Matlystatin A

Metalloproteinase inhibitors often feature hydroxamate moieties to facilitate the chelation of metal ions in the catalytic center of target enzymes. Actinonin and matlystatins are potent metalloproteinase inhibitors that comprise rare N-hydroxy-2-pentyl-succinamic acid warheads. Here we report the identification and characterization of their biosynthetic pathways. By gene cluster comparison and a combination of precursor feeding studies, heterologous pathway expression and gene deletion experiments we are able to show that the N-hydroxy-alkyl-succinamic acid warhead is generated by an unprecedented variation of the ethylmalonyl-CoA pathway. Moreover, we present evidence that the remarkable structural diversity of matlystatin congeners originates from the activity of a decarboxylase-dehydrogenase enzyme with high similarity to enzymes that form epoxyketones. We further exploit this mechanism to direct the biosynthesis of non-natural matlystatin derivatives. Our work paves the way for follow-up studies on these fascinating pathways and allows the identification of new protease inhibitors by genome mining.

To investigate a series of new inhibitors of gelatinases based on matlystatin B (1b), extensive structure-activity relationship studies were performed. The new derivatives were evaluated in vitro for the ability to inhibit gelatinases. The inhibitory activities against thermolysin were also assayed to test the compounds' selectivity. Among the compounds modified at the P'3 moiety, the N-methylamide derivative 5 g was virtually twice as effective on gelatinase B as the parent compound 1b (5g, IC50 = 0.27 microM vs. 1b, IC50 = 0.57 microM). Other derivatives, including 1) esters 7a and 7b having the ester portions P'2 and P'3, 2) the cyclic amino acids, L-proline or L-pipecolinic acid (13a and 13b) bearing P'2, and 3) compounds 29a and 29b representing an attachment of the pentyl side chain at C3' (P'1 side chain) instead of C2', all showed decreased potencies. The key discovery was the observation that the introduction of a nonyl group at the P'1 position yielded a compound (31f, IC50 = 0.0012 microM) with high inhibitory activity against gelatinases and high selectivity over thermolysin. This result suggested that the S'1 subsites of the gelatinases have a locally deep hydrophobic structure, since on the basis of the optimum inhibitory activity in the alkyl series, the nonyl group seems to fit best into this hydrophobic pocket. Thus 31f exhibited a 475-fold more potent inhibitory activity than 1b towards gelatinase B.

We investigated the effects of several types of aminopeptidase inhibitors on tumor cell-associated aminopeptidase activity and invasion. The aminopeptidase expressed by the human metastatic HT1080 fibrosarcoma cells was effectively suppressed by actinonin A, bestatin, leuhistin and matlystatin A, which are capable of inhibiting the purified aminopeptidase N, but not by arphamenine B specific for aminopeptidase B. The aminopeptidase N inhibitors inhibited HT1080 cells from degrading the subendothelial matrix and from invading into Matrigel in parallel with their aminopeptidase inhibitory activities. Matlystatin A, with multiple inhibitory activity against both aminopeptidase N and matrix metalloproteinases (MMP), was the most effective inhibitor of invasion. However, leuhistin and bestatin, without MMP inhibitory activity, also exhibited significant inhibition of invasion. The results suggest that aminopeptidase N plays a crucial role in the degradation and invasion of extracellular matrices by fibrosarcoma cells and that aminopeptidase inhibitors may be useful for preventing the spread of malignant tumors.