Leflunomide Inhibits Pyrimidine de Novo Synthesis in Mitogen-stimulated T-lymphocytes from Healthy Humans

Katarzyna Rückemann,Lynette D Fairbanks,Elizabeth A Carrey,Catherine M Hawrylowicz,David F Richards,Bernhard Kirschbaum,H Anne Simmonds

doi:10.1074/jbc.273.34.21682

Katarzyna Rückemann, Lynette D Fairbanks + Show 5 more

Open Access

https://doi.org/10.1074/jbc.273.34.21682

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Abstract

The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated. This study, using 14C-labeled de novo purine and pyrimidine synthesis precursors, proves conclusively that the prime target in proliferating human T-lymphocytes is pyrimidine biosynthesis at the level of dihydroorotic-acid dehydrogenase. Leflunomide (25 and 50 microM), like Brequinar (0.5 and 1 microM), a demonstrated dihydroorotic-acid dehydrogenase inhibitor, was cytostatic, not cytotoxic, with proliferation being halted in the G1 phase. Both drugs restricted the normal 4-8-fold mitogen-induced expansion of pyrimidine pools over 72 h to concentrations found in nonstimulated T-cells and [14C]bicarbonate incorporation into UTP, ATP, and GTP. Uridine (50 microM) restored expansion of all pools, but [14C]bicarbonate incorporation into ATP and GTP only, not UTP. [14C]Hypoxanthine salvage was also restricted, indicating that purine salvage pathways are compromised likewise by both inhibitors. [14C]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 microM Leflunomide. 100 microM Leflunomide markedly depleted ATP and GTP pools also, which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidine-related effects reported for Leflunomide at 100 microM and above.

Highlights

The mode of action of Leflunomide, an immunomodulatory drug used in rheumatoid arthritis, is debated
Uridine (50 ␮M) restored expansion of all pools, but [14C]bicarbonate incorporation into ATP and GTP only, not UTP. [14C]Hypoxanthine salvage was restricted, indicating that purine salvage pathways are compromised likewise by both inhibitors. [14C]Glycine studies confirmed that restriction of de novo purine synthesis occurred secondary to inhibition of proliferation since this was reversed by uridine rescue, except at 100 ␮M Leflunomide. 100 ␮M Leflunomide markedly depleted ATP and GTP pools which would have serious consequences for ATP-dependent enzymes essential to the immune response, thereby explaining non-pyrimidinerelated effects reported for Leflunomide at 100 ␮M and above
Inhibition of various tyrosine kinases has been implicated from animal models [21,22,23,24], but the inhibitory concentrations of LFM (Ͼ100 ␮M) are much higher than the IC50 values for rat lymphocytes, 86 nM, and the 3.5 ␮M reported for mouse lymphocytes, or the 12.5 ␮M reported for human lymphocytes [5]

Summary

EXPERIMENTAL PROCEDURES

Chemicals—PHA, trichloroacetic acid, all chemicals and standards for HPLC (analar or Aristar grade), and RPMI 1640 medium without bicarbonate were purchased from Sigma (Poole, United Kingdom). [14C]Bicarbonate (54 mCi/mmol) was from Amersham Life Science Ltd. (Little Chalfont, UK) [8-14C]Hypoxanthine (1.07 mM, 49.6 Ci/mol) and [2-14C]uridine (0.89 mM, 56 Ci/mol) were purchased from Sigma. [U-14C]Glycine (0.47 mM, 106 Ci/mol) was purchased from ICN Biochemicals Ltd. (Thame, UK). After incubation at 37 oC for 2 h, reactions were stopped by centrifugation at 1000 ϫ g for 1 min, and the cell pellet and supernatant medium were processed as described below. T-cells were washed once with Hanks’ balanced salt solution and centrifuged at 1000 ϫ g, and the cell pellet was disrupted with 200 ␮l of 10% trichloroacetic acid Both medium and cell extracts were centrifuged for 1 min at 12,000 ϫ g, and trichloroacetic acid in the supernatants was removed by back-extraction with water-saturated diethyl ether to pH 5. Statistical Analysis—Statistical analysis of ribonucleotide concentration changes in response to PHA in the presence of inhibitors compared with control lymphocytes alone was performed using Student’s t test available in the Lotus 1-2-3 spreadsheet. Slides were stained with Grunwald-Giemsa stain, fixed with D.P.X., and examined by light microscopy

RESULTS

24 Ϯ 5 88 Ϯ 18 15 Ϯ 1 95 Ϯ 27 33 Ϯ 6 46 Ϯ 1

DISCUSSION