Phosphinic peptides, the first potent inhibitors of astacin, behave as extremely slow-binding inhibitors.
TL;DR: The phosphinic peptides may provide a rational basis for the design of drugs directed towards other members of the astacin family which, like bone morphogenetic protein 1 (BMP1), have become targets of pharmacological research.
Abstract: A series of phosphinic pseudo-peptides varying in length and composition have been designed as inhibitors of the crayfish zinc endopeptidase astacin, the prototype of the astacin family and of the metzincin superfamily of metalloproteinases. The most efficient phosphinic peptide, fluorenylmethyloxycarbonyl-Pro-Lys-PhePsi(PO2CH2)Ala-P ro-Leu-Val, binds to astacin with a Ki value of 42 nM, which is about three orders of magnitude below the corresponding values for previously used hydroxamic acid derivatives. However, the rate constants for association (kon = 96.8 M-1.s-1) and dissociation (koff = 4.1 x 10(-6) s-1) are evidence for the extremely slow binding behaviour of this compound. N-terminally or C-terminally truncated phosphinic analogues of this parent molecule are much less potent, indicating a critical role of the peptide size on the potency. In particular, omission of the N-terminal proline residue leads to a 40-fold increase in Ki which is mostly due to a 75-fold higher koff value. These findings are consistent with the previously solved crystal structure of astacin complexed with one of the phosphinic peptides, benzyloxycarbonyl-Pro-Lys-PhePsi(PO2CH2)Ala-Pro-O-methyl, Ki = 14 microM [Grams, Dive, Yiotakis, Yiallouros, Vassiliou, Zwilling, Bode and Stocker (1996) Nature Struct. Biol. 3, 671-675]. This structure also reveals that the phosphinic group binds to the active site as a transition-state analogue. The extremely slow binding behaviour of the phosphinic peptides is discussed in the light of the conformational changes involving a unique 'tyrosine switch' in the structure of astacin upon inhibitor binding. The phosphinic peptides may provide a rational basis for the design of drugs directed towards other members of the astacin family which, like bone morphogenetic protein 1 (BMP1; i.e. the procollagen C-proteinase), have become targets of pharmacological research.
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TL;DR: A structure-based derivation of the AHNP represents a novel strategy for the design of receptor-specific tumor therapies for p185HER2/neu-expressing human cancers.
Abstract: Monoclonal antibodies specific for the p185HER2/neu growth factor receptor represent a significant advance in receptor-based therapy for p185HER2/neu-expressing human cancers. We have used a structure-based approach to develop a small (1.5 kDa) exocyclic anti-HER2/neu peptide mimic (AHNP) functionally similar to an anti-p185HER2/neu monoclonal antibody, 4D5 (Herceptin). The AHNP mimetic specifically binds to p185HER2/neu with high affinity (KD=300 nM). This results in inhibition of proliferation of p185HER2/neu-overexpressing tumor cells, and inhibition of colony formation in vitro and growth of p185HER2/neu-expressing tumors in athymic mice. In addition, the mimetic sensitizes the tumor cells to apoptosis when used in conjunction with ionizing radiation or chemotherapeutic agents. A comparison of the molar quantities of the Herceptin antibody and the AHNP mimetic required for inhibiting cell growth and anchorage-independent growth showed generally similar activities. The structure-based derivation of the AHNP represents a novel strategy for the design of receptor-specific tumor therapies.
213 citations
TL;DR: The in vivo substrates of meprins are unknown, but the abundant expression of these proteinases in the epithelial cells of the intestine, kidney and skin provide clues to their functions.
178 citations
TL;DR: RXP 407, a highly potent and selective inhibitor of the N-terminal active site of wild ACE, may lead to a new generation of ACE inhibitors able to block in vivo only a subset of the different functions regulated by ACE.
Abstract: The human somatic angiotensin converting enzyme (ACE) contains two homologous domains, each bearing a zinc-dependent active site. All of the synthetic inhibitors of this enzyme used in clinical applications interact with these two active sites to a similar extent. Recently, several lines of evidence have suggested that the N-terminal active site of ACE might be involved in specific hydrolysis of some important physiological substrates, like Acetyl-Seryl-Aspartyl-Lysyl-Proline, a negative regulator of hematopoietic stem cell differentiation and proliferation. These findings have stimulated studies aimed at identifying new ACE inhibitors able to block only one of the two active sites of this enzyme. By screening phosphinic peptide libraries, we discovered a phosphinic peptide Ac-Asp-(L)Pheψ(PO2-CH2)(L)Ala-Ala-NH2, called RXP 407, which is able to differentiate the two ACE active sites, with a dissociation constant three orders of magnitude lower for the N-domain of the enzyme. The usefulness of a combinatorial chemistry approach to develop new lead structures is underscored by the unusual chemical structure of RXP 407, as compared with classical ACE inhibitors. As a highly potent and selective inhibitor of the N-terminal active site of wild ACE (Ki = 12 nM), RXP 407, which is metabolically stable in vivo, may lead to a new generation of ACE inhibitors able to block in vivo only a subset of the different functions regulated by ACE.
165 citations
TL;DR: This review is focused on the most promising sortase A inhibitor compounds that are currently in development as leads towards a new class of anti-infective drugs that are urgently needed to help combat the alarming increase in antimicrobial resistance.
139 citations
01 Dec 2014
TL;DR: The most promising sortase A inhibitor compounds that are currently in development as leads towards a new class of anti-infective drugs that are urgently needed to help combat the alarming increase in antimicrobial resistance are presented in this paper.
Abstract: Sortase A is a membrane enzyme responsible for the anchoring of surface-exposed proteins to the cell wall envelope of Gram-positive bacteria. As a well-studied member of the sortase subfamily catalysing the cell wall anchoring of important virulence factors to the surface of staphylococci, enterococci and streptococci, sortase A plays a critical role in Gram-positive bacterial pathogenesis. It is thus considered a promising target for the development of new anti-infective drugs that aim to interfere with important Gram-positive virulence mechanisms, such as adhesion to host tissues, evasion of host defences, and biofilm formation. The additional properties of sortase A as an enzyme that is not required for Gram-positive bacterial growth or viability and is conveniently located on the cell membrane making it more accessible to inhibitor targeting, constitute additional reasons reinforcing the view that sortase A is an ideal target for anti-virulence drug development. Many inhibitors of sortase A have been identified to date using high-throughput or in silico screening of compound libraries (synthetic or natural), and while many have proved useful tools for probing the action model of the enzyme, several are also promising candidates for the development into potent inhibitors. This review is focused on the most promising sortase A inhibitor compounds that are currently in development as leads towards a new class of anti-infective drugs that are urgently needed to help combat the alarming increase in antimicrobial resistance.
118 citations
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TL;DR: The Xenopus laevis DNA fragment encoding a protein homologous with human bone morphogenetic protein-1 (BMP-1) was amplified by polymerase chain reaction (PCR) from cDNA derived from stage 26 (st.26) embryonic RNA to isolate cDNA clones by screening of a X.laevis st.24 embryonic cDNA library.
42 citations
TL;DR: Pseudo-substrate analogues of collagenase from Corynebacterium rathayii, in which the scissile peptide bond is replaced by a phosphinic moiety, were synthesized and evaluated as inhibitors of this enzyme.
Abstract: Pseudo-substrate analogues of collagenase from Corynebacterium rathayii, in which the scissile peptide bond is replaced by a phosphinic moiety, were synthesized and evaluated as inhibitors of this enzyme The phosphinic tetrapeptide, Z-Phe-psi(PO2CH2)-Gly-Pro-Nle (1), was found to be a potent inhibitor of collagenase with a Ki value of 8 nM Increasing the length of the phosphinic-containing inhibitors from tetra- to hepta-peptide size further improves the potency of these compounds The heptapeptide analogue, Z-Phe-Gly-Pro-Phe-psi(PO2CH2)-Gly-Pro-Nle-OMe, with a Ki value of 06 nM, is the most potent inhibitor reported to date for bacterial collagenases A comparison between the phosphinic analogue Z-Phe-psi(PO2CH2)-Gly-Pro-Nle (1) and the phosphonamide peptide Z-Phe-psi(PO2NH)-Gly-Pro-Nle (2) shows that for bacterial collagenase the replacement of a CH2 by an NH group results only in a modest increase in affinity from Ki = 8 nM for compound 1 to Ki = 6 nM for compound 2 Most of the phosphorus-containing inhibitors of this series are slow- or slow-tight-binding inhibitors with second-order rate constants for association and dissociation varying respectively for the kon values from 1 x 10(3) to 26 x 10(3) M-1s-1 and for the koff values from 3 x 10(-4) to 2 x 10(-5) s-1 Interestingly, the lower affinity of the molecule containing a D residue in the P1 position of the inhibitor, compared with the molecule with an L residue in this position, is mainly the consequence of a lower rate constant for association of these D stereoisomers with the enzyme This study demonstrates that phosphinic peptide analogues are potent inhibitors of a bacterial collagenase The development of new phosphinic peptides should lead to the discovery of potent inhibitors of other zinc metalloproteases Details of how the analogues were synthesized are given in Supplementary Publication SUP 50176 (14 pages), which has been deposited with the British Library Document Supply Centre, Boston Spa, Wetherby, W Yorkshire LS23 7BQ, from whom copies can be obtained on the terms indicated in Biochem J (1994) 297, 9
30 citations
TL;DR: Reversible synthetic inhibitors are characterized for Astacus protease, a 22,614-Da zinc containing neutral endopeptidase from the digestive tract of crayfish and it was determined that cysteine was acting as a weak chelator and slowly establishing an equilibrium between metallo- and apoenzyme.
15 citations