Molecular mechanisms of action of new xenobiotic immunosuppressive drugs: Tacrolimus (FK506), sirolimus (rapamycin), mycophenolate mofetil and leflunomide

Current Opinion in Immunology

Volumn 8, Issue 5, 1996, Pages 710-720

  Brazelton, Timothy R ^a   Randall E, Morris ^a  

^a STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLOSPORIN; IMMUNOSUPPRESSIVE AGENT; LEFLUNOMIDE; MYCOPHENOLIC ACID 2 MORPHOLINOETHYL ESTER; RAPAMYCIN; TACROLIMUS; TERIFLUNOMIDE;

AUTOIMMUNITY; BONE MARROW TOXICITY; CLINICAL TRIAL; DRUG MECHANISM; DRUG TARGETING; DRUG THERAPY; GASTROINTESTINAL TOXICITY; GRAFT REJECTION; HUMAN; IMMUNOSUPPRESSIVE TREATMENT; LEUKOPENIA; NEPHROTOXICITY; NONHUMAN; PSORIASIS; REVIEW; RHEUMATOID ARTHRITIS; T LYMPHOCYTE;

EID: 0030272487     PISSN: 09527915     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0952-7915(96)80090-2     Document Type: Article

References (74)

1. of outstanding interest Morris RE. New immunosuppressive drugs. Busuttil RW, Klintmalm GB. Transplantation of the Liver. 1995;760-786 WB Saunders Company, Philadelphia, This textbook chapter is a detailed review of the pharmacology and clinical status of new xenobiotic immunosuppressants.
2. of outstanding interest Morris RE. Mechanism of action of new immunosuppressive drugs. Kidney Int Suppl. 49:1996;S26-S38 An article that is an updated version of the textbook chapter [1] but is less detailed.
3. of special interest Goto T, Kino T, Hatanaka H, Nishiyama M, Okuhara M, Kohsaka M, Aoki H, Imanaka H. Discovery of FK-506, a novel immunosuppressant isolated from Streptomyces tsukubaensis. Transplant Proc. 19:1987;4-8.
4. Ochiai T, Nakajima K, Nagata M, Suzuki T, Asano T, Uematsu T, Goto T, Hori S, Kenmochi T, Nakagoori T, et al. Effect of a new immunosuppressive agent, FK 506, on heterotopic cardiac allotransplantation in the rat. Transplant Proc. 19:1987;1284-1286.
5. of outstanding interest Griffith JP, Kum JL, Kum EE, Sintchak MD, Thomson JA, Fitzgibbon MJ, Fleming MA, Caron PR, Hsaio K, Navia MA. X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell. 82:1995;507-522 This study concludes, based on the structural information these authors obtained, that the tacrolimus - FKBP binary complex sterically, rather than directly, inhibits dephosphorylation of macromolecular substrates within the active site of the catalytic subunit (calcineurin A). These investigators further predict that the tacrolimus - FKBP complex prevents the binding of calcineurin's physiological substrate to this enzyme's active site.
6. of outstanding interest Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, Tempczyk A, Kalish VJ, Tucker KD, Showalter RE, Moonmaw EW, et al. Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature. 378:1995;641-644 This publication concludes that the tacrolimus - FKBP complex neither sterically hinders peptide binding to the active site nor induces large conformational changes in calcineurin that would account for its inhibition of calcineurin's catalytic activity. Rather, allosteric changes in the active site's geometry (undetectable at the resolution of this study) caused by the tacrolimus - FKBP complex are proposed as the mechanism by which calcineurin's enzymatic activity is inhibited. Furthermore, it is suggested that the tacrolimus - FKBP complex does not exclude substrate binding to the active site.
7. of outstanding interest Loh C, Shaw KT, Carew J, Viola JP, Luo C, Perrino BA, Rao A. Calcineurin binds the transcription factor NFAT1 and reversibly regulates its activity. J Biol Chem. 271:1996;10884-10891 This research provides the most detailed account yet of the phosphorylation state of NFAT and its interaction with calcineurin.
8. of special interest Cardenas ME, Muir RS, Breuder T, Heitman J. Targets of immunophilin-immunosuppressant complexes are distinct highly conserved regions of calcineurin A. EMBO J. 14:1995;2772-2783 To more precisely understand the tacrolimus - FKBP binding sites with calcineurin, a genetic approach was used to isolate dominant tacrolimus- or cyclosporine-resistant mutants from a yeast strain that is normally sensitive to both drugs. Mutations were single amino acid substitutions in the catalytic domain of the calcineurin A subunit. This finding confirms the conclusion from the crystallographic studies of the tacrolimus - FKBP - calcineurin complex that inhibition of enzyme activity is indirect and noncompetitive. Since cyclosporine - and tacrolimus - immunophilin complexes bind to yeast calcineurin at different sites, it was suggested that patients who are resistant to immunosuppression by cyclosporine but sensitive to tacrolimus may have somatic or germline cyclophilin A or calcineurin A mutations that confer their differential sensitivity to the immunosuppressive effects of these two drugs.
9. of special interest Lam E, Martin MM, Timerman AP, Sabers C, Fleischer S, Lucas T, Abraham RT, O'Keefe SJ, O'Neill EA, Wiederrecht GJ. A novel FK506 binding protein can mediate the immunosuppressive effects of FK506 and is associated with the cardiac ryanodine receptor. J Biol Chem. 270:1995;26511-26522 The biological function of the FKBP family has never been clear. This paper investigates a new function of this family of immunophilins: modulation of the ryanodine receptor. Ryanodine receptors are channels that mediate calcium flux in many tissues. The newly discovered FKBP, FKBP12.6, modulates a different isoform of the ryanodine receptor than FKBP12.
10. 3R complexes, leading to increased calcium flux. Since FKBP12 is abundant in cells, it is unlikely that doses of tacrolimus would produce tissue levels high enough to limit the binding of FKBP12 to either of these calcium channels.
11. of outstanding interest Baughman G, Wiederrecht GJ, Campbell NF, Martin MM, Bourgeois S. FKBP51, a novel T-cell-specific immunophilin capable of calcineurin inhibition. Mol Cell Biol. 15:1995;4395-4402 The intriguing speculation from this study is that it may be possible to target FKBP-binding drugs selectively to T cells. Since FKBP12 is so abundant and binds with such high affinity to tacrolimus, it will be important to insure that any new drug binds with high avidity to FKBP51. The largest obstacle to exploiting the findings from this work remains the identification of an FKBP that is selectively expressed in human lymphocytes.
12. of special interest Bennett WM. The nephrotoxicity of immunosuppressive drugs. Clin Nephrol. 43:1995;3-7 This general review of immunosuppressive drug-induced nephrotoxicity indicates preglomerular afferent arteriolar vasoconstriction is a cause of acute cyclosporine nephrotoxicity, interstitial fibrosis and tubular atrophy which is a manifestation of chronic effects of cyclosporine on the kidney. Similar histopathological findings have been seen in patients treated with tacrolimus. In no clinical or experimental studies has inhibition of calcineurin phosphatase activity been proven to be the lynch pin for nephrotoxicity caused by either cyclosporine or tacrolimus.
13. Goodall T, Kind CN, Hammond TG. FK506-induced endothelin release by cultured rat mesangial cells. J Cardiovasc Pharmacol. 26:1995;482-485.
14. of outstanding interest Andoh TF, Burdmann EA, Lindsley J, Houghton DC, Bennett WM. Functional and structural characteristics of experimental FK 506 nephrotoxicity. Clin Exp Pharmacol Physiol. 22:1995; 646-654 These authors emphasize the need for sodium depletion in this model to produce a nephrotoxic effect indistinguishable from those noted in patients treated with tacrolimus. Since sodium depletion is essential for this model, the following pathway leading to tacrolimus nephrotoxicity was hypothesized: renal hypoperfusion caused by increased endothelin and thromboxane activity activates the intrarenal renin - angiotensin system, thereby causing additional vasoconstriction and resulting in medullary ischemia. Induction of TGFβ expression and collagen synthesis by angiotensin II may be another avenue leading to tubulointerstitial fibrosis. Finally, in this rat model, as in humans, tacrolimus induced hypomagnesemia. This defect was also suggested as a mechanism leading to tubular dysfunction. The question remains: what initial molecular events are triggered by tacrolimus that then lead to the series of pathways causing acute and chronic nephrotoxicity? The answer to the question should have taken priority over the extensive investigations of this drug's molecular actions on immune cells conducted over the past several years.
15. of outstanding interest Hadad SJ, Souza ER, Ferreira AT, Oshiro ME, Boim MA, Razvickas CV, Moura LA, Schor N. FK 506: effects on glomerular hemodynamics and on mesangial cells in culture. Kidney Int. 48:1995;56-64 These investigators found that tacrolimus increases calcium flux in mesangial cells in vitro. Increased intracellular calcium in mesangial cells may be responsible for the reduction in their surface area after treatment with tacrolimus in vitro. This in vivo finding may explain the reduction in glomerular filtration caused by tacrolimus in vivo, but the molecular mechanism responsible for the effect of tacrolimus on calcium flux remains to be defined.
16. of outstanding interest Muraoka K, Fujimoto K, Sun X, Yoshioka K, Shimizu K, Yagi M, Bose H, Miyazaki I, Yamamoto K. Immunosuppressant FK506 induces interleukin-6 production through the activation of transcription factor nuclear factor (NF)-kB. J Clin Invest. 97:1996;2433-2439 These workers showed that treatment of rats with tacrolimus increases IL-6 expression in the kidney, and since IL-6 has been implicated as responsible for mesagioproliferative glomerulonephritis, these effects of tacrolimus need to be studied further to determine whether are causally related to this drug's nephrotoxicity. These effects of tacrolimus and their relationship to nephrotoxicity are particularly interesting, since this mechanism of nephrotoxicity appears to be completely independent of the drug's effect on calcineurin.
17. Calne RY, Collier DS, Lim S, Pollard SG, Samaan A, White DJ, Thiru S. Rapamycin for immunosuppression in organ allografting [letter]. Lancet. 2:1989;227.
18. Morris RE, Meiser BM. Identification of a new pharmacologic action for an old compound. Med Sci Res. 17:1989;609-610.
19. Morris RE. Repamycin: FK506's fraternal twin or distant cousin? Immunol Today. 12:1991;137-140.
20. Morris RE. Rapamycin: antifungal, antitumor antiproliferative, and immunosuppressive macrolides. Transplant Rev. 6:1992;39-87.
21. of outstanding interest Sehgal SN, Camardo JS, Scarola JA, Maida BT. Rapamycin (sirolimus, rapamune). Curr Opin Nephrol Hypertens. 4:1995;482-487 This is an up to date review of this drug's effects on cells in vitro as well as a comprehensive summary of contemporary research of the drug's actions in vivo.
22. of special interest Lorenz MC, Heitman J. TOR mutations confer rapamycin resistance by preventing interaction with FKBP12-rapamycin. J Biol Chem. 270:1995;27531-27537 These studies of sirolimus-resistant TOR and FPR1 (FKBP12) mutant yeast strains have shown that, firstly, drug resistance results from the inability of the sirolimus - FKBP12 complex to interact through its composite surface directly with TOR proteins, secondly, FKBP12 isomerase activity is unnecessary for binding of the sirolimus - FKBP12 complex to TOR proteins, and thirdly, inhibition of calcineurin and TOR proteins by both tacrolimus - FKBP12 and sirolimus - FKBP12 complexes, respectively, depends on the same surface amino acids in FKBP12.
23. 1 effector molecule(s) rather than inhibit TOR2 kinase activity in general.
24. of outstanding interest Sabers CJ, Martin MM, Brunn GJ, Williams JM, FJ Dumont, Wiederrecht G, Abraham RT. Isolation of a protein target of the FKBP12 - rapamycin complex in mammalian cells. J Biol Chem. 270:1995;815-822 In this study only small amounts of mTOR bound to the affinity matrix when the mTOR originated from tissue extracts derived from sirolimus-resistant cell lines. Amino acid sequencing of isolated mTOR indicates it is a mammalian homologue of yeast TOR proteins.
25. Chen J, Zheng XF, Brown EJ, Schreiber SL. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc Natl Acad Sci USA. 92:1995;4947-4951.
26. of special interest Sabatini DM, Pierchala BA, Barrow RK, Schell MJ, Snyder SH. The rapamycin and FKBP12 target (RAFT) displays phosphatidylinositol 4-kinase activity. J Biol Chem. 270:1995;20875-20878 This study's failure to show that sirolimus - FKBP12 complex inhibits the kinase activity of mTOR is at odds with data from other groups in which kinase activity is inhibited by the drug - immunophilin complex. Nevertheless, in the discussion of this paper, the authors suggest ways that the kinase might be inhibited but would not have been detected in their assay system. Therefore, sirolimus - FKBP12 inhibition of kinase activity is not completely excluded as a mechanism of action.
27. Choi J, Chen J, Schreiber S, Clardy J. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science. 273:1996;239-242.
28. 1 progression, it was found that sirolimus lengthens the time at this stage of the cell-cycle. The authors suggest that previous investigators' conclusion that sirolimus halts cell-cycle progression is not correct and may have resulted from failure to assess cells for longer times.
29. Mahalingam M, Templeton DJ. Constitutive activation of S6 kinase by deletion of amino-terminal autoinhibitory and rapamycin sensitivity domains. Mol Cell Biol. 16:1996;405-413.
30. s6k.
31. 1/S, this suggests that inhibition of CREB/ATF transcription factors by sirolimus may be caused by inhibition of kinase activity which ultimately leads to reduced expression of PCNA, causing cell-cycle delay or arrest.
32. of outstanding interest Gregory CR, Huang X, Pratt RE, Dzau VJ, Shorthouse R, Billingham ME, Morris RE. Treatment with rapamycin and mycophenolic acid reduces arterial intimal thickening produced by mechanical injury and allows endothelial replacement. Transplantation. 59:1995;655-661 This paper extends earlier work by our group that had initially demonstrated the efficacy of sirolimus for prevention of intimal thickening in transplanted vascular grafts. To prove that the efficacy of sirolimus is not limited to its effects on immune cells, it was shown that treatment with sirolimus also inhibits intimal thickening after mechanical injury to native arteries caused by balloon catheter injury.
33. Morris RE, Cao W, Huang X, Gregory CR, Billingham ME, Rowan R, Shorthouse RA. Rapamycin (sirolimus) inhibits vascular smooth muscle DNA synthesis in vitro and suppresses narrowing in arterial allografts and in balloon-injured carotid arteries: evidence that rapamycin antagonizes growth factor action on immune and nonimmune cells. Transplant Proc. 27:1995;430-431.
34. of special interest Cao W, Mohacsi P, Shorthouse R, Pratt R, Morris RE. Effects of rapamycin on growth factor-stimulated vascular smooth muscle cell DNA synthesis. Inhibition of basic fibroblast growth factor and platelet-derived growth factor action and antagonism of rapamycin by FK506. Transplantation. 59:1995;390-395 This paper is a more detailed examination of the earlier finding of our group that sirolimus suppresses growth factor-induced arterial smooth muscle cell proliferation in vitro. The effects of sirolimus provide a mechanistic foundation for its ability to inhibit arterial smooth muscle proliferation in vivo. In addition, since tacrolimus antagonized the inhibitory actions of sirolimus in this system, it was suggested antagonized the inhibitory actions of sirolimus in this system, muscle cells depend on sirolimus - FKBP complexes.
35. of special interest Marx SO, Jayaraman T, Go LO, Marks AR. Rapamycin-FKBP inhibits cell cycle regulators of proliferation in vascular smooth muscle cells. Circ Res. 76:1995;412-417 These investigators independently found the actions of sirolimus on smooth muscle cells were as described in the article by Cao [34]. In addition, it was suggested that a target of the antiproliferative actions of sirolimus is cell-cycle kinases.
36. of special interest Ransom JT. Mechanism of action of mycophenolate mofetil. Ther Drug Monit. 17:1995;681-684 A concise but detailed review of the preclinical studies done on MMF.
37. Wu JC. Mycophenolate mofetil: molecular mechanisms of action. Perspect Drug Discovery Res. 2:1994;185-204.
38. Morris RE. New immunosuppressive drugs: mycophenolate mofetil. New Dev Transplant Med. 2:1995;10-12.
39. of special interest Laurent AF, Dumont S, Poindrom P, Muller CD. Mycophenolic acid suppresses protein N-linked glycosylation in human monocytes and their adhesion to endothelial cells and to some substrates. Exp Hematol. 24:1996;59-67 A very good study of MMF's ability to decrease adhesion molecule function.
40. Morris RE, Hoyt EG, Eugui EM, Allison AC. Prolongation of rat heart allograft survival by RS-61443. Surg Forum. 40:1989;337-338.
41. Kimball JA, Pescovitz MD, Book BK, Normal DJ. Reduced human IgG anti-ATGAM antibody formation in renal transplant recipient receiving mycophenolate mofetil. Transplantation. 60:1995;1379-1383.
42. Fraser-Smith EB, Rosete JD, Schatzman RC. Suppression by mycophenolate mofetil of the neointimal thickening caused by vascular injury in a rat arterial stenosis model. J Pharmacol Exp Ther. 275:1995;1204-1208.
43. of special interest Hager PW, Collart FR, Huberman E, Mitchell BS. Recombinant human inosine monophosphate dehydrogenase type I and type II proteins. Purification and characterization of inhibitor binding. Biochem Pharmacol. 49:1995;1323-1329 Following mitogen stimulation of lymphocytes, production of type II IMPDH increases rapidly. Thus, modulation of IMPDH activity in proliferating cells appears to be controlled by synthesis of the type II isoform. This has led to the speculation that specific inhibition of the type II isoform may selectively achieve MPA's immunosuppressive effects. In this paper, however, Hager find that MPA inhibits both isoforms of IMPDH.
44. Makara GM, Keseru GM, Kajtar-Peredy M, Anderson WK. Nuclear magnetic resonance and molecular modeling study on mycophenolic acid: implications for binding to inosine monophosphate dehydrogenase. J Med Chem. 39:1996;1236-1242.
45. of outstanding interest Langman LJ, Shapiro AM, Lakey JR, LeGatt DF, Knetman NM, Yatscoff RW. Pharmacodynamic assessment of mycophenolic acid-induced immunosuppression by measurement of inosine monophosphate dehydrogenase activity in a canine model. Transplantation. 61:1996;87-92 An excellent description of the interaction between IMPDH and MPA at 2.6 Å resolution.
46. of special interest Langman LJ, Shapiro AM, Lakey JR, LeGatt DF, Kneteman NM, Yatscoff RW. Pharmacodynamic assessment of mycophenolic acid-induced immunosuppression by measurement of inosine monophosphate dehydrogenase activity in a canine model. Transplantation. 61:1996;87-92 Demonstrates that IMPDH inhibition occurs following in vivo administration of NMF.
47. Allison AC, Eugui EM. Preferential suppresion of lymphocyte proliferation by mycophenolic acid and predicted long-term effects of mycophenolate mofetil in transplantation. Transplant Proc. 26:1994;3205-3210.
48. 1 events are not inhibited by mizoribine including the expression of c-myb, c-myc, IL-2, and cdc2 kinase.
49. Senda M, DeLustro B, Eugui E, Natsumeda Y. Mycophenolic acid, an inhibitor of IMP dehydrogenase that is also an immunosuppressive agent, suppresses the cytokine-induced nitric oxide production in mouse and rat vascular endothelial cells. Transplantation. 60:1995;1143-1148.
50. Görlich D, Mattaj IW. Nucleocytoplasmic transport. Science. 271:1996;1513-1518.
51. of special interest Sollinger HW. Mycophenolate mofetil for the prevention of acute rejection in primary cadaveric renal allograft recipients. Transplantation. 60:1995;225-232 Presents first results of clinical efficacy with MMF from US Phase II clinical trials.
52. Placebo-controlled study of mycophenolate mofetil combined with combined with cyclosporin and corticosteroids for the prevention of acute rejection. Lancet. 345:1995;1321-1325.
53. of outstanding interest Cherwinski HM, McCarley D, Schatzman R, Devens B, Ransom JT. The immunosuppressant leflunomide inhibits lymphocyte progression through cell cycle by a novel mechanism. J Pharmacol Exp Ther. 272:1995;460-468 An excellent paper that explores the effect of LFM on cell-cycle, proliferation of various cell types, and initial signal transduction events.
54. Bartlett RR, Campion G, Musikic P, Schleyerbach R, Zielinski T, Schorlemmer H-U. Leflunomide: a novel immunomodulating drug. Lewis AJ, Furst DE. Nonsteroidal Anti-inflammatory Drugs. 1994;349-366 Marcel Dekker Incorporated, New York.
55. of outstanding interest Siemasko KF, Chong AS, Williams JW, Bremer EG, Finnegan A. Regulation of B cell function by the immunosuppressive agent leflunomide. Transplantation. 61:1996;635-642 A thorough exploration of A77 1726's substantial inhibitory effects on B cells and antibody production.
56. Morris RE, Huang X, Cao W, Zheng B, Shorthouse RA. Leflunomide (HWA 486) and its analog suppress T- and B-cell proliferation in vitro, acute rejection, ongoing rejection, and antidonor antibody synthesis in mouse, rat, and cynomolgus monkey transplant recipients as well as arterial intimal thickening after balloon catheter injury. Transplant Proc. 27:1995;445-447.
57. of special interest Cao WW, Kao PN, Chao AC, Gardner P, Ng J, Morris RE. Mechanism of the antiproliferative action of leflunomide. J Heart Lung Transplant. 14:1995;1016-1030 This is a detailed paper comparing various molecular mechanisms of A77 1726 that have been proposed. It provides evidence that inhibition of pyrimidine synthesis is a key mechanism and reviews prior literature on A77 1726's in vitro mechanism of action.
58. Morris RE, Huang X, Gregory CR, Billingham ME, Rowan R, Shorthouse R, Berry GJ. Studies in experimental models of chronic rejection: use of rapamycin (sirolimus) and isoxazole derivatives (leflunomide and its analog) for the suppression of graft vascular disease and obliterative bronchiolitis. Transplant Proc. 27:1995;2068-2069.
59. Nair RV, Cao W, Morris RE. The antiproliferative effects of leflunomide on vascular smooth muscle cells in vitro is mediated by selective inhibition of pyrimidine synthesis. Transplant Proc. 1996;. in press.
60. of special interest Mladenovic V, Domljan Z, Rozman B, Jajic I, Mihajlovic D, Dordevic J, Popovic M, Dimitrijevic M, Zivkovic M, Campion G, et al. Safety and effectiveness of leflunomide in the treatment of patients with active rheumatoid arthritis. Results of a randomized, placebo-controlled, phase II study. Arthritis Rheum. 38:1995;1595-1603 Documents the clinical efficacy and minimal toxicity of leflunomide.
61. of outstanding interest Williamson RA, Yea CM, Robson PA, Curnock A, Gadher S, Hambleton AB, Woodward K, Bruneau JM, Hambleton P, Moss D, et al. Dihydroorotate dehydrogenase is a high affinity binding protein for A77 1726 and mediator of a range of biological effects of the immunomodulatory compound. J Biol Chem. 270:1995;22467-22472 Reported several important observations including the high affinity binding of A77 1726 to DHODH and uridine reversal of the malononitriloamide in vivo.
62. Cao WW, Kao PN, Aoki Y, Xu JC, Shorthouse R, Morris RE. A novel mechanism of action of the immunomodulatory drug leflunomide: augmentation of the immunosuppressive cytokine TGF-β1 and suppression of the immunostimulatory cytokine IL-2. Transplant Proc. 1996;. in press.
63. Lang R, Wagner H, Heeg K. Differential effects of the immunosuppressive agents cyclosporine and leflunomide in vivo. Leflunomide blocks clonal T cell expansion yet allows production of lymphokines and manifestation of T cell mediated shock. Transplantation. 59:1995;382-389.
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67. Chong AS, Rezai K, Gebel HM, Finnegan A, Foster P, Xu X, Williams JW. Effects of leflunomide and other immunosuppressive agents on T cell proliferation in vitro. Transplantation. 61:1996;140-145.
68. of special interest Silva HT, Cao W, Shorthouse R, Morris RE. Mechanism of action of leflunomide in vivo uridine administration reverses LFM's inhibition of lymphocyte proliferation. Transplant Proc. 1996; An important paper that demonstrated that inhibition of DHODH may be the most significant mechanism of action of LFM in vivo.
69. of special interest Greene S, Watanabe K, Braatz-Trulson J, Lou L. Inhibition of dihydroorotate dehydrogenase by the immunosuppressive agent leflunomide. Biochem Pharmacol. 50:1995;861-867 Identified DHODH as a target of leflunomide by measuring its effects on all six enzymes in the de novo pathway of pyrimidine synthesis.
70. of special interest Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorolate dehydrogenase. Biochemistry. 35:1996;1270-1273 This study was the first to report the molecular interaction by which LFM may bind to and inhibit DHODH.
71. of special interest Cherwinski HM, Byers N, Ballaron SJ, Nakano GM, Young JM, Ransom JT. Leflunomide interferes with pyrimidine nucleotide biosynthesis. Inflamm Res. 44:1995;317-322 Identifies many of the cellular effects of A77 1726 inhibition of DHODH including decreased DNa synthesis and inhibition of cell-cycle progression.
72. Cherwinski HM, Byars N, Ballaron SJ, Nakano GM, Young JM, Ransom JT. Leflunomide interferes with pyrimidine nucleotide biosynthesis. Inflamm Res. 44:1995;317-322.
73. Peters GJ, Veerkamp JH. Purine and pyrimidine metabolism in peripheral blood lymphocytes. Int J Biochem. 15:1983;115-123.
74. Fairbanks LD, Bofill M, Ruckemann K, Simmonds HA. Importance of ribonucleotide availability to proliferating T lymphocytes from healthy humans. J Biol Chem. 270:1995;29682-29689.