We demonstrated previously that the switch from nonmetastatic to highly metastatic phenotype of human melanoma cells is directly related to secretion of procathepsin L form. This cysteine proteinase was identified on the basis of its property to cleave human C3, the third component of complement. In an attempt to control procathepsin L secretion, we have recently generated an anti-cathepsin L single chain variable fragment (ScFv) from an anti-cathepsin L monoclonal antibody generated against recombinant cathepsin L. We herein selected clones stably transfected with this anti-cathepsin L ScFv and analyzed them for changes in tumor growth and metastasis. We show that in stably transfected clones, anticathepsin L ScFv strongly inhibited the secretion of procathepsin L without modifying the intracellular amount or processing pattern of cathepsin L forms. Confocal analysis demonstrated colocalization of endogenous cathepsin L and anti-cathepsin L ScFv. In addition, expression of this ScFv strongly inhibited generation of tumor and metastasis by these human melanoma clones in nude mice. In vivo, the anti-cathepsin L ScFv-transfected cells produced tumors with decreased vascularization (angiogenesis) concomitant with increased apoptosis of tumor cells. Matrigel assay also demonstrated that melanoma invasiveness was completely abolished. Thus, this is the first demonstration that anti-cathepsin L ScFv could be used to inhibit the tumorigenic and metastatic phenotype of human melanoma, depending on procathepsin L secretion, and could therefore be used as a molecular tool in a therapeutic cellular approach.

In the treatment of cardiovascular diseases, it could be of therapeutic interest to associate the hypotensive effects resulting from the inhibition of angiotensin II formation, ensured by endothelial angiotensin-converting enzyme (ACE), with the diuretic and natriuretic responses due to the protection of the endogenous atrial natriuretic peptide (ANP) from inactivation by epithelial neutral endopeptidase (NEP). However, an investigation of this hypothesis requires an orally active compound able to jointly inhibit ACE and NEP. Dual inhibitors have therefore been designed by a rational approach, based on the characteristics of the active sites of both enzymes, which belong to the same family of zinc metallopeptidases, and on the structures of their most potent and selective inhibitors. As both NEP and ACE contain a large S'1-S'2 domain able to accommodate aromatic residues, the cyclic ACE inhibitor 3-(mercaptomethyl)-3,4,5,6-tetrahydro-2-oxo-1H-1-benzazocine-1-ace tic acid was selected as a template. Various aliphatic constraints were introduced on the benzyl moiety of the potent NEP inhibitor N-[2-(mercaptomethyl)-3-phenylpropanoyl]-L-tyrosine (IC50 NEP = 2 nM, IC50 ACE = 25 nM) to improve the fit between the computed most stable conformers of these molecules and the ACE template. New dual inhibitors, of general formula, N-[2(R,S)-(mercaptomethyl)-3(R,S)-phenylbutanoyl]-L-amino acid with IC50 values in the nanomolar range for both enzymes were generated by this approach. The separation of the four stereoisomers using chiral amines and the stereoselective synthesis of the 2-(mercaptomethyl)-3-phenylbutanoyl moiety showed that inhibitors with the 2S,3R configuration are the most potent on both NEP and ACE. The "in vivo" potency of various prodrugs of these inhibitors to inhibit ACE activity in lung and NEP activity in kidney was measured after oral administration in mice. From this pharmacokinetical study the most potent dual inhibitor RB 105 (N-[(2S,3R)-2-(mercaptomethyl)-3-phenylbutanoyl-L-alanine (compound 44c) (KI NEP 1.7 nM, KI ACE 4.5 nM) and its most efficient in vivo prodrug mixanpril, [N-[(2S,3R)-2-[(benzoylthio)methyl]-3-phenylbutanoyl]-L-alan ine (compound 18) (ED50 NEP approximately 1 mg/kg, ED50 ACE approximately 7 mg/kg) were selected. Competition experiments with a tritiated inhibitor of ACE or NEP bound to mouse lung and kidney membranes respectively showed that mixanpril has a long duration of action (> 8 h). As expected, after i.v. administration in the spontaneously hypertensive rat (SHR), RB 105 decreased blood pressure and increased diuresis and natriuresis.(ABSTRACT TRUNCATED AT 400 WORDS)

In the zinc metallopeptidases produced by the genus Bacillus, an active site histidine has been proposed to either stabilize the transition state in catalysis by donating a hydrogen bond to the hydrated peptide (Matthews, B. W. (1988) Acc. Chem. Res. 21, 333-340) or to polarize a water molecule, which subsequently attacks the peptidyl bond (Mock, W. L., and Aksamawati, M. (1994) Biochem. J. 302, 57-68). Site-directed mutagenesis techniques have been used to change this residue in the zinc endopeptidase from Bacillus stearothermophillus to either phenylalanine or alanine. At pH 7.0, the kcat/Km values of the substrate leucine enkephalin for the phenylalanine and alanine mutants were reduced by factors of 430- and 500-fold, respectively, as compared with the wild-type enzyme, mostly due to changes in kcat. In addition, the enzymatic activities of the mutant enzymes showed little pH dependence in the alkaline range, unlike the wild-type enzyme. The mutations did not greatly alter the binding affinities of inhibitors containing sulfydryl groups to chelate the active site zinc, while those of inhibitors containing hydroxamate or carboxylate zinc-chelating groups were increased between 80- and 250-fold. The largest change in the binding affinity of an inhibitor (> 5 orders of magnitude) was found with the proposed transition state mimic, phosphoramidon. The results are generally in agreement with x-ray crystallography studies and favor the involvement of the active site histidine in transition state binding.

Cystic fibrosis results from reduced cystic fibrosis transmembrane conductance regulator protein activity leading to defective epithelial ion transport. Ca2+-activated Cl− channels mediate physiological functions independently of cystic fibrosis transmembrane conductance regulator. Anoctamin 1 (ANO1/TMEM16A) was identified as the major Ca2+-activated Cl− channel in airway epithelial cells, and we recently demonstrated that downregulation of the anoctamin 1 channel in cystic fibrosis patients contributes to disease severity via an unknown mechanism. Here we show that microRNA-9 (miR-9) contributes to cystic fibrosis and downregulates anoctamin 1 by directly targeting its 3′UTR. We present a potential therapy based on blockage of miR-9 binding to the 3′UTR by using a microRNA target site blocker to increase anoctamin 1 activity and thus compensate for the cystic fibrosis transmembrane conductance regulator deficiency. The target site blocker is tested in in vitro and in mouse models of cystic fibrosis, and could be considered as an alternative strategy to treat cystic fibrosis.