Metalloproteases

Zinc proteases are metalloenzymes containing tightly bound zinc examples are carboxypeptidases A and B, collagenase, and thermolysin. The zinc atom is bound to the imidazole moiety of two histidines and the carboxylate of Glu the fourth ligand is a molecule of H20. 

The peptide carbonyl oxygen interacts with zinc, withdrawing electrons from carbon. Water is displaced from zinc. Glu270 accepts a proton from water - it acts as a general base - and the hydroxyl anion of water acts as a nucleophile to attack the carbonyl carbon of the peptide bond and forms a negatively charged tetrahedral 

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Systematic screening experiments have identified more than 100 synthetic compounds with potent antiangiogenic activity. The mode of action for most of these molecules is not well understood, but some of the 40 compounds are well advanced in clinical trials (Table 3). The first substance to have entered clinical trials was the Fumagillin-derivative AGM 1470. Fumagillin is an antibiotic which inhibits bFGF- and PDGF-induced endothelial cell proliferation. The mechanism of action of AGM 1470 is poorly understood, but it was shown that it binds and inhibits the metalloprotease methionine aminopeptidase (MetAp-2). 

The anthrax toxin is a tripartite toxin and consists ofthe binding component protective antigen (PA), the lethal factor (LF), which is a metalloprotease, and the edema factor (EF), which is a calmodulin-dependent adenylyl-cyclase. Both enzyme components are translocated via PA into target cells. PA is activated by furin-induced cleavage and forms heptamers, which are similar to the binding components of C2 toxin and iota toxin. In the low pH compartment of endosomes, the heptamers form pores to allow translocation of LF and EF. LF cleaves six of the seven MEKs (MAPK-kinases) thereby inhibiting these enzymes. The functional consequence is the blockade of the MAPK pathways that control cell proliferation, differentiation, inflammation, stress response, and survival. Whether this is the reason for the LT-induced cell death of macrophages is not clear [1]. 

One intensively investigated feature of the inflammatory process in COPD is the release of proteases from neutrophils and monocytic cells that destroy elastin and other components of the interstitial matrix (Table 1). The best studied protease is neutrophil elastase. Independent of its elastolytic activity, neutrophil elastase is a potent secretagogue. More recently matrix metalloproteases (MMP) have received increasing attention, in particular MMP 12 (macrophages elastase). To which extent and how exactly these proteases become activated is not clear at present. 

ROS, reactive oxygen species AHR, airway hyperresponsiveness MMP, matrix metalloprotease. Correlates with cough and chronic bronchitis. bNumber increases with disease severity. 

The light chains of the clostridial neurotoxins are metalloproteases with exclusive specificity for neuronal SNAREs. TeNT, BoNTs B,D,F, and G cleave synapto-brevin, BoNTs A and E SNAP-25, and BoNT/Cl syntaxin, and to a lesser extent also SNAP-25. Cleavage of any of the SNAREs causes complete and irreversible block of synaptic transmission. 

Endothelin Concerting Enzymes (ECEs) belong to the family of metalloproteases that catalyze the proteolytic activation of big endothelins. 

In some cases, receptor inactivation, e.g., of the V2 vasopressin receptor, is mediated by agonist-induced enzymatic cleavage of the GPCR. This nonendocytic proteolysis is promoted by a plasma membrane-associated metalloprotease. Proteinase-activated receptors (PARs) such as the thrombin receptor also follow a distinctly different pathway. PARs require the enzymatic cleavage of their N terminus, and the newly generated N terminus activates the receptor. Once... 

A peptide, once released, is not subject to reuptake like most transmitters, but is broken down by membrane peptidases. There are no known peptide transporters so that reuptake and re-use are not likely. The peptidases are predominantly membrane bound at the synapse and many are metalloproteases in that they have a metal moiety, most often zinc, near the active site. These enzymes are generally selective for particular... 

The second example of property space applications concerns the dipephde camo-sine (P-alanine-L-histidine, see Fig. 1.4) which represents the archetype of a series of histidine-containing dipeptides whose full physiological role remains poorly understood despite extensive studies in recent years [18-20]. Carnosine is synthesized by carnosine synthetase and hydrolyzed by dipeptidases (also called camosi-nases) which belong to the metalloproteases [21]. 

Comparison with other Metalloproteases Substrate-binding Pockets... 

While PDF was originally proposed to be a zinc-metalloprotease [51], it is now generally accepted that Fe is the physiologically relevant metal ion occupying the active site in vivo [52], The native forms of most PDF enzymes are highly unstable due to propensity to oxidation, rendering them difficult to purify [53, 54], However, the Fe can be suitably replaced by either or Co, both of which provide a stable enzyme and main-... 

Although native PDFs utilise Fe rather than Zn, the PDF metal-binding site is still very representative of a traditional zinc-metalloprotease [57], containing a conserved HEXXH motif. Crystal structures of the Fe ", Ni ", ... 

In the active site of PDF proteins, three substrate-binding pockets exist along with the metal-binding site. Using standard metalloprotease nomenclature. 

Another PDF inhibitor series derived from comparisons with known metalloprotease inhibitor classes has been reported by Merck [68]. Their study investigated a small set of peptide aldehyde inhibitors, postulating that the aldehyde might bind to the metal centre in the form of a hydrate,... 

More recently, screening efforts at Novartis have identified a hydroxamic acid containing a benzothiazinone ring system (32) [108]. This inhibitor is very potent versus S. aureus Ni -PDF (<5nM) and displays good selectivity versus matrix metalloprotease-2 (MMP-2) and MMP-13. Unfortunately (32), and all other analogues prepared, such as carbon isosteres (33), sulfones (34), N-substituted analogues (35) and N-formyl-N-hydroxylamines (36), lacked appreciable antibacterial activity in spite of their potent enzyme inhibitory activity. Further studies performed by Novartis suggest that these molecules are unable to penetrate the outer cell membrane of E. coli, and may bind to the cell membrane of S. aureus [108]. 

Kanbe K, Takagishi K, Chen Q. Stimulation of matrix metalloprotease 3 release from human chondrocytes by the interaction of stromal cell-derived factor 1 and CXC chemokine receptor 4. Arthritis Rheum 2002 46(1) 130-137. 

Browner MF, Smith WW, Castelhano AL. Matrilysin-inhibitor complexes common themes among metalloproteases. Biochemistry 1995 34 6602-6610. 

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