inhibition-associated side effects. Hence, targeting microsomal prostaglandin E synthase-1 (mPGES-1) and 5-LOX can be an ideal approach while sparing COX-1/2 activities for development of the next generation of anti-inflammatory drugs with better efficacy and safety.

Materials and methods: In silico (molecular modelling) studies were used to design a mPGES-1/5-LOX dual inhibitory and COX-1/2 sparing lead molecule licofelone analogue-9 (LFA-9) by modifying the pharmacophore of licofelone. In vitro cell-free enzymatic (mPGES-1, 5-LOX, COX-1/2) assays using fluorometric/colorimetric methods and cell-based assays (LPS-induced PGE₂, LTB₄, and PGI₂ productions from macrophages) using ELISA technique, isothermal calorimetry, and circular dichroism techniques were performed to determine the mPGES-1/5-LOX inhibitory efficacy and selectivity. Anti-inflammatory efficacy of LFA-9 was evaluated using a carrageenan (inflammogen)-induced rat paw edema model. Infiltration/expression of CD68 immune cells and TNF-α in paw tissues were evaluated using confocal microscope and immunoblot analysis. Anti-cancer effect of LFA-9 was evaluated using colon spheroids in vitro.

Results: LFA-9 inhibited mPGES-1/5-LOX and their products PGE₂ and LTB₄, spared COX-1/2 and its product PGI₂. LFA-9 bound strongly with human mPGES-1/5-LOX enzymes and induced changes in their secondary structure, thereby inhibited their enzymatic activities. LFA-9 inhibited carrageenan-induced inflammation (70.4%) in rats and suppressed CD68 immune cell infiltration (P ≤ 0.0001) and TNF-α expression. LFA-9 suppressed colon tumor stemness (60.2%) in vitro through inhibition of PGE₂ (82%) levels.

Conclusion: Overall study results suggest that LFA-9 is a mPGES-1/5-LOX dual inhibitor and showed anti-inflammatory and colorectal cancer preventive activities, and warranted detailed studies.

Keywords: LFA-9; anti-inflammatory agent; cancer chemoprevention; drug design; mPGES-1/5-LOX dual inhibitor.

Figure 1

(A) Arachidonic acid pathway and its inhibitors in clinical use for inflammatory and oncological diseases. (B) Chemical structures of licofelone (i) and LFA-9 (ii).

Figure 2

Molecular docking studies represent binding interaction of LFA-9 (i) with three dimensional structures of mPGES-1 (A) (PDB ID: 4YL3), 5-LOX (PDB ID: 3O8Y) (B), COX-1 (modelled) (C), and COX-2 (PDB ID: 5KIR) (D) in comparison with licofelone (ii). Highlighted square area in white color (ribbon model) represented the interacting amino acids in three-dimensional structures.

Figure 3

mPGES-1 and 5-LOX dual inhibitory activity of LFA-9 in a cell-free system. (A). Effect of LFA-9 and licofelone on human (i), rat (ii), mouse (iii) specific mPGES-1 (A), 5-LOX (B), COX-1 (C), COX-2 (D) and on an in vitro cell-free system. IC₅₀ values were calculated based on the dose-response curve vs log inhibitor concentration though variable slope (four parameters) of non-linear regression analysis.

Figure 4

PGE₂ and LTB₄ inhibitory, and PGI₂ sparing effects of LFA-9 in a cell-based system. Effect of LFA-9 on PGE₂ (i) and LTB₄, (ii) and PGI₂ (iii) production in LPS-stimulated human (A), rat (B), and mouse (C) macrophages (cell-based assays). IC₅₀ values were calculated based on the dose-response curve vs log inhibitor concentration though variable slope (four parameters) of non-linear regression analysis. P-values ≤0.05 for data sets were considered significant.

Figure 5

Anti-inflammatory activity of LFA-9. Paw thickness (A) and percent inhibition (B) using inflammogen (carrageenan (CAR))-induced rat paw edema. (C) Effect of LFA-9 on PGE₂, LTB₄, PGI_2, and TXB₂ levels in exudates of carrageenan (CAR))-induced rat paw edema. (D) Effect of LFA-9 on expression of CD68 and TNF-α in CAR-injected paw tissue using immunoblot analysis. (E and F) Effect of LFA-9 on CD68-positive inflammatory cell infiltration and TNF-α expression in CAR-injected paw tissue sections using immunofluorescence technique. P-values ≤0.05 for data sets were considered significant. (NS) Not significant (P>0.05); *(P ≤ 0.05); **(P ≤ 0.01); *** (P ≤ 0.001); **** (P ≤ 0.0001). (G) Schematic representation of mechanism of action of LFA-9 for its anti-inflammatory activity with safety.

Figure 6

LFA-9 prevents colorectal cancer stemness. The effect of LFA-9 and licofelone on colon tumor stemness and colonic spheroids formation of APC^pirc rats in vitro (A (i and ii)) and its inhibitory effect on PGE₂ production (B) in its culture supernatants. (C) Schematic representation of mechanism of action of LFA-9 for its preventive efficacy of colon tumor stemness. P-values ≤0.05 for data sets were considered significant. Not significant (P>0.05); *(P ≤ 0.05); **(P ≤ 0.01); **** (P ≤ 0.0001).