Abstracts of the 24th international isotope society (UK group) symposium: synthesis and applications of labelled compounds 2015.

The 24th annual symposium of the International Isotope Society's United Kingdom Group took place at the Møller Centre, Churchill College, Cambridge, UK on Friday 6th November 2015. The meeting was attended by 77 delegates from academia and industry, the life sciences, chemical, radiochemical and scientific instrument suppliers. Delegates were welcomed by Dr Ken Lawrie (GlaxoSmithKline, UK, chair of the IIS UK group). The subsequent scientific programme consisted of oral presentations, short 'flash' presentations in association with particular posters and poster presentations. The scientific areas covered included isotopic synthesis, regulatory issues, applications of labelled compounds in imaging, isotopic separation and novel chemistry with potential implications for isotopic synthesis. Both short-lived and long-lived isotopes were represented, as were stable isotopes. The symposium was divided into a morning session chaired by Dr Rebekka Hueting (University of Oxford, UK) and afternoon sessions chaired by Dr Sofia Pascu (University of Bath, UK) and by Dr Alan Dowling (Syngenta, UK). The UK meeting concluded with remarks from Dr Ken Lawrie (GlaxoSmithKline, UK).

Covalent modification of matrix metalloproteinases by a photoaffinity probe: influence of nucleophilicity and flexibility of the residue in position 241.

A photoaffinity probe, developed for the specific labeling of matrix metalloproteinase (MMP) active sites, was recently shown to covalently modify a single residue in human MMP-12, namely, Lys(241), by reacting selectively with the side chain epsilon-amino group of that residue. The residue in position 241 of MMPs is not conserved; thus, variability in this position may be responsible for the dispersion in cross-linking yield observed between MMPs when labeled by this photoaffinity probe. By studying the pH dependence of the labeling properties of this probe toward different MMPs (MMP-12, MMP-3, MMP-9, and various mutants of human MMP-12) and identifying the site of covalent modification of MMP-3 by this probe, our new data demonstrated that the nucleophilicity of the residue in position 241 plays a key role in determining the cross-linking yield of MMP modification by the probe. However, these studies also reveal that subtle additional structural parameters, including local conformation and flexibility, of the residue in position 241 should also be taken into consideration, a property adding a further degree of complexity in our understanding of the photolabeling probe reactivity and in designing optimal photoaffinity probes for performing functional proteomic studies of zinc proteinases like MMPs.

Delineation of chicken cathepsin L secondary structure; relationship between pH dependence activity and helix content.

The secondary structure of the recently sequenced chicken liver cathepsin L (EC 3.4.22.15) has been studied both by circular dichroism and a predictive method. The structural data provided by these approaches allow us to underline the extent of the structural similarities between cathepsin L and papain, one of the best known proteins in the cysteine proteinase family. The predictive method of Garnier et al. (J. Mol. Biol. 120 (1978) 97-120) is used to locate alpha-helix and beta-sheet segments in the cathepsin L sequence. An optimization of decision constants has been performed, using circular dichroism data, to improve good predictions. The combination of these approaches lead us to suggest that the location of ordered structures observed in papain is maintained in cathepsin L, but with an additional alpha-helix in the middle region (residues 85-108) of cathepsin L. Furthermore, we show that cathepsin L inactivation at neutral pH is correlated to the lost of alpha-helix content (40% at pH 5.8 and 17% at pH 7.0) in this protein. It appears that such an effect can be related to the change in the ionization state of histidine side-chains which are shown to be mainly located in the predicted alpha-helix regions.

Future challenges facing the development of specific active-site-directed synthetic inhibitors of MMPs.

Despite a deep knowledge on the 3D-structure of several catalytic domains of MMPs, the development of highly specific synthetic active-site-directed inhibitors of MMPs, able to differentiate the different members of this protease family, remains a strong challenge. Due to the flexible nature of MMP active-site, the development of specific MMP inhibitors will need to combine sophisticated theoretical and experimental approaches to decipher in each MMP the specific structural and dynamic features that can be exploited to obtain the desired selectivity.

Substrate recognition and selectivity of peptide deformylase. Similarities and differences with metzincins and thermolysin.

The substrate specificity of Escherichia coli peptide deformylase was investigated by measuring the efficiency of the enzyme to cleave formyl- peptides of the general formula Fo-Xaa-Yaa-NH2, where Xaa represents a set of 27 natural and unusual amino acids and Yaa corresponds to a set of 19 natural amino acids. Substrates with bulky hydrophobic side-chains at the P1' position were the most efficiently cleaved, with catalytic efficiencies greater by two to five orders of magnitude than those associated with polar or charged amino acid side-chains. Among hydrophobic side-chains, linear alkyl groups were preferred at the P1' position, as compared to aryl-alkyl side-chains. Interestingly, in the linear alkyl substituent series, with the exception of norleucine, deformylase exhibits a preference for the substrate containing Met in the P1' position. Next, the influence in catalysis of the second side-chain was studied after synthesis of 20 compounds of the formula Fo-Nle-Yaa-NH2. Their deformylation rates varied within a range of only one order of magnitude. A 3D model of the interaction of PDF with an inhibitor was then constructed and revealed indeed the occurrence of a deep and hydrophobic S1' pocket as well as the absence of a true S2' pocket. These analyses pointed out a set of possible interactions between deformylase and its substrates, which could be the ground driving substrate specificity. The validity of this enzyme:substrate docking was further probed with the help of a set of site-directed variants of the enzyme. From this, the importance of residues at the bottom of the S1' pocket (Ile128 and Leu125) as well as the hydrogen bond network that the main chain of the substrate makes with the enzyme were revealed. Based on the numerous homologies that deformylase displays with thermolysin and metzincins, a mechanism of enzyme:substrate recognition and hydrolysis could finally be proposed. Specific features of PDF with respect to other members of the enzymes with motif HEXXH are discussed.

Crystal structure of the stromelysin-3 (MMP-11) catalytic domain complexed with a phosphinic inhibitor mimicking the transition-state.

Stromelysin-3 (ST3) is a matrix metalloproteinase (MMP-11) whose proteolytic activity plays an important role in tumorigenicity enhancement. In breast cancer, ST3 is a bad prognosis marker: its expression is associated with a poor clinical outcome. This enzyme therefore represents an attractive therapeutic target. The topology of matrix metalloproteinases (MMPs) is remarkably well conserved, making the design of highly specific inhibitors difficult. The major difference between MMPs lies in the S(1)' subsite, a well-defined hydrophobic pocket of variable depth. The present crystal structure, the first 3D-structure of the ST3 catalytic domain in interaction with a phosphinic inhibitor mimicking a (d, l) peptide, clearly demonstrates that its S(1)' pocket corresponds to a tunnel running through the enzyme. This open channel is filled by the inhibitor P(1)' group which adopts a constrained conformation to fit this pocket, together with two water molecules interacting with the ST3-specific residue Gln215. These observations provide clues for the design of more specific inhibitors and show how ST3 can accommodate a phosphinic inhibitor mimicking a (d, l) peptide. The presence of a water molecule interacting with one oxygen atom of the inhibitor phosphinyl group and the proline residue of the Met-turn suggests how the intermediate formed during proteolysis may be stabilized. Furthermore, the hydrogen bond distance observed between the methyl of the phosphinic group and the carbonyl group of Ala182 mimics the interaction between this carbonyl group and the amide group of the cleaved peptidic bond. Our crystal structure provides a good model to study the MMPs mechanism of proteolysis.

Three-dimensional structure of cyclohexapeptides containing a phosphinic bond in aqueous solution: a template for zinc metalloprotease inhibitors. A NMR and restrained molecular dynamics study.

The 3D structures of two phosphinic cyclic hexapeptide inhibitors of bacterial collagenase, cyclo-(Gly1-Pro2-Phe3 psi[PO2-CH2]Gly4-Pro5-Nle6) (compound I) and cyclo(Gly1-Pro2-D-Phe3 psi[PO2-CH2]-Gly4-Pro5-Nle6) (compound II), in aqueous solution, as derived from NMR spectroscopy and molecular dynamics simulations, are described. The general structures of these cyclic hexapeptides closely resemble the "canonic" two-reverse-turn structure, with the proline occupying the (i + 1) position of the turns and the glycine the connecting positions. The phosphinic bond is located between the (i + 2) and (i + 3) positions of one of these turns. However, a striking feature of the backbone structure of these peptides is the presence of double type VIII-turns in compound I, and in compound II of type VIII- and tentatively named type IX-turns. The comparison of the 3D structures of these two cyclic hexapeptides shows that the stereochemistry of the phenylalanylphosphinyl residue influences not only the local conformation but also the global topology of the peptide macrocycle. The differences in the 3D structure of these compounds are discussed in relation to their inhibitory potencies and with the view of using these constrained cyclic peptides as a scaffold for the development of rigid metalloproteases inhibitors.

Cyclic peptides with a phosphinic bond as potent inhibitors of a zinc bacterial collagenase.

A series of cyclic peptides containing a phosphinic bond were synthesized and evaluated as inhibitors of a zinc bacterial collagenase from Corynebacterium rathaii. Among this series of pseudopeptides of different sizes of cycles, only two molecules Ia (cyclo[Gly-Pro-Phe psi(PO2CH2)-Gly-Pro-Ahx]) and Va (cyclo[beta Ala-Pro-Phe psi (PO2CH2)Gly-Pro-Ahx]) were found to be rather potent inhibitors of this protease, with Ki values of 120 and 90 nM, respectively. Besides the influence of the peptide ring size, this study suggests that both the stereochemical and the conformational properties of the pseudophenylalanine residue in these cyclic peptides may determine their potency. Interestingly, the kinetic analysis for the binding of the cyclic peptide inhibitors Ia and Va to the collagenase, as compared to a linear parent compound, reveals that the lower potency of the cyclic peptides is mostly the consequence of a lower rate constant for association to the enzyme. To our knowledge, this is the first report on cyclic phosphinic peptides and on their activities as inhibitors of a zinc protease.

Phosphinic pseudo-tripeptides as potent inhibitors of matrix metalloproteinases: a structure-activity study.

Several phosphinic pseudo-tripeptides of general formula R-XaaPsi(PO(2)-CH(2))Xaa'-Yaa'-NH(2) were synthesized and evaluated for their in vitro activities to inhibit stromelysin-3, gelatinases A and B, membrane type-1 matrix metalloproteinase, collagenases 1 and 2, and matrilysin. With the exception of collagenase-1 and matrilysin, phosphinic pseudo-tripeptides behave as highly potent inhibitors of matrix metalloproteinases, provided they contain in P(1)' position an unusual long aryl-alkyl substituent. Study of structure-activity relationships regarding the influence of the R and Xaa' substituents in this series may contribute to the design of inhibitors able to block only a few members of the matrix metalloproteinase family.

Role of endopeptidase 3.4.24.16 in the catabolism of neurotensin, in vivo, in the vascularly perfused dog ileum.

1. The degradation of tritiated and unlabelled neurotensin (NT) following close intra-arterial infusion of the peptides in ileal segments of anaesthetized dogs was examined. 2. Intact NT and its catabolites recovered in the venous effluents were purified by chromatography on Sep-Pak columns followed by reverse-phase h.p.l.c. and identified by their retention times or by radioimmunoassay. 3. The half-life of neurotensin was estimated to be between 2 and 6 min. Four labelled catabolites, corresponding to free tyrosine, neurotensin (1-8), neurotensin (1-10) and neurotensin (1-11), were detected. 4. Neurotensin (1-11) was mainly generated by a phosphoramidon-sensitive cleavage, probably elicited by endopeptidase 24-11. 5. Two endopeptidase 3.4.24.16 inhibitors, phosphodiepryl 03 and the dipeptide Pro-Ile, dose-dependently potentiated the recovery of intact neurotensin. Furthermore, both agents inhibited the formation of neurotensin (1-10), the product that results from the hydrolysis of neurotensin by purified endopeptidase 3.4.24.16. In contrast, the endopeptidase 3.4.24.15 inhibitor Cpp-AAY-pAB neither protected neurotensin from degradation nor modified the production of neurotensin (1-10). 6. Our study is the first evidence to indicate that endopeptidase 3.4.24.16 contributes to the catabolism of neurotensin, in vivo, in the dog intestine.

Phosphorus-containing peptides as mixed inhibitors of endopeptidase 3.4.24.15 and 3.4.24.16: effect on neurotensin degradation in vitro and in vivo.

1. We have examined several phosphorus-containing peptides as potential mixed inhibitors of two neurotensin-degrading zinc metallopeptidases, endopeptidase 3.4.24.15 and endopeptidase 3.4.24.16. 2. Among a series of 13 phosphonamide peptides, N-(2-(2-naphtyl)ethylphosphonyl-glycyl-prolyl-norleucine (phosphodiepryl 08) was found to inhibit potently the hydrolysis of neurotensin by purified endopeptidase 3.4.24.15 and 3.4.24.16 with an identical Ki value of 0.4 nM. 3. Phosphodiepryl 08 displayed a strong selectivity towards the two peptidases since it failed to inhibit several other zinc-containing peptidases such as endopeptidase 3.4.24.11, angiotensin-converting enzyme, aminopeptidase M, leucine aminopeptidase and carboxypeptidases A and B. 4. The protective effect of phosphodiepryl 08 on neurotensin degradation was examined in vitro and in vivo in central and peripheral bioassays. 5. Phosphodiepryl 08 virtually abolished neurotensin degradation by 4-day-old plated pure cultured neurones from mouse embryos and greatly potentiated neurotensin-induced antinociception in the mouse hot plate test. 6. In the periphery, phosphodiepryl 08 inhibited neurotensin degradation by membranes prepared from isolated longitudinal smooth muscle of guinea-pig ileum and greatly potentiated the neurotensin-induced contraction of the same longitudinal smooth muscle preparation. 7. Our study indicates that phosphodiepryl 08 behaves as a potent and selective mixed inhibitor of endopeptidase 3.4.24.15 and 3.4.24.16 and can be used as a powerful agent to prevent neurotensin degradation, in vitro and in vivo, in central and peripheral assays.

Examination of the role of endopeptidase 3.4.24.15 in A beta secretion by human transfected cells.

1. We have taken advantage of our recent development of highly potent and specific phosphinic inhibitors of endopeptidase 3.4.24.15 to examine the putative contribution of the enzyme in the secretion of A beta by HK293 transfected cells overexpressing the wild type and the Swedish (Sw) double mutated form of beta APP751. 2. First, we showed that HK293 cells contain a peptidase activity, the inhibition profile of which fully matches that of purified endopeptidase 3.4.24.15. Second, we established that the treatment of HK293 cells with specific phosphinic inhibitors leads to about 80% inhibition of intracellular endopeptidase 3.4.24.15 activity, indicating that these inhibitors penetrate the cells. 3. Metabolic labelling of wild type and Sw beta APP751-expressing cells, followed by immunoprecipitation of A beta-containing peptides, revealed the secretion of A beta and the intracellular formation of an A beta-containing 12 kDa product. 4. A beta secretion by Sw beta APP751 transfected cells was drastically enhanced when compared to cells expressing wild type beta APP751. This production was not affected by endopeptidase 3.4.24.15 inhibitors in either cell type. This correlates well with the observation that endopeptidase 3.4.24.15 does not cleave recombinant baculoviral Sw beta APP751, in vitro. 5. Our previous data indicated that endopeptidase 3.4.24.15 activity was reduced in the parietal cortex of Alzheimer's disease affected brains and that the enzyme probably participated, in this brain area, to the catabolism of somatostatin 1-14. However, the present work indicates that endopeptidase 3.4.24.15 does not seem to behave as a beta-secretase in HK293 transfected cells. Therefore, it is suggested that endopeptidase 3.4.24.15 could participate in the symptomatology, but probably not in the aetiology of Alzheimer's disease.

Effect of a novel selective and potent phosphinic peptide inhibitor of endopeptidase 3.4.24.16 on neurotensin-induced analgesia and neuronal inactivation.

1. We have examined a series of novel phosphinic peptides as putative potent and selective inhibitors of endopeptidase 3.4.24.16. 2. The most selective inhibitor, Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 displayed a Ki value of 12 nM towards endopeptidase 3.4.24.16 and was 5540 fold less potent on its related peptidase endopeptidase 3.4.24.15. Furthermore, this inhibitor was 12.5 less potent on angiotensin-converting enzyme and was unable to block endopeptidase 3.4.24.11, aminopeptidases B and M, dipeptidylaminopeptidase IV and proline endopeptidase. 3. The effect of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2, in vitro and in vivo, on neurotensin metabolism in the central nervous system was examined. 4. Pro-Phe-psi(PO2CHH2)-Leu-Pro-NH2 dose-dependently inhibited the formation of neurotensin 1-10 and concomittantly protected neurotensin from degradation by primary cultured neurones from mouse embryos. 5. Intracerebroventricular administration of Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 significantly potentiated the neurotensin-induced antinociception of mice in the hot plate test. 6. Altogether, our study has established Pro-Phe-psi(PO2CH2)-Leu-Pro-NH2 as a fully selective and highly potent inhibitor of endopeptidase 3.4.24.16 and demonstrates, for the first time, the contribution of this enzyme in the central metabolism of neurotensin.

Potency and selectivity of RXP407 on human, rat, and mouse angiotensin-converting enzyme.

By screening phosphinic peptide libraries, we recently reported the discovery of RXP407 (Ac-Asp-PheY(PO2-CH2)LAla-Ala-NH2), a potent N-domain-selective inhibitor of recombinant human angiotensin-converting enzyme (ACE). Preliminary studies to evaluate the in vivo activity of RXP407 in rat led us to suspect possible differences in the binding property of RXP407 between human and rat ACE. The aim of the present study was thus to determine the potency of RXP407 toward rat and mouse ACEs, as compared to non-recombinant human ACE, and to assess the efficacy of this inhibitor in discriminating between the N- and C-domains of these ACE enzymes. By comparing the ability of RXP407 to block purified somatic and germinal ACE from mice, RXP407 was shown to be a potent N-domain-selective inhibitor of mouse somatic ACE, a behavior similar to that observed with human somatic ACE. In contrast, RXP407 appeared less potent toward purified ACE from rat and furthermore was unable to block ACE activity present in crude rat plasma. This study demonstrated that for further evaluation of the in vivo efficacy of RXP407, mice rather than rats should be used as the animal model. Thus, following the change in the Ac-S-D-K-P plasmatic levels, after i.v. injection of RXP407 to mice, will permit the potency and selectivity of this novel ACE inhibitor to be assessed.

Convenient synthesis and diversification of dehydroalaninyl phosphinic peptide analogues.

[structure: see text]. Dehydroalaninyl phosphinic dipeptide analogues were synthesized, via an efficient tandem Arbuzov addition/allylic rearrangement, in high yields. The susceptibility of the conjugate system to 1,4 nucleophilic additions was investigated. C-Elongation of the dipeptides was performed, and the efficiency of 1,4 addition to the resulting acrylamidic moiety was evaluated. Derivatization of such phosphinic templates is a powerful approach for rapid access to large number of phosphinic pseudopeptides bearing various side chains in the P1' position.

Contribution of endopeptidase 3.4.24.15 to central neurotensin inactivation.

The tridecapeptide, neurotensin elicits naloxone-insensitive analgesia after its intracebroventricular administration in mice. We used this central pharmacological effect to assess the putative contribution of the endopeptidase 3.4.24.15 to central inactivation of the peptide. By means of combinatorial chemistry, we previously designed the first potent endopeptidase 3.4.24.15 inhibitor. This agent, Z-(L,D)Phe psi(PO2CH2)(L,D)Ala-Lys-Met (phosphodiepryl 21), is shown here to behave as a fully specific endopeptidase 3.4.24.15 inhibitor, as demonstrated by the absence of effect on a series of other exo- and endopeptidases belonging to various classes of proteolytic activities present in murine brain membranes. Furthermore, central administration of phosphodiepryl 21 drastically prolongs the forepaw licking latency of mice tested on the hot plate and injected with sub-maximally active doses of neurotensin. Altogether, our results demonstrated that, in addition to endopeptidase 3.4.24.16, endopeptidase 3.4.24.15 likely contributes to the physiological termination of the neurotensinergic message in murine brain.

[Peptide inhibitors of a bacterial collagenase]. / Inhibiteurs peptidiques d'une collagénase bactérienne.

The properties of the cleavage site of collagen by collagenases remain discussed. The authors report their studies on the active site of the collagenase of a bacteria, achromobacter, based on two types of methods. The first type uses synthetic substrates, the second one enzymatic inhibitors. The results of both methods are reviewed. The inhibitors seem more sensitive than substrates to study the relation between structure and activity of the enzyme.

New thiol inhibitor of Achromobacter iophagus collagenase. Specificity of the enzyme's S3' subsite.

New synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) which inhibit Achromobacter iophagus collagenase were produced in order to obtain more powerful bacterial collagenase inhibitors than currently available, and to investigate the specificity of the S3' subsite of the enzyme. Since similar binding constants were found for inhibitors carrying uncharged residues of various sizes in the P3' position (Yaa = Ala, Leu, Phe, Pro, Hyp) steric hindrance at the collagenase S3' appears relatively limited. The compound (HS-CH2-CH2-CO-Pro-Arg), which carries an arginine residue in the position P3' and had the highest inhibition constant of the series tested (Ki = 0.5 microM), proved to be the strongest inhibitor so far reported in the literature. The weakest in the present series was the compound (HS-CH2-CH2-CO-Pro-Asp) which carries an aspartic residue in position P3' and had a Ki = 70 microM. The present work revealed that the charged groups in the P3' position play a key role in the interaction of the inhibitors with the enzyme.