l-683590 and Hyperplasia

C24-Deoxyascomycin was prepared in a two-step process from ascomycin and evaluated for its immunosuppressant activity relative to ascomycin and FK506. An intermediate in the synthetic pathway, Delta(23,24)-dehydroascomycin, was likewise evaluated. Despite lacking the hydrogen-bonding interactions associated with the C24-hydroxyl moiety of ascomycin, C24-deoxyascomycin was found to be equipotent to the parent compound both in its immunosuppressive potency and in its interaction with the immunophilin, FKBP12. Conversely, Delta(23,24)-dehydroascomycin which also lacks the same hydrogen-bonding interactions did not exhibit this potency. NMR studies were conducted on the FKBP12/C24-deoxyascomycin complex in an attempt to understand this phenomenon at the molecular level. The NMR structures of the complexes formed between FKBP12 and ascomcyin or C24-deoxyascomcyin were very similar, suggesting that hydrogen-bonding interactions with the C24 hydroxyl moiety are not important for complex formation.

Topics: Amino Acid Sequence; Animals; Humans; Hyperplasia; Immunophilins; Immunosuppressive Agents; Lymph Nodes; Lymphocyte Culture Test, Mixed; Magnetic Resonance Spectroscopy; Male; Models, Molecular; Molecular Sequence Data; Nucleotidyltransferases; Peptidylprolyl Isomerase; Protein Binding; Rats; Rats, Inbred Lew; Rats, Sprague-Dawley; Recombinant Fusion Proteins; Tacrolimus; Tacrolimus Binding Proteins

The potent immunosuppressant ascomycin (1b) was selectively alkylated at the C-32 carbinol, thus providing esters and amides of 32-ascomycinyloxyacetic acid (4, AOAA). These compounds present structural variation at the FKBP/calcineurin interface. While the native carboxylic acid 4 shows no activity in vitro, esters and simple amides of 4 exhibit potent immunosuppression in the human MLR assay. Moreover, amides show inhibitory activity in the rat popliteal lymph node hyperplasia assay. Surprisingly, FKBP binding was weakened by several orders of magnitude when secondary hydrophobic aryl amides of 4 were tested, while maintaining potent immunosuppressive efficacy in vitro.

Topics: Animals; Calcineurin; Carrier Proteins; DNA-Binding Proteins; Heat-Shock Proteins; Humans; Hyperplasia; Immunosuppressive Agents; Lymph Nodes; Lymphocyte Culture Test, Mixed; Rats; Rats, Inbred Lew; Structure-Activity Relationship; Tacrolimus; Tacrolimus Binding Proteins

Ascomycin 2, a close analogue of the immunosuppressant FK506 1, was modified to incorporate a hydroxyl group at the C-33 position. This increased the aqueous solubility of ascomycin by a hundred-fold at pH 7.4 and by approximately 300-fold at pH 6.5. Ascomycin 3 also exhibited an excellent immunosuppressive activity in vitro, as tested in a human mixed lymphocyte proliferation (HuMLR) assay, and in vivo using a rat popliteal lymph node (rPLN) hyperplasia assay.

Topics: Animals; Humans; Hydrogen-Ion Concentration; Hyperplasia; Immunosuppressive Agents; Indicators and Reagents; Lymph Nodes; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Molecular Conformation; Molecular Structure; Rats; Solubility; Structure-Activity Relationship; Tacrolimus

The immunosuppressive agent FK-506 has received much attention due to its efficacy and potency in the areas of transplant rejection and autoimmune disease. Calcineurin, a Ca(2+)-calmodulin activated phosphatase, was recently implicated in the immunosuppressive mechanism of FK-506. In our ongoing search for superior immunosuppressive agents, we have synthesized several analogues of FK-506 and tested their mechanistic and immunosuppressive actions. It was found that C-18 hydroxyl analogues of ascomycin, an analogue of FK-506 also called FR900520, bound tightly to immunophilin FKBP-12, but do not show any immunosuppressive activity in vitro or in vivo despite good bioavailability. Further, they reverse the inhibition of calcineurin caused by FK-506/FKBP-12 complex.

Topics: Carrier Proteins; Cell Division; Cells, Cultured; Humans; Hyperplasia; Interleukin-2; Lymph Nodes; Lymphocytes; Structure-Activity Relationship; Tacrolimus; Tacrolimus Binding Proteins