Leupeptin

More than 50 endogenous and exogenous inhibitors of the calpains have been described as either transition-state reversible or irreversible inhibitors. The first transition-state inhibitors were the peptide aldehydes (e.g., leupeptin). Using this compound, new ones were synthesized that exhibited improved membrane permeability and calpain specificity (e.g., calpeptin). Other groups of inhibitors have since been discovered a-dicarbonyls (originally developed as serine protease inhibitors), nonpeptide quinolinecarboxamides,... 

The action of a peptidase can be neutralized by an inhibitor. Some inhibitors are very broad in their action and are capable of inhibiting many different peptidases, including peptidases of different catalytic types. Some inhibitors are assumed to be specific for a particular catalytic type, but can inhibit peptidases of different types. Leupeptin, for example, is widely used as an inhibitor of serine peptidases from family SI, but it is also known to inhibit cysteine peptidases from family Cl. Cysteine pqrtidase inhibitors such as iodoacetic acid interact with the thiol of the catalytic cysteine. However, this reduction can occur on any thiol group and can affect other, predominantly intracellular, peptidases with a thiol dependency. One example is thimet oligopepti-dase. Metal chelators such as EDTA can inhibit meta-llopeptidases, but can also affect peptidases that have a requirement for metal ions that is indq>endent of their catalytic activity, such as the calcium-dependent cysteine endopqrtidase calpain 1. 

As shown in Table I, free HRP is poorly transported across MDCK cells but, when conjugated to a PLL carrier, HRP transport is increased considerably. The existence of a proteolytic compartment involved in the transcytotic digestion of HRP-S-PLL conjugate was further confirmed by the finding that when PLL was replaced by PDL, the transport of HRP was completely abolished (Table I) (8). In addition, when protease inhibitors such as leupeptin were added to the basal medium, the transcytosis of HRP was also significantly decreased (Table I). We have previously reported that the partial degradation of HRP-S-PLL was not inhibited by lysosomotropic amines (<8), indicating that this proteolytic process does not occur in lysosomes. 

Smith, R. S. and Snyder, R. E. Reversal of rapid axonal transport at a lesion leupeptin inhibits reversed protein transport, but does not inhibit reversed organelle transport. Brain Res. 552 215-227,1991. 

Roots, B. Neurofilament accumulation induced in synapses by leupeptin. Science 221 971-972,1983. 

A strong link between the phosphorylation of nucleolin, its proteolysis and the production of ribosomal RNA has been observed (Bouche et al, 1984 Bourbon et al, 1983 Warrener and Petryshyn, 1991). The inhibition of proteolysis using leupeptin leads to a lower rRNA transcription in an in vitro transcription system (Bouche et al, 1984). In another series of experiments, the injection of nucleolin antiserum leads to 2-3.5 fold stimulation of pre-rRNA synthesis in Chironomus tentans salivary glands (Egyhazi et al, 1988), although it was not clearly demonstrated that these antibodies blocked specifically the homolog of nucleolin in this species. A model was proposed based on these observations where nucleolin was... 

Fig. 5.5 On-line HPLC bioactivity screening of a mixture of five flavonoids spiked with two cathepsin B inhibitors, E-64 and leupeptin using acetylcholinesterase as biological target. MS instrument Shimadzu LCMS-2010 single-stage quadrupole mass spectrometer, (a) TIC chromatogram of the mixture, scan range m/z 75-750 (b) mass chromatogram of AMC (m/z 176) (c) mass...

Mammalian Cell Protease Inhibitor CocktaiL These should contain AEBSF, pepstatin A, E-64, bestatin, leupeptin, and aprotinin. (Metal chelators can be added to suppress the activity of calcium ion-dependent proteases such as calpain. Again, one must determine whether the protein or enzyme being purified does not require a divalent metal cofactor for stabihty or activity.)... 

Figure 17.3 Schematic representation of the design of the symmetric cathepsin K inhibitor diacylaminomethyl ketone (1,3-bis[[A/-[(phenylmethoxy)carbonyl]-L-leucyl]amino]-2-propanone), based on the crystal structures of papain bound to leupeptin (Leu-Leu-Arg-aldehyde) and to Cbz-Leu-Leu-Leu-aldehyde, and an example of its further optimization.

Three preliminary experiments were carried out the first done as described above, the second utilized a modified buffer containing protectants, and the third utilized both the modified buffer and a hemoglobin gel step to remove proteases. The modified buffer had the following recipe 50 mM EPPS-KOH buffer at pH 8.6, 20% (v/v) glycerol, 1.0 mM EDTA, 1.0 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.05 mM leupeptin, and 0.05% / -mercaptoethanol. 

Other outstanding structures from marine Alteromonads are thiomarinol [98] (6) and the related holothines, or the bromoalterochromides (7) [99]. The latter are yellow chromopeptides due to their polyene chain however, simple peptides (e.g. the leupeptins) have also been described. 

Levtpeptin acid is redviced to leupeptin by leupeptin acid reductase which requires ATP and NAOPH. This enzyme is located in cell membranes. 

Leqpeptln-acld reductase is Inhibited by leupeptin, but not by elastatlnal (42). This specific inhibition indicates that leppeptln acid which has no antiprotease activity is produced within cells on the other h uld, the letq>eptin prodviced from leupeptin acid is not accvimulated in cells, but is rapidly released extracellularly. 

Compounds like lev ptln acid, which are the precursors of secondary metabolites, should not have cytotoxicity, and inmany cases, the enzyme involved in the last step of the biosynthesis may be inhibited by the last product as in the case of leupeptin acid reductase. It is also possible that multifunctional enzyme conplexes may be involved in the synthesis of many secondcury metabolites. 

A number of inhibitors of carcinogen- and radiation-induced ceU transformation have been identified. These include protease inhibitors (antipain, leupeptin, soybean tryspin inhibitor), vitamin A analogs (retinoids), vitamin C (ascorbic add), glucocorticoids, and lymphotoxins (Kennedy, 1984). 

A group of inhibitors such as leupeptin have C-terminal aldehyde groups. Small oligopeptides with this structure and with appropriate specificitydetermining side chains form tetrahedral hemiace-tals, which may mimic transition state structures, at the active sites of the target enzymes/"1 ... 

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