Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory and immunomodulatory cytokine implicated in inflammatory conditions such as rheumatoid arthritis, Crohn's disease, multiple sclerosis and the cachexia associated with cancer or human immunodeficiency virus infection. TNF-alpha is initially expressed as a 233-amino-acid membrane-anchored precursor which is proteolytically processed to yield the mature, 157-amino-acid cytokine. The processing enzyme(s) which cleave TNF-alpha are unknown. Here we show that the release of mature TNF-alpha from leukocytes cultured in vitro is specifically prevented by synthetic hydroxamic acid-based metalloproteinase inhibitors, which also prevent the release of TNF-alpha into the circulation of endotoxin challenged rats. A recombinant, truncated TNF-alpha precursor is cleaved to biologically active, mature TNF-alpha by several matrix metalloproteinase enzymes. These results indicate that processing of the TNF-alpha precursor is dependent on at least one matrix metalloproteinase-like enzyme, inhibition of which represents a novel therapeutic mechanism for interfering with TNF-alpha production.

Tumor necrosis factor-alpha (TNF-alpha) is released from a cell membrane-anchored precursor by proteolytic cleavage. We have shown that broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs) prevent the processing of the TNF precursor but do not inhibit the release of other cytokines. Purified MMPs, stromelysin, matrilysin, collagenase, and the gelatinases can all cleave a recombinant pro-TNF substrate to yield mature TNF. MMP inhibitors prevent the rise in blood levels of TNF after endotoxin administration in rats and are effective in animal models of inflammatory disease such as adjuvant arthritis. Drugs that inhibit MMP action and TNF release show great promise for the treatment of autoimmune inflammatory diseases.

The a-amino acids with long alkyl side chains, the so-called lipidic amino acids 1 a -e, and their homo-oligomers, the lipidic peptides 1 p -aj, represent a class of compounds which combine the structural properties of peptides and proteins with the characteristics of lipids and membranes. The amino acids were synthesised from the appropriate alkyl bromide and diethyl acetamidomalonate. Resolution was made chemically, by forming diastereomers of the amino acid esters with an optically pure a-pinene derivative. The protected homooligomers were synthesised in solution with the assistance of a water-soluble carbodiimide coupling agent. In order to modify the physical and chemical properties of the peptides, a series of protected hetero-oligomers were prepared, by similar methods, incorporating either other amino acids (3a-d, 7a-i) or side-chain-substituted lipidic amino acids (6a -d).

The solution and crystal structure of the alkaloid cytisine has been studied using n.m.r. techniques and Xray crystallography respectively. ' H and 13C relaxation parameters and coupling constants have been used to (i) study the motion of cytisine in solution and (ii) measure interatomic distance and dihedral angles. Agreement, within experimental error, has been found between the solution and crystal parameters.Quinolizidine alkaloids are polycyclic molecules well known for their toxicity to man and livestock.' Among these alkaloids, cytisine (l), which contains a pyridone moiety, is more acutely toxic than the corresponding saturated alkaloids and its toxicological responses include nausea, convulsions, and death by respiratory failure. The structure of cytisine has been elucidated by chemical degradation and by ~y n t h e s i s . ~. ~ 13 * Atoms not included in least-square plane calculation.

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