Leupeptin is a reversible competitive protease inhibitor, which was shown to inhibit cathepsins B, H, L, and S, calpain and trypsin.

In 1969 Puri et al., identified leupeptins, new protease inhibitors from Actinomycetes 1. They isolated and characterized the biological activities of leupeptins 2.  In 1980, Knight et al., described the characteristics of the inhibition of cathepsin B by leupeptin. Leupeptin was a purely competitive inhibitor of human cathepsin B.  Human liver contains cathepsin B and also proteinases that are analogous to rat liver cathepsins H and L (in their action on synthetic substrates). These cathepsins B, H and L were all  inhibited by leupeptin 3.

Structural Characteristics
Leupeptins were isolated from culture filtrates of several strains of streptomyces on the basis of their antiplasmin activity. Their structures were determined to be propionyl- and acetyl- L-leucyl-L-leucyl-L-argininal (a mixture of N-acetyl- and N-propyl-L-leucyl-L-leucyl-DL- 1- amino-4-guanidinovaleraldehyde) and the analogs are usually are those in which one or both leucine residues are replaced by isoleucine or valine residues 4.  Biosynthesis of leupeptins, primarily of acetyl-leucyl-leucylargininal, has been studied with Streptomyces roseus MA839-A1. Kawamura et al., reported the structures of two major components, acetyl-L-leucyl- leucyl- DL-argininal (leupeptin Ac-LL) and propionyl-L-leucyl-L-leucyl-DL-argininal (leupeptin Pr-LL), confirmed by chemical syntheses 5. A series of leupeptin analogs R-L-leucyl-L-leucyl-L-argininal with variable N-terminal substituents has been synthesized using N-alpha-tert-butyl-oxycarbonyl-NG-benzyloxycarbonyl-L-arginine-delta-lactam as the starting material 6.

Mode of Action
Leupeptins produced by various species of Actinomycetes, strongly inhibit proteases such as plasmin, trypsin and papain. Leupeptin is a purely competitive inhibitor of human cathepsin B and interact directly with the active site. Similarly, competitive inhibition has been observed with trypsin and with rat liver cathepsins B and H. NMR studies have shown the presence of three forms of leupeptin in aqueous solutions. Trace amounts of the free aldehyde existed in equilibrium with almost equal quantities of the covalent aldehyde hydrate and an intramolecular cyclic addition product. The apparent dissociation constant for the binding of leupeptin by cathepsin B was, however, several orders of magnitude smaller than that for the structurally related substrate Bz-Arg- NH2 7. The formation of tetrahedral intermediates during catalysis is implied by the demonstration that cathepsin B cleaves Z-Lys-ONp by an acyl-enzyme mechanism8.

Leupeptin as a calpain inhibitor: the effect of treatment with leupeptin, a calpain inhibitor, on motoneuron survival and muscle function was examined in in vitro and in vivo models of motoneuron degeneration. Treatment with leupeptin, rescues motoneurons from cell death and improves muscle function following nerve injury 9.

Effect on intestinal reperfusion: Delayed fluid resuscitation during burn shock is thought to compromise the integrity of gut mucosa and allow enteric bacteria to cross the luminal wall and infect other sterile organ systems. Data indicate that intestinal reperfusion injury in burned rats can be effectively modulated with leupeptin therapy 10.

Inhibition of endoprotease acrosin: Two of the leupeptin derivatives (R = trifluoroacetyl, R = tert-butyloxycarbonyl) were found to be more effective than the natural leupeptins from microbial sources as antienzymatic contraceptives. The modified leupeptins proved to be strong competitive inhibitors of the endoprotease acrosin from mammalian spermatozoa 6.

Effect on ventilation-induced diaphragmatic contractile dysfunction: Controlled mechanical ventilation (CMV) has been shown to result in elevated diaphragmatic proteolysis and atrophy together with diaphragmatic contractile dysfunction. Administration of the protease inhibitor leupeptin concomitantly with mechanical ventilation completely prevented ventilation-induced diaphragmatic contractile dysfunction and atrophy 11.


1.     Aoyagi T,  Takeuchi T, Matsuzaki A, Kawamura K,  Kondo S, Hamada M, Maeda K, Umezawa H (1969). Leupeptins, new protease inhibitors from Actinomycetes. J. Antibiot., 22:283-286.

2.     Aoyagi T, Miyata S, Nanbo M, Kojima F, Matsuzaki M, Ishizuka M, Takeuchi T,  Umezawa H (1969). Biological activities of leupeptins., J. Antibiotics., 22:558-568.

3.     Knight CG (1980). Human cathepsin B. Biochein J., 189:447-453.

4.     Hori M, Hemmi M, Suzukake M, Hayashi H, Uehara Y, Takeuchi T, Umezawa H (1978). Biosynthesis of leupeptin.  J. Antibiotics., 31:95-98.

5.     Kawamura K, Kondo S, Maeda K, Umezawa H (1969). Structures and syntheses of leupeptins Pr-LL and Ac-LL. Chem. Pharm. Bull., 17:1902-1909.

6.     Borin G, Chessa G, Cavaggion G, Marchiori F, Müller-Esterl W (1981). Synthesis of leupeptins and inhibition of proteinases. Hoppe Seylers Z Physiol Chem., 362(11): 1435-1445.

7.     Book: Barrett, A. J. (1977). Proteinases in Mammalian Cells and Tissues p. 181-207, North- Holland Publishing Co., Amsterdam.

8.     Book: Bajkowski AS, Frankfater A (1976). Proteolysis and Physiological Regulation (Ribbons, D. W. & Brew, K., eds.), p. 132, Academic Press, New York.

9.     Kieran D, Greensmith L (2004). Inhibition of calpains, by treatment with leupeptin, improves motoneuron survival and muscle function in models of motoneuron degeneration. Neuroscience, 125(2):427-439.

10.  Xia ZF, Hollyoak M, Barrow RE, He F, Muller MJ, Herndon DN (1995). Superoxide dismutase and leupeptin prevent delayed reperfusion injury in the rat. J Burn Care Rehabil., 16:111-117.

11.  Maes K, Testelmans D, Powers S, Decramer M, Ghislaine Gayan-Ramirez G (2007). Leupeptin Inhibits Ventilator-induced Diaphragm Dysfunction in Rats. American Journal of Respiratory and Critical Care Medicine, 175:1134-1138.

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