Peptide inhibitors of angiotensin-I converting enzyme based on angiotensin (1-7) with selectivity for the C-terminal domain.

The renin-angiotensin system (RAS) is a key regulator of human arterial pressure. Several of its effects are modulated by angiotensin II, an octapeptide originating from the action of angiotensin-I converting enzyme (ACE) on the decapeptide angiotensin-I. ACE possess two active sites (nACE and cACE) that have their own kinetic and substrate specificities. ACE inhibitors are widely used as the first-line treatment for hypertension and other heart-related diseases, but because they inactivate both ACE domains, their use is associated with serious side effects. Thus, the search for domain-specific ACE inhibitors has been the focus of intense research. Angiotensin (1-7), a peptide that also belongs to the RAS, acts as a substrate of nACE and an inhibitor of cACE. We have synthetized 15 derivatives of Ang (1-7), sequentially removing the N-terminal amino acids and modifying peptides extremities, to find molecules with improved selectivity and inhibition properties. Ac-Ang (2-7)-NH2 is a good ACE inhibitor, resistant to cleavage and with improved cACE selectivity. Molecular dynamics simulations provided a model for this peptide's selectivity, due to Val3 and Tyr4 interactions with ACE subsites. Val3 has an important interaction with the S3 subsite, since its removal greatly reduced peptide-enzyme interactions. Taken together, our findings support ongoing studies using insights from the binding of Ac-Ang (2-7)-NH2 to develop effective cACE inhibitors.

Comparison of morphological changes of corneal collagen fibers treated with collagen crosslinking agents using second harmonic generation images.

Corneal cross-linking (CXL) is a common surgical procedure used to modify corneal biomechanics and stabilize keratoconus progression which is still under discussion. Its side effects, which are mostly related to anatomical unpredictability and stromal exposure, are the reason for the search for new CXL agents. In this work we have quantitatively evaluated the porcine corneal stroma architecture treated with collagen crosslinking agents such as riboflavin solutions and açai extract, using second harmonic generation microscopy. Aimed at evaluating the morphological changes in the corneal stroma after collagen crosslinking under a CXL chemical agent, a tubeness filter based Hessian matrix to obtain a 3D fiber characterization of the SHG images was applied. The results showed a curling effect and shortening of the collagen fibers treated with açai as compared to the control. They also showed a higher degree of clustering of the collagen fibers with larger empty spaces when compared to the other two groups. We believe that studies such as these presented in this paper are a good direct nondestructive and free labeling evaluation technique that allows the observation of morphologic features of corneas treated with new CXL agents.

Characterization of Rabbit Corneas Subjected to Stromal Stiffening by the Açaí Extract (Euterpe oleracea).

PURPOSE: In this study, we characterized rabbit corneas subjected to corneal cross-linking (CXL) with açaí extract compared with a riboflavin photo-stimulated procedure. MATERIALS AND METHODS: The corneas of the slaughterhouse rabbits were divided into three groups: control, consisting of untreated corneal samples; riboflavin/UVA, where corneas were treated with 0.1% riboflavin photo-stimulated at 365 nm as the standard protocol; and açaí, where the samples were subjected to 4% açaí extract for 0.5-2 h. After the CXL procedure, corneas of the three groups were characterized by analyzing their elastic modulus and thermal denaturation profile. RESULTS: The elastic modulus at 3% strain showed an approximately threefold increase in the riboflavin/UVA group and 10.5 times in the corneas treated with 4% açaí extract for 2 h, compared with the control group (p < 0.01). The denaturation temperature values of the two groups of crosslinked corneas increased significantly (p < 0.05) and were more pronounced in the açaí group. CONCLUSIONS: The açaí extract was effective in promoting CXL in rabbit corneas as characterized by the different techniques.

Conformational Properties of Seven Toac-Labeled Angiotensin I Analogues Correlate with Their Muscle Contraction Activity and Their Ability to Act as ACE Substrates.

Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC0-AngI and TOAC1-AngI maintained partial potency in guinea pig ileum and rat uterus. Kinetic parameters revealed that only derivatives labeled closer to the N-terminus (positions 0, 1, 3, and 5) were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different ß-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.

Characterization of angiotensin I-converting enzyme from anterior gills of the mangrove crab Ucides cordatus.

Angiotensin I-converting enzyme (ACE) is a well-known metallopeptidase that is found in vertebrates, invertebrates and bacteria. We isolated from the anterior gill of the crab Ucides cordatus an isoform of ACE, here named crab-ACE, which presented catalytic properties closely resembling to those of mammalian ACE. The enzyme was purified on Sepharose-lisinopril affinity chromatography to apparent homogeneity and a band of about 72 kDa could be visualized after silver staining and Western blotting. Assays performed with fluorescence resonance energy transfer (FRET) selective ACE substrates Abz-FRK(Dnp)P-OH, Abz-SDK(Dnp)P-OH and Abz-LFK(Dnp)-OH, allowed us to verify that crab-ACE has hydrolytic profile very similar to that of the ACE C-domain. In addition, we observed that crab-ACE can hydrolyze the ACE substrates, angiotensin I and bradykinin. The enzyme was strongly inhibited by the specific ACE inhibitor lisinopril (Ki of 1.26 nM). However, in contrast to other ACE isoforms, crab-ACE presented a very particular optimum pH, being the substrate Abz-FRK(Dnp)-P-OH hydrolyzed efficiently at pH 9.5. Other interesting characteristic of crab-ACE was that the maximum hydrolytic activity was reached at around 45°C. The description of an ACE isoform in Ucides cordatus is challenging and may contribute to a better understanding of the biochemical function of this enzyme in invertebrates.

Theoretical basis, laboratory evidence, and clinical research of chemical surgery of the cornea: cross-linking.

Corneal cross-linking (CXL) is increasingly performed in ophthalmology with high success rates for progressive keratoconus and other types of ectasia. Despite being an established procedure, some molecular and clinical aspects still require additional studies. This review presents a critical analysis of some established topics and others that are still controversial. In addition, this review examines new technologies and techniques (transepithelial and ultrafast CXL), uses of corneal CXL including natural products and biomolecules as CXL promoters, and evidence for in vitro and in vivo indirect effectiveness.

Corneal absorption of a new riboflavin-nanostructured system for transepithelial collagen cross-linking.

Corneal collagen cross-linking (CXL) has been described as a promising therapy for keratoconus. According to standard CXL protocol, epithelium should be debrided before treatment to allow penetration of riboflavin into the corneal stroma. However, removal of the epithelium can increase procedure risks. In this study we aim to evaluate stromal penetration of a biocompatible riboflavin-based nanoemulsion system (riboflavin-5-phosphate and riboflavin-base) in rabbit corneas with intact epithelium. Two riboflavin nanoemulsions were developed. Transmittance and absorption coefficient were measured on corneas with intact epithelia after 30, 60, 120, 180, and 240 minutes following exposure to either the nanoemulsions or standard 0.1% or 1% riboflavin-dextran solutions. For the nanoemulsions, the epithelium was removed after measurements to assure that the riboflavin had passed through the hydrophobic epithelium and retained within the stroma. Results were compared to de-epithelialized corneas exposed to 0.1% riboflavin solution and to the same riboflavin nanoemulsions for 30 minutes (standard protocol). Mean transmittance and absorption measured in epithelialized corneas receiving the standard 0.1% riboflavin solution did not reach the levels found on the debrided corneas using the standard technique. Neither increasing the time of exposure nor the concentration of the riboflavin solution from 0.1% to 1% improved riboflavin penetration through the epithelium. When using riboflavin-5-phosphate nanoemulsion for 240 minutes, we found no difference between the mean absorption coefficients to the standard cross-linking protocol (p = 0.54). Riboflavin nanoemulsion was able to penetrate the corneal epithelium, achieving, after 240 minutes, greater stromal concentration when compared to debrided corneas with the standard protocol (p = 0.002). The riboflavin-5-phosphate nanoemulsion diffused better into the stroma than the riboflavin-base nanoemulsion.

Characterization of angiotensin I-converting enzyme N-domain selectivity using positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides.

Somatic angiotensin I-converting enzyme (ACE)has two homologous active sites (N and C domains) that show differences in various biochemical properties.In a previous study, we described the use of positionals canning synthetic combinatorial (PS-SC) libraries of fluorescence resonance energy transfer (FRET) peptides to define the ACE C-domain versus N-domain substrate specificity and developed selective substrates for the C-domain(Bersanetti et al., 2004). In the present work, we used the results from the PS-SC libraries to define the N-domain preferences and designed selective substrates for this domain. The peptide Abz-GDDVAK(Dnp)-OH presented the most favorable residues for N-domain selectivity in the P 3 to P 1 ' positions. The fluorogenic analog Abz-DVAK(Dnp)-OH (Abz = ortho -aminobenzoic acid; Dnp = 2,4-dinitrophenyl)showed the highest selectivity for ACE N-domain( k cat /K m = 1.76 µ m -1 · s -1) . Systematic reduction of the peptide length resulted in a tripeptide that was preferentially hydrolyzed by the C-domain. The binding of Abz-DVAK(Dnp)-OH to the active site of ACE N-domain was examined using a combination of conformational analysis and molecular docking. Our results indicated that the binding energies for the N-domain-substrate complexes were lower than those for the C-domain-substrate, suggesting that the former complexes are more stable.

ACE activity is modulated by kinin B2 receptor.

Angiotensin-converting enzyme (ACE) is an ectoprotein able to modulate the activity of a plethora of compounds, among them angiotensin I and bradykinin. Despite several decades of research, new aspects of the mechanism of action of ACE have been elucidated, expanding our understanding of its role not only in cardiovascular regulation but also in different areas. Recent findings have ascribed an important role for ACE/kinin B(2) receptor heterodimerization in the pharmacological properties of the receptor. In this work, we tested the hypothesis that this interaction also affects ACE enzymatic activity. ACE catalytic activity was analyzed in Chinese hamster ovary cell monolayers coexpressing the somatic form of the enzyme and the receptor coding region using as substrate the fluorescence resonance energy transfer peptide Abz-FRK(Dnp)P-OH. Results show that the coexpression of the kinin B(2) receptor leads to an augmentation in ACE activity. In addition, this effect could be blocked by the B(2) receptor antagonist icatibant. The hypothesis was also tested in endothelial cells, a more physiological system, where both proteins are naturally expressed. Endothelial cells from genetically ablated kinin B(2) receptor mice showed a decreased ACE activity when compared with wild-type mice cells. In summary, this is the first report showing that the ACE/kinin B(2) receptor interaction modulates ACE activity. Taking into account the interplay among ACE, ACE inhibitors, and kinin receptors, we believe that these results will shed new light into the arena of the controversial search for the mechanism controlling these interactions.

Neprilysin carboxydipeptidase specificity studies and improvement in its detection with fluorescence energy transfer peptides.

We examined the substrate specificity of the carboxydipeptidase activity of neprilysin (NEP) using fluorescence resonance energy transfer (FRET) peptides containing ortho-aminobenzoyl (Abz) and 2,4-dinitrophenyl (Dnp) as a donor/acceptor pair. Two peptide series with general sequences Abz-RXFK(Dnp)-OH and Abz-XRFK(Dnp)-OH (X denotes the position of the altered amino acid) were synthesized to study P1 (cleavage at the X-F bond) and P2 (cleavage at R-F bond) specificity, respectively. In these peptides a Phe residue was fixed in P1' to fulfill the well-known NEP S1' site requirement for a hydrophobic amino acid. In addition, we explored NEP capability to hydrolyze bradykinin (RPPGFSPFR) and its fluorescent derivative Abz-RPPGFSPFRQ-EDDnp (EDDnp=2,4-dinitrophenyl ethylenediamine). The enzyme acts upon bradykinin mainly as a carboxydipeptidase, preferentially cleaving Pro-Phe over the Gly-Phe bond in a 9:1 ratio, whereas Abz-RPPGFSPFRQ-EDDnp was hydrolyzed at the same bonds but at an inverted proportion of 1:9. The results show very efficient interaction of the substrates' C-terminal free carboxyl group with site S2' of NEP, confirming the enzyme's preference to act as carboxydipeptidase at substrates with a free carboxyl-terminus. Using data gathered from our study, we developed sensitive and selective NEP substrates that permit continuous measurement of the enzyme activity, even in crude tissue extracts.

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides for defining substrate specificity of the angiotensin I-converting enzyme and development of selective C-domain substrates.

Positional-scanning combinatorial libraries of fluorescence resonance energy transfer peptides were used for the analyses of the S(3) to S(1)' subsites of the somatic angiotensin I-converting enzyme (ACE). Substrate specificity of ACE catalytic domains (C- and N-domains) was assessed in an effort to design selective substrates for the C-domain. Initially, we defined the S(1) specificity by preparing a library with the general structure Abz-GXXZXK(Dnp)-OH [Abz = o-aminobenzoic acid, K(Dnp) = N(epsilon)-2,4-dinitrophenyllysine, and X is a random residue], where Z was successively occupied with one of the 19 natural amino acids with the exception of Cys. The peptides containing Arg and Leu in the P(1) position had higher C-domain selectivity. In the sublibraries Abz-GXXRZK(Dnp)-OH, Abz-GXZRXK(Dnp)-OH, and Abz-GZXRXK(Dnp)-OH, Arg was fixed at P(1) so we could define the C-domain selectivity of the S(1)', S(2), and S(3) subsites. On the basis of the results from these libraries, we synthesized peptides Abz-GVIRFK(Dnp)-OH and Abz-GVILFK(Dnp)-OH which contain the most favorable residues for C-domain selectivity. Systematic reduction of the length of these two peptides resulted in Abz-LFK(Dnp)-OH, which demonstrated the highest selectivity for the recombinant ACE C-domain (k(cat)/K(m) = 36.7 microM(-1) s(-1)) versus the N-domain (k(cat)/K(m) = 0.51 microM(-1) s(-1)). The substrate binding of Abz-LFK(Dnp)-OH with testis ACE using a combination of conformational analysis and molecular docking was examined, and the results shed new light on the binding characteristics of the enzyme.