Target: Cyclophilin A (inhibits cis-trans isomerase activity) [1]
27 times more potent than cyclosporine A (exact IC50 not provided) [1]

Cyclophilin A protein expression was reduced by 75.9% when TMN 355 (0.5–10 μM) was administered for three to nine hours. After six hours of activation, 1 μM TMN 355 inhibits cyclophilin A [1].

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In Vitro: TMN355 at 1 μM for 6 hours reduced cyclophilin A protein expression by 75.9% in THP-1 differentiated macrophages as determined by western blotting. It also decreased cyclophilin A mRNA levels measured by quantitative real-time PCR. [1]
TMN355 treatment (1 μM for 6 h) markedly decreased lipid uptake in macrophages as evident from immunofluorescence assay and oil red O staining, indicating reduced foam cell formation. [1]
TMN355 reduced the expression of scavenger receptor markers CD36 and LOX-1 in monocyte-derived macrophages treated with oxidized LDL. [1]
TMN355 significantly reduced the levels of proinflammatory cytokines TNF-α and MCP-1 as well as extracellular cyclophilin A in conditioned medium of monocyte-derived macrophages as measured by ELISA. [1]
In human primary monocytes isolated from blood, treatment with TMN355 (1 μM) resulted in 77% reduction of Dil-oxLDL uptake compared to cyclophilin A-treated cells (p<0.005). [1]

Western Blot Analysis[1]
Cell Types: THP Cell Line
Tested Concentrations: 0.5, 1, 2.5, 5 and 10 μM
Incubation Duration: 3, 6 and 9 hrs (hours)
Experimental Results: Resulted in 75.9% reduction in cyclophilin A protein expression.

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Cell Assay: For cell viability assessment, THP-1 differentiated macrophages were treated with TMN355 in a time- and dose-dependent manner using MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. A dose of 1 μM TMN355 was found to inhibit cyclophilin A expression without affecting cell viability, and this concentration was used for subsequent experiments (treatment for 6 hours). [1]
For silencing/inhibition experiments, THP-1 cells (2-4×10⁵) were transfected with mission siRNA for 48 h or treated with TMN355 at 1 μM for 6 h at 37°C. After treatment, cells were differentiated into macrophages with PMA (50 ng/mL) for 96 h in high glucose (20 mM) RPMI 1640 medium, then starved for 24 h and incubated with oxidized LDL (50 μg/mL) for 24 h to induce foam cell formation. Lipid uptake was quantified by oil red O staining, immunofluorescence (Dil-oxLDL), and flow cytometry. [1]
For western blot analysis, after treatment with OxLDL and TMN355, protein lysates were prepared and separated on SDS-PAGE. Proteins were transferred to nitrocellulose membranes and incubated with primary antibodies (anti-cyclophilin A, anti-CD36, anti-LOX-1, anti-β-actin) at 1:1000 dilution overnight at 4°C, followed by HRP-conjugated secondary antibodies (1:5000). Proteins were visualized with ECL substrate. [1]
For ELISA, levels of TNF-α, MCP-1, and cyclophilin A in conditioned medium after treatment were determined using sandwich immunoassay kits according to manufacturer’s instructions. All samples were analyzed in duplicate. [1]
For quantitative real-time PCR, total RNA was isolated using TRIzol, reverse transcribed with random hexamers and M-MLV reverse transcriptase. PCR was performed using specific primers for cyclophilin A and beta-2 microglobulin (as endogenous control) on an ABI Prism 7900HT system with 40 cycles (95°C for 15 s, 60°C for 1 min). Ct values were used to calculate relative expression. [1]
For human primary monocyte experiments, CD14+ monocytes were isolated from healthy volunteers using anti-CD14 microbeads and magnetic separation. Monocytes were cultured for 96 h in RPMI 1640 with PMA (50 ng/mL) in the presence or absence of TMN355 (1 μM) and/or cyclophilin A (100 ng/mL), then treated with Dil-oxLDL (10 μg/mL) for 4 h. Dil-oxLDL uptake was analyzed by confocal microscopy (559 nm filter) and quantified using NIS-Elements software. [1]

Toxicity/Toxicokinetics: TMN355 at 1 μM did not affect cell viability in THP-1 differentiated macrophages as determined by MTT assay. No other toxicity data reported. [1]

[1]. Cyclophilin A enhances macrophage differentiation and lipid uptake in high glucose conditions: a cellular mechanism for accelerated macro vascular disease in diabetes mellitus. Cardiovasc Diabetol. 2016 Nov 3;15(1):152.

Additional Info: TMN355 is a chemical inhibitor of cyclophilin A that is 27 times more potent than cyclosporine A but lacks immunosuppressive function. It inhibits the cis-trans isomerase activity of cyclophilin A by forming 2-3 hydrogen bonds with residues Arg55, Gln63, and Asn102. In this study, TMN355 was used to demonstrate that cyclophilin A plays a pivotal role in monocyte adhesion, transmigration, differentiation into macrophages, and foam cell formation under high glucose conditions. Inhibition of cyclophilin A by TMN355 reduced lipid uptake, scavenger receptor expression (CD36 and LOX-1), and proinflammatory cytokine secretion (TNF-α, MCP-1), suggesting that targeting cyclophilin A may retard progression of atherosclerotic lesions in diabetes. [1]

C21H14CLFN2O2

380.8034

380.073

1186372-20-2

44249042

White to yellow solid powder

5.47

2

3

2

27

552

0

C1=CC=C2C(=C1)C(C3=CC=CC=C32)NC(=O)NC(=O)C4=C(C=CC=C4Cl)F

SFNLLCUAISZNRV-UHFFFAOYSA-N

InChI=1S/C21H14ClFN2O2/c22-16-10-5-11-17(23)18(16)20(26)25-21(27)24-19-14-8-3-1-6-12(14)13-7-2-4-9-15(13)19/h1-11,19H,(H2,24,25,26,27)

2-chloro-N-[(9H-fluoren-9-ylamino)carbonyl]-6-fluoro-benzamide

Compound 3i TMN 355 TMN-355 TMN355

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~31.25 mg/mL (~82.06 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.46 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)