Abstract 6364: LFA-9, a dual mPGES-1 and 5-LOX inhibitor, suppresses colon cancer stemness and inflammogen-induced inflammatory response
Authors: Yarla Nagendra Sastri , Pathuri Gopal , Gali Hariprasad , Madka Venkateshwar , Panneerselvam Janani , Chandrakesan Parthasarathy , Houchen Courtney W. , Glaze Elizabeth R. , Fox Jennifer , McCormick David , Rao Chinthalapally V. 
Published: September 2020

Chronic inflammation triggers tumorigenesis of the colorectum; hence several non-steroidal anti-inflammatory drugs (NSAIDs) have been explored for prevention and treatment of inflammatory diseases, including colorectal cancer (CRC). Although traditional NSAIDs and COX-2 inhibitors are effective chemopreventive agents for CRC, unwanted side effects (gastrointestinal and renal) hinder their use. Mechanistic studies suggest that the side effects are in part due to a COX-2 inhibition-associated increase in 5-lipoxygenase (5-LOX) metabolites and reduced PGI2 levels. Hence, agents targeting microsomal prostaglandin synthase-1 (mPGES-1) and 5-LOX inhibitors may prove to be more effective and safer agents for chemoprevention. LFA-9 was designed based on the pharmacophore of licofelone by adding 2-aminoethanoic acid (Glycine) at its carboxylic end to enhance renal clearance and selectivity towards dual mPGES-1 and 5-LOX inhibition.

Here we characterized LFA-9 in detail for its mPGES1/5-LOX selectivity over COX-1/2 using in silico molecular modelling, in vitro enzyme activity assays, and ex vivo inhibition studies with human/mouse/rat mPGES-1 and 5-LOX enzymes to facilitate pre-clinical experimentation and clinical translation. In silico, LFA-9 showed strong binding affinity against 5-LOX (binding energies -199.85 and -215.3 Kcal/mol) and mPGES-1 (binding energies -238.27 and -257.76 Kcal/mol), but not against COX-1/2. LFA-9 substantially inhibited mPGES-1 (IC50 0.52 - 1.40 µM) and 5-LOX (IC50 0.89 - 2.75 µM) but not COX-1/2 (IC50&gt;1mM) in cell-free assays. Ex-vivo, LFA-9 inhibited PGE2 (IC50 0.47 - 0.78 µM) and LTB4 (IC50 0.66 - 1.24 µM) production and spared PGI2 (IC50&gt;1 mM) and TXB2 (IC50&gt;1 mM) production in LPS-stimulated human/mouse/rat macrophages and whole blood (cell-based system). Circular dichroism and isothermal calorimetric-based studies demonstrated that LFA-9 strongly binds and induces changes in the secondary structure of human mPGES-1 and 5-LOX enzymes, thereby inhibiting their activities.

Since PGE2 from mPGES1 activity promotes colon tumor stemness, LFA-9 was evaluated on colonic cancer organoids (spheroids) derived from PIRC (Polyposis in Rat Colon) rats that replicate human familial adenomatous polyposis. LFA-9 significantly inhibited the growth of colonic spheroids at 25 µM as compared to that of untreated as well as licofelone-treated spheroids. Anti-inflammatory properties of LFA-9 were evaluated in vivo using an inflammogen (carrageenan)-induced rat paw edema model. Dietary LFA-9 inhibited edema formation after carrageenan injection. In summary, LFA-9 showed anti-inflammatory and anti-tumor properties through mPGES1/5-LOX inhibition. Our data supports LFA-9, a novel mPGES-1 and 5-LOX inhibitor, as a safer drug candidate for prevention and therapy of colorectal cancer. (Supported by R01CA 213987, NIH/NCI HHSN261201500024I and VA merit).

Citation Format: Nagendra Sastri Yarla, Gopal Pathuri, Hariprasad Gali, Venkateshwar Madka, Janani Panneerselvam, Parthasarathy Chandrakesan, Courtney W. Houchen, Elizabeth R. Glaze, Jennifer Fox, David McCormick, Chinthalapally V. Rao. LFA-9, a dual mPGES-1 and 5-LOX inhibitor, suppresses colon cancer stemness and inflammogen-induced inflammatory response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6364.