The serine protease Corin is a novel modifier of the Agouti pathway

The hair follicle is a model system for studying epithelial-mesenchymal interactions during organogenesis. Although analysis of the epithelial contribution to these interactions has progressed rapidly, the lack of tools to manipulate gene expression in the mesenchymal component, the dermal papilla, has hampered progress towards understanding the contribution of these cells. In this work, Corin was identified in a screen to detect genes specifically expressed in the dermal papilla. It is expressed in the dermal papilla of all pelage hair follicle types from the earliest stages of their formation, but is not expressed elsewhere in the skin. Mutation of the Corin gene reveals that it is not required for morphogenesis of the hair follicle. However, analysis of the ;dirty blonde' phenotype of these mice reveals that the transmembrane protease encoded by Corin plays a critical role in specifying coat color and acts downstream of agouti gene expression as a suppressor of the agouti pathway.     

Structure-activity relationship within the serine protease inhibitors of the pacifastin family

The members of the Pacifastin family are serine protease inhibitors found in insects and crustacean. They are either small inhibitors (made of one consensus cysteine-rich motif) or proteins (4-9 motifs). Some of these inhibitors are characterized by a species selectivity for the trypsin inhibition. Structural data discriminate the small inhibitors that apparently look very similar into two groups. Interestingly, the inhibitors that display species selectivity fall in the same structural group.     

Squamous cell carcinoma antigen is a new member of the serine protease inhibitors

We have cloned cDNA of squamous cell carcinoma antigen. Sequence analysis of the complete 1711 basepairs SCC antigen cDNA revealed that it coded 390 amino acids and no typical signal sequence in the NH2-terminus. Northern blot analysis of human squamous cell poly(A)+ RNA using this cDNA insert as the probe showed a single RNA species of about 1.7 kilobases. The cDNA was expressed in Escherichia coli and the product was detected by immunological methods using antibodies against SCC antigen, indicating that this cDNA encodes the entire SCC antigen sequence. The amino acid homology search revealed that SCC antigen was a member of the serine protease inhibitors family.     

Quinolylhydrazones as novel inhibitors of Plasmodium falciparum serine protease PfSUB1

Plasmodium falciparum subtilisin-like protease 1 (PfSUB1) is a serine protease that plays key roles in the egress of the parasite from red blood cells and in preparing the released merozoites for the subsequent invasion of new erythrocytes. The development of potent and selective PfSUB1 inhibitors could pave the way to the discovery of potential antimalarial drugs endowed with an innovative mode of action and consequently able to overcome the current problems of resistance to established chemotherapies. Through the screening of a proprietary library of compounds against PfSUB1, we identified hydrazone 2 as a hit compound. Here we report a preliminary investigation of the structure-activity relationships for a class of PfSUB1 inhibitors related to our identified hit.     

The insect immune protein scolexin is a novel serine proteinase homolog

Scolexin is a coagulation-provoking plasma protein induced in response to bacterial or viral infection of larval Manduca sexta, a large lepidopterous insect. Here we report the isolation and sequencing of two cDNA clones that code for scolexin isoforms sharing 80% sequence identity. The scolexin sequences have low but recognizable sequence similarity to members of the chymotrypsin family and represent a new subfamily of chymotrypsin-like serine proteinases. Comparison with known structures reveals the conservation of key catalytic residues and a possible specificity for small nonpolar residues. Most remarkable is the absence of a canonical activation peptide cleavage site. This suggests that the regulation of scolexin activity will involve a novel activation mechanism.     

The novel inhibitors of serine proteases

Thirty optically active nonprotein α-amino acids and peptides based thereon have been screened for their ability to interact with bovine trypsin and proteinase K from Tritirachium album Limber, which belong to the group of serine proteases. Both structure-based drug design approach and determination of enzyme activity have been used to identify low molecular weight inhibitors of trypsin and proteinase K. Compounds have been selected that according to the docking analysis were able to interact with trypsin and proteinase K. Following the docking analysis measurement of enzymes activity (2R,3S)-β-hydroxyleucine and (2S,3R)-β-hydroxyleucine inhibited both enzymes activity, whereas (S)-α-methyl-β-phenylalanine, (R)-α-methyl-β-phenylalanine, (S)-allylglycine, (R)-allylglycine, (S)-α-allylalanine, (R)-α-allylalanine and allo-O-ethylthreonine inhibited only proteinase K; and N-formyl-(S)-methionyl-(2S,3R)-hydroxyleucine, N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine, N-formyl-(S)-methionyl-(S)-allylglycine and N-formyl-(S)-methionyl-(R)-allylglycine inhibited trypsin. It has been shown that inhibition of trypsin by (2R,3S)-β-hydroxyleucine and N-formyl-(S)-methionyl-(2R,3S)-hydroxyleucine is of a competitive mode.     

Macrocyclic inhibitors for the serine protease plasmin

Macrocyclic inhibitors for the serine protease plasmin were synthesized and evaluated. The inhibitors were constructed starting from a cyclohexanone core. This core was linked to either the C- or N-terminus of a peptide so that the inhibitors were designed to interact with the non-primed or primed binding sites of the protease. Macrocycles were prepared by connecting the side chain of Tyr or Trp, via a short linker, to one end of the peptide. The activities of the macrocyclic inhibitors, while modest, were up to 10-fold more potent than a related non-cyclic analog.     

Clonidine displacement from type 1 imidazoline receptor by p-aminobenzamidine,the prototype of trypsin-like serine protease inhibitors

p-Aminobenzamidine inhibits competitively the catalytic activity of enzymes that recognize preferentially the L-arginyl side chain and related structures. Notably, p-aminobenzamidine is considered as the prototype of trypsin-like serine protease inhibitors. Furthermore, p-aminobenzamidine inhibits the catalytic activity of nitric oxide synthase type I and type II as well as copper amine oxidase. Taking into account the structural similarity between p-aminobenzamidine, agmatine (the putative endogenous ligand of the membrane type 1 imidazoline receptor (I1-R)), and N-amidino-2-hydroxypyrrolidine (the product of agmatine oxidation by copper amine oxidase), the [3H]clonidine displacement from I1-R in rat heart membranes by p-aminobenzamidine was investigated. p-Aminobenzamidine is as effective as agmatine and N-amidino-2-hydroxypyrrolidine and more effective than the antihypertensive drug clonidine to displace [3H]clonidine from I1-R. Therefore, trypsin-like serine protease inhibitors structurally related to p-aminobenzamidine should be administrated under careful control.     

Structural and functional properties of a novel serine protease inhibiting peptide family in arthropods

Recently, several arthropod peptides that belong to a new serine protease inhibitor family were discovered. Three members (HI, PMP-D2=LMCI-1 and PMP-C=LMCI-2) were isolated from the migratory locust, Locusta migratoria. Five additional members (SGPI-1-5) were identified in the desert locust Schistocerca gregaria, and a heterodimeric serine protease inhibitor (pacifastin) was isolated from the hemolymph of the crayfish Pacifastacus leniusculus. The light chain of pacifastin constitutes the inhibitory subunit that has nine cysteine-rich domains (PLDs) that are homologous with the locust inhibitors. These locust inhibitors and PLDs share a conserved array of six cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa(3)-Cys), which are involved in an identical disulfide bridge pattern (Cys(1)-Cys(4), Cys(2)-Cys(6), Cys(3)-Cys(5)). The solution structures of LMCI-1 and LMCI-2 showed a similar, compact, globular folding, which is unique within the group of the small 'canonical' inhibitors. Moreover, the reactive site, including the P1-P'1 bond was thoroughly investigated by means of synthetic variants. However, the biological function(s) of the locust inhibitors is (are) not fully understood. LMCI-1 and LMCI-2 were shown to inhibit the endogenous proteolytic activating cascade of prophenoloxidase. Northern blot analysis indicated that the genes encoding the SGPI precursors are differentially expressed in a time-, stage- and hormone-dependent manner.     

Kunitz型丝氨酸蛋白酶抑制剂研究进展

Kunitz型丝氨酸蛋白酶抑制剂是一类普遍存在的蛋白酶抑制剂,在体内各项生命活动中扮演着重要角色。这类抑制剂结构稳定且富有特色,通常具有一个或几个串联存在的Kunitz结构域,能够以类似底物的方式与丝氨酸蛋白酶结合,从而抑制酶的活性。在功能方面,Kunitz型丝氨酸蛋白酶抑制剂参与凝血和纤维蛋白溶解、肿瘤免疫、炎症调节以及抵抗细菌、真菌感染等过程。文中就Kunitz型丝氨酸蛋白酶抑制剂研究进展作一综述,为新型Kunitz型丝氨酸蛋白酶抑制剂的开发提供研究思路。     

Macrocyclic inhibitors for the serine protease plasmin

Macrocyclic inhibitors for the serine protease plasmin were synthesized and evaluated. The inhibitors were constructed starting from a cyclohexanone core. This core was linked to either the C- or N-terminus of a peptide so that the inhibitors were designed to interact with the non-primed or primed binding sites of the protease. Macrocycles were prepared by connecting the side chain of Tyr or Trp, via a short linker, to one end of the peptide. The activities of the macrocyclic inhibitors, while modest, were up to 10-fold more potent than a related non-cyclic analog.     

The native metastability and misfolding of serine protease inhibitors

The native metastability of serine protease inhibitors (serpins) is believed to facilitate the conformational change required for biological function. However, energetically unfavorable structural features that contribute to metastability of the native serpin conformation, such as buried polar groups, cavities, and over-packing of side-chains, also appear to hinder proper folding. Hence, folding of serpin polypeptides appears prone to error; in particular, the folding polypeptides are readily diverted toward a non-productive folding pathway culminating in a more stable but inactive conformation. In a survey of deficient serpin mutants, various folding defects, such as retarded protein folding, destabilized native conformation, and spontaneous conversion into more stable, inactive conformations such as the latent form and loop-sheet polymers, have been discovered.     

Prostasin is a glycosylphosphatidylinositol-anchored active serine protease

A recombinant human prostasin serine protease was expressed in several human cell lines. Subcellular fractionation showed that this serine protease is synthesized as a membrane-bound protein while a free-form prostasin is secreted into the culture medium. Prostasin was identified in nuclear and membrane fractions. Membrane-bound prostasin can be released by phosphatidylinositol-specific phospholipase C treatment, or labeled by [(3)H]ethanolamine, indicating a glycosylphosphatidylinositol anchorage. A prostasin-binding protein was identified in mouse and human seminal vesicle fluid. Both the secreted and the membrane-bound prostasin were able to form a covalently linked 82-kDa complex when incubated with seminal vesicle fluid. The complex formation between prostasin and the prostasin-binding protein was inhibited by a prostasin antibody, heparin, and serine protease inhibitors. Prostasin's serine protease activity was inhibited when bound to the prostasin-binding protein in mouse seminal vesicle fluid. This study indicates that prostasin is an active serine protease in its membrane-bound form.     

NMR studies of fluorinated serine protease inhibitors

Powers and co-workers have provided evidence that thiobenzyl N-heptafluorobutyrylanthranilate (I) is an extremely potent inhibitor of serine proteases, especially alpha-chymotrypsin (Teshima, T., Griffin, J. C., and Powers, J. C. (1982) J. Biol. Chem. 257, 5085-5091). We have prepared additional derivatives of this structure in which fluorine substitutions have been made on the aromatic rings and have attempted to carry out fluorine NMR studies of the interaction of Powers' compound and these new derivatives with chymotrypsin. The solubility of all inhibitors examined in solvent systems compatible with the retention of native enzyme structure is extremely low. While some nmr evidence for complex formation could be obtained, preparations of the complexes examined were metastable and precipitation of the inhibitor eventually limits the amount of complex that can be present in solution to such low levels that nmr experiments are impractical. An unusual effect of solvent composition on fluorine chemical shifts suggests that the conformation of the inhibitors in aqueous solution and when bound to the enzyme is different from that in organic solvents.     

Parallel synthesis of isatin-based serine protease inhibitors

The synthesis of N-functionalised isatins using parallel, solution synthesis is described. Functionalised polymers were employed as stoichiometric and catalytic reagents as well as purification media in the exercise, and the derivatives were screened against a panel of serine proteases; high percentage inhibition was observed in several cases.     

Factor VIIa inhibitors: target hopping in the serine protease family using X-ray structure determination

Selective factor VIIa-tissue factor complex (FVIIa/TF) inhibition is regarded as a promising target for developing new anticoagulant drugs. Compound 1 was discovered from focused screening of serine protease-directed compounds from our internal collection. Using parallel synthesis supported by structure-based drug design, we identified peptidemimetic FVIIa/TF inhibitors (compounds 4-11) containing L-Gln or L-Met as the P2 moiety. However, these compounds lacked the selectivity of other serine proteases in the coagulation cascade, especially thrombin. Further optimization of these compounds was carried out with a focus on the P4 moiety. Among the optimized compounds, 12b-f showed improved selectivity.     

Neutral inhibitors of the serine protease factor Xa.

A neutral inhibitor of the serine protease factor Xa was identified via a high-throughput screen of a commercial library. The initial lead 1 demonstrated reversible and competitive inhibition kinetics for factor Xa and possessed a high degree of selectivity versus other related serine proteases. Initial modeling efforts and the generation of a series of analogues of 1 are described.     

Reelin is a serine protease of the extracellular matrix.

Reelin is an extracellular matrix protein that plays a pivotal role in development of the central nervous system. Reelin is also expressed in the adult brain, notably in the cerebral cortex, where it might play a role in synaptic plasticity. The mechanism of action of reelin at the molecular level has been the subject of several hypotheses. Here we show that reelin is a serine protease and that proteolytic activity is relevant to its function, since (i) Reelin expression in HEK 293T cells impairs their ability to adhere to fibronectin-coated surfaces, and adhesion to fibronectin is restored by micromolar concentrations of diisopropyl phosphorofluoridate, a serine hydrolase inhibitor; (ii) purified Reelin binds FP-Peg-biotin, a trap probe which irreversibly binds to serine residues located in active catalytic sites of serine hydrolases; (iii) purified Reelin rapidly degrades fibronectin and laminin, while collagen IV is degraded at a much slower rate; fibronectin degradation is inhibited by inhibitors of serine proteases, and by monoclonal antibody CR-50, an antibody known to block the function of Reelin both in vitro and in vivo. The proteolytic activity of Reelin on adhesion molecules of the extracellular matrix and/or receptors on neurons may explain how Reelin regulates neuronal migration and synaptic plasticity.