Aneta Lukaszuk

β-Homo-amino Acid Scan of Angiotensin IV

Angiotensin IV, a metabolite of angiotensin II, inhibits the enzyme insulin regulated aminopeptidase or IRAP and also, although with lower potency, aminopeptidase-N (AP-N). When both beta (2)-homo amino acid- and beta (3)-homo amino acid substitutions were used, allowed the identification of H-( R)beta (2)hVal-Tyr-Ile-His-Pro-beta (3)hPhe-OH as a potent and stable Ang IV analog with high selectivity for IRAP versus AP-N and the AT1 receptor.

Pressor and Renal Hemodynamic Effects of the Novel Angiotensin A Peptide Are Angiotensin II Type 1A Receptor Dependent

Recently, a new derivative of angiotensin (Ang) II, called “Ang A,” has been discovered to be present in plasma of healthy humans and, in increased concentrations, in end-stage renal failure patients. The objectives of the study were to investigate the blood pressure and renal hemodynamic responses to Ang A in normotensive and hypertensive rats and in genetically modified mice and the binding properties of Ang A to Ang II type 1 (AT1) or Ang II type 2 (AT2) receptors. Intravenous and intrarenal administration of Ang A induced dose-dependent pressor and renal vasoconstrictor responses in normotensive rats, which were blocked by the AT1 receptor antagonist candesartan but were not altered by the AT2 receptor ligands PD123319, CGP42112A, or compound 21. Similar responses were observed after intravenous administration in spontaneously hypertensive rats. Deletion of AT1a receptors in mice almost completely abolished the pressor and renal vasoconstrictor responses to Ang A, indicating that its effects are mediated via AT1a receptors. Ang A was less potent than Ang II in vivo. The in vitro study demonstrated that Ang A is a full agonist for AT1 receptors, with similar affinity for AT1 and AT2 receptors as Ang II. Overall, the responses to Ang A and Ang II were similar. Ang A has no physiological role to modulate the pressor and renal hemodynamic effects of Ang II.

The replacement of His(4) in angiotensin IV by conformationally constrained residues provides highly potent and selective analogues.

The histidine residue in angiotensin IV was replaced by various conformationally constrained amino acids. The substitution of the His(4)-Pro(5) dipeptide sequence by the constrained Trp analogue Aia-Gly, in combination with beta(2)hVal substitution at the N-terminus, provided a new stable analogue H-(R)-beta(2)hVal-Tyr-Ile-Aia-Gly-Phe-OH (AL-40) that is a potent ligand for the Ang IV receptor IRAP and selective versus AP-N and the AT1 receptor.

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

We report on the characteristics of the radical-ion-driven dissociation of a diverse array of β-amino acids incorporated into α-peptides, as probed by tandem electron-capture and electron-transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β-amino acids than for their α-form counterparts. In particular, only aromatic (e.g., β-Phe), and to a substantially lower extent, carbonyl-containing (e.g., β-Glu and β-Gln) amino acid side chains, lead to N-Cβ bond cleavage in the corresponding β-amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical-driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α-amino acids, ECD of peptides composed mainly of β-amino acids reveals a shift in cleavage priority from the N-Cβ to the Cα-C bond. The incorporation of CH2 groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical-driven β-amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.

Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors

SummaryThe vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug–target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

Conformational constraints in angiotensin IV to probe the role of Tyr2, Pro5 and Phe6

The aromatic amino acids Tyr and Phe in angiotensin IV (Ang IV) were conformationally constrained by the use of β‐Me substituted analogs, or cyclic constrained analogs. None of these modifications was allowed for Tyr1, while only e‐β‐MePhe6 substitution resulted in an AngIV analog with high IRAP potency and selectivity versus AP‐N or the AT1 receptor. This indicates an important role of the orientation of the Phe6 for inducing selectivity. Pro5 replacement with 2‐aminocyclopentanecarboxylic acid maintained IRAP potency and abolished AT1 affinity. These results confirm the importance of conformational constrained amino acids to generate selectivity in bioactive peptides. Copyright

Angiotensin IV displays only low affinity for native insulin-regulated aminopeptidase (IRAP)

Radioligand binding studies revealed that Ang IV binds to insulin‐regulated aminopeptidase (IRAP)/’AT4 receptors’ with high affinity. Yet, as these experiments were routinely carried out in the presence of chelators, only the catalytic zinc‐depleted apo‐form of IRAP was labelled. While the chelators remove the catalytic zinc from IRAP and protect Ang IV from proteolytic degradation, the aminopeptidase N selective inhibitor ‘7B’ only exerts the latter effect. By using 7B along with the new stable Ang IV‐analog [3H]AL‐11, we here show that the native enzyme is only a low‐affinity target for Ang IV.

Differential effects of hydroxamate histone deacetylase inhibitors on cellular functionality and gap junctions in primary cultures of mitogen-stimulated hepatocytes

Histone deacetylase (HDAC)-inhibitors are well known to induce proliferative blocks and concomitant differentiation boosts in a plethora of tumor cells. Despite their promising potential as clinical therapeutics, however, the biological outcome of HDAC-inhibitors in non-tumorous cells has been poorly documented. We previously reported that the HDAC-inhibitor trichostatin A (TSA) and its metabolically more stable structural analogue 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me2N-BAVAH) cause cell cycle arrests in primary cultures of mitogen-stimulated hepatocytes. The present study was set up to explore whether this proliferative block in non-tumorous cells is also associated with inducing effects on the differentiated hepatocellular phenotype, a scenario that is usually observed in tumorous cells. In particular, the molecular actions of TSA and 4-Me2N-BAVAH on hepatic functionality and gap junctions, gatekeepers of liver homeostasis, in primary cultures of mitogen-stimulated hepatocytes are investigated. Both HDAC-inhibitors were found to promote albumin secretion and CYP1A1 gene transcription and functionality, whereas CYP2B1 gene transcription and activity were only slightly enhanced. The protein production of the gap junction component Cx26 was downregulated, whereas Cx32 expression was upregulated in response to HDAC-inhibition. Furthermore, TSA increased protein levels of the non-specific hepatocellular Cx43, whereas 4-Me2N-BAVAH rather diminished its expression. These data provide new insight into the biological impact of HDAC-inhibitors on the homeostatic balance in hepatocytes, being major executors of xenobiotic biotransformation and primary targets of drug-induced toxicity.

Preservation of hepatocellular functionality in cultures of primary rat hepatocytes upon exposure to 4-Me2N-BAVAH, a hydroxamate-based HDAC-inhibitor.

Great efforts are being put in the development/optimization of reliable and highly predictive models for high-throughput screening of efficacy and toxicity of promising drug candidates. The use of primary hepatocyte cultures, however, is still limited by the occurrence of phenotypic alterations, including loss of xenobiotic biotransformation capacity. In the present study, the differentiation-stabilizing effect of a new histone deacetylase inhibitor 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), a structural Trichostatin A (TSA)-analogue with a more favourable pharmaco-toxicological profile, was studied at a genome-wide scale by means of microarray analysis. Several genes coding for xenobiotic biotransformation enzymes were found to be positively regulated upon exposure to 4-Me(2)N-BAVAH. For CYP1A1/2B1/3A2, these observations were confirmed by qRT-PCR and immunoblot analysis. In addition, significantly higher 7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-dealkylase activity levels were measured. These effects were accompanied by an increased expression of CCAAT/enhancer binding protein alpha and hepatic nuclear factor (HNF)4α, but not of HNF1α. Finally, 4-Me(2)N-BAVAH was found to induce histone H3 acetylation at the proximal promoter of the albumin, CYP1A1 and CYP2B1 genes, suggesting that chromatin remodelling is directly involved in the transcriptional regulation of these genes. In conclusion, histone deacetylase inhibitors prove to be efficient agents for better maintaining a differentiated hepatic phenotype in rat hepatocyte cultures.