Jan Hlaváček

The multiple T-maze in vivo testing of the neuroprotective effect of humanin analogues.

Humanin (HN) and its analogues have been shown to protect cells against death induced by various Alzheimers disease (AD) genes and amyloid-beta-peptides in vitro; the analogues [Gly(14)]-HN and colivelin have also been shown to be potent in reversing learning and memory impairment induced by scopolamine or quinuclidinyl benzilate (QNB) in mice or rats in vivo using the Y-maze or multiple T-maze tests. This paper describes the activity of new peptides of the HN family, after i.p. administration, on QNB-induced impairment of spatial memory in the multiple T-maze test in rats. The following peptides have been studied: HN analogues truncated either on the C- or N-terminus, or analogues having a tert-Leu in place of Leu in the central part of the molecule, the active HN core PAGASRLLLLTGEIDLP (RG-PAGA) and its analogues having three or five leucines instead of four, and finally the recently described hybrid peptide colivelin (i.e. a peptide having the activity-dependent neurotrophic factor SALLRSIPA attached to the N-terminus of the active RG-PAGA) and its des-Leu- and plus-Leu-analogues. While the truncated analogues and most of the tert-Leu containing analogues were devoid of activity, the analogues of the RG-PAGA were active, i.e. they reversed the impairment of spatial memory irrespective of the number of Leu present in their sequence. The highest activity was shown by colivelin and its des-Leu-analogue. These results demonstrate the potential of HN analogues in the modulation of the cholinergic system, which plays an important role in the cognitive deficits associated with AD and other neurodegenerative diseases.

A Role of the 9-Aminoacridines and their Conjugates in a Life Science

The 9-aminoacridines play an important role in medicine. They were applied first in a treatment of protozoal infections in the beginning of the last century. Recently, it has been shown that the 9-aminoacridines are successful candidates for treatment of cancer, viral and prion diseases. Their conjugation with biomolecules such as peptides and proteins may modulate their activity, bioavailability and applicability. This review deals with the synthesis of 9-aminoacridine, its conjugation with variety of molecules and utilization of such conjugates in several fields of science.

Structure–activity relationship of CART (cocaine- and amphetamine-regulated transcript) peptide fragments

CART (cocaine- and amphetamine-regulated transcript) peptides are neuropeptides abundant in the central nervous system and periphery found to be involved in the regulation of food intake behavior and other physiological processes. Recently, we reported specific binding of (125)I-CART(61-102) to the rat adrenal pheochromocytoma cell line PC12, both intact cells and cell membranes. In this study, several fragments of CART(61-102) corresponding to its structural loops were synthesized and tested for their potency in binding experiments using PC12 intact cells and cell membranes and in feeding test with fasted mice. From all shorter peptides tested, only CART(74-86) and CART(62-86) containing disulfide bridges kept partial binding potency of the original molecule with K(i) in 10(-5) and 10(-4)M range. However, these fragments were not able to inhibit food intake after their central administration up to a dose of 4 nmol/mouse. The results showed that a compact structure containing three disulfide bridges is necessary for preservation of full biological activity of CART peptides.

Application of capillary and free-flow zone electrophoresis and isotachophoresis to the analysis and preparation of the synthetic tetrapeptide fragment of growth hormone-releasing peptide

The use of high-performance electromigration separation methods, capillary zone electrophoresis (CZE) and capillary isotachophoresis (CITP) and continuous free-flow arrangements of these two separation principles, free-flow zone electrophoresis (FFZE) and free-flow isotachophoresis (FFITP), was investigated in the analysis and purification of the synthetic C-terminal tetrapeptide fragment (H2N-Ala-Trp-D-Phe-Lys.NH2) of the growth hormone-releasing peptide. CZE and CITP were used for microanalysis of peptide preparations after different steps of their purification. The homogeneity of the peptide preparations, including fractions of preparative separations, was quantified by relative zone length (CITP) and/or relative peak height (CZE). In addition, the data obtained by CZE and CITP (electrophoretic and electroosmotic flow migration velocities) were utilized for conversion of micro-scale capillary separations (nano- to picomole level) into the preparative separations realized by FFZE and FFITP with a capacity from tens to hundreds of milligrams per hour.

Pharmacological characterization of new cholecystokinin analogues

New analogues of cholecystokinin-7 (CCK-7) modified at amino acid residues 5 and 7 were assayed for their effect on gall bladder, pancreatic secretion, food intake (anorectic activity), amount of rearing (sedative activity) and analgesia, as well as their ability to inhibit 125I-CCK-8 binding to pancreatic cell membrane receptors and brain membrane receptors. The results were compared to the activities of standard compounds, CCK-8, cerulein, BOC-CCK-7 (BOC = tertbutyloxycarbonyl) and BOC-[Nle2,Nle5]CCK-7. All analogues exhibited agonistic effects. Their anorectic activity was significantly prolonged.

Synthesis and utilization of 13C and 15N backbone-labeled proline: NMR study of synthesized oxytocin with backbone-labeled C-terminal tripeptide amide

Summary.The 13C and 15N backbone-labeled proline was prepared using Oppolzer’s method based on application of a sultam as chiral auxiliary. This isotopomer was used in the synthesis of the 13C, 15N backbone-labeled C-terminal tripeptide amide fragment of neurohypophyseal hormone oxytocin. Finally, this tripeptide amide was coupled by segment condensation with N-Boc- or N-Fmoc-tocinoic acid, followed by N-deprotection with TFA or piperidine. The labeled oxytocin exhibited biological activity identical with that of natural oxytocin. A detailed 1H, 13C and 15N NMR study confirmed the assigned oxytocin conformation containing a β-turn in the cyclic part of the molecule, stabilized by H-bond(s) that can be perturbed by the C-terminal tripeptide amide moiety as indicated by comparison of NMR data for both the tocine ring in oxytocin and tocinoic acid.

Quinacrine reactivity with prion proteins and prion-derived peptides

Quinacrine is a drug that is known to heal neuronal cell culture infected with prions, which are the causative agents of neurodegenerative diseases called transmissible spongiform encephalopathies. However, the drug fails when it is applied in vivo. In this work, we analyzed the reason for this failure. The drug was suggested to “covalently” modify the prion protein via an acridinyl exchange reaction. To investigate this hypothesis more closely, the acridine moiety of quinacrine was covalently attached to the thiol groups of cysteines belonging to prion-derived peptides and to the full-length prion protein. The labeled compounds were conveniently monitored by fluorescence and absorption spectroscopy in the ultraviolet and visible spectral regions. The acridine moiety demonstrated characteristic UV–vis spectrum, depending on the substituent at the C-9 position of the acridine ring. These results confirm that quinacrine almost exclusively reacts with the thiol groups present in proteins and peptides. The chemical reaction alters the prion properties and increases the concentration of the acridine moiety in the prion protein.

Evaluation of carrier ampholyte-based capillary electrophoresis for separation of peptides and peptide mimetics

Carrier ampholyte‐based capillary electrophoresis (CABCE) has recently been introduced as an alternative to CE (CZE) in the classical buffers. In this study, isoelectric BGEs were obtained by fractionation of Servalyt pH 4–9 carrier ampholytes to cuts of typical width of 0.2 pH unit. CABCE feasibility was examined on a series of insect oostatic peptides, i.e. proline‐rich di‐ to decapeptides, and phosphinic pseudopeptides – tetrapeptide mimetics synthesized as a mixture of four diastereomers having the –P(O)(OH)–CH2– moiety embedded into the peptide backbone. With identical selectivity, the separation efficiency of CABCE proved to be as good as classical CE for the insect oostatic peptides and better for diastereomers of the phosphinic pseudopeptides. In addition, despite the numerous species present in the narrow pH cuts of carrier ampholytes, CABCE seems to be free of system zones that could hamper the analysis. Peak symmetry was good for moderately to low mobile peptides, whereas some peak distortion due to electromigration dispersion, was observed for short peptides of rather high mobility.

Cholecystokinin analogs with suppressed central activities.

Several analogs of Boc-protected C-terminal heptapeptide of cholecystokinin (Boc-CCK-7) with modified C-end Phe were pharmacologically characterized. The influence of the number of methyl groups on aromatic side chain of Phe was investigated in following tests: binding to pancreatic and brain membrane receptors, gall bladder contraction, amylase secretion, anorexia, sedation and analgesia. Two analogs seem to be promising selective anorectic agents with strongly protracted effect: Boc-[Phe(triMe)7]CCK-7 and Boc-[Phe(pentaMe)7]CCK-7. The first analog exhibits the same spectrum of activities as CCK-8, however partially decreased central effects, the second one shows partially decreased peripheral activities and totally suppressed central ones. Our study supports the idea that C-terminal residue of CCK is more important for biological potency than for binding to CCK receptors.

Cationic oligopeptides with the repeating sequence L-lysyl-L-alanyl-L-alanine: conformational and thermal stability study using optical spectroscopic methods.

The infrared (IR), vibrational circular dichroism (VCD), and electronic circular dichroism (ECD) spectra of short cationic sequential peptides (L‐Lys‐L‐Ala‐L‐Ala)n (n = 1, 2, and 3) were measured over a range of temperatures (20–90 °C) in aqueous solution at near‐neutral pH values in order to investigate their solution conformations and thermally induced conformational changes. VCD spectra of all three oligopeptides measured in the amide I′ region indicate the presence of extended helical polyproline II (PPII)‐like conformation at room temperature. UV‐ECD spectra confirmed this conclusion. Thus, the oligopeptides adopt a PPII‐like conformation, independent of the length of the peptide chain. However, the optimized dihedral angles ϕ and ψ are within the range −82 to −107° and 143–154°, respectively, and differ from the canonical PPII values. At elevated temperatures, the observed intensity and bandshape variations in the VCD and ECD spectra show that the PPII‐like conformation of the Lys‐Ala‐Ala sequence is still preferred, being in equilibrium with an unordered conformer at near‐neutral pH values within the range of temperatures from 20 to 90 °C. This finding was obtained from analysis of the temperature‐dependent spectra using the singular value decomposition method. The study presents KAA‐containing oligopeptides as conformationally stable models of biologically important cationic peptides and proteins. Copyright