Dermorphin-based potential affinity labels for mu-opioid receptors.

Dermorphin and [Lys7]dermorphin, selective micro -opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the 'message' or 'address' sequences as potential peptide-based affinity labels for micro -receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe3 and Phe5 was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine-containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing micro - and delta-opioid receptors. In dermorphin, introduction of the electrophilic groups in the 'message' domain lowered the binding affinity by > 1000-fold; only [Phe(p-NH2)3]dermorphin retained nanomolar affinity for micro -receptors. Modifications in the 'address' region of both dermorphin and [Lys7]dermorphin were relatively well tolerated. In particular, [Phe(p-NH2)5,Lys7]dermorphin showed similar affinity to dermorphin, with almost 2-fold higher selectivity for micro -receptors. [Phe(p-NHCOCH2Br)5]- and [Phe(p-NHCOCH2Br)5,Lys7]dermorphin exhibited relatively high affinity (IC50 = 27.7 and 15.1 nm, respectively) for micro -receptors. However, neither of these peptides inhibited [3H]DAMGO binding in a wash-resistant manner.     

Haloacetol phosphates as affinity labels for methylglyoxal synthase

3-Bromo- and 3-iodoacetol phosphates irreversibly inactivate methylglyoxal synthase. The substrate, dihydroxyacetone phosphate, and inorganic phosphate protect against the inhibition. Although the 3-chloro derivative does not inactivate the enzyme, it is a competitive inhibitor. Reduction of the enzyme-inactivator complex with [${}^3\text{H}$]-NaBH₄ indicates the incorporation of four haloacetol phosphates per mole of enzyme. These studies suggest the bromo- and iodoacetol phosphates inactivate the enzyme by reacting with a nucleophilic group located in the active center.     

Synthesis and evaluation of derivatives of leucine enkephalin as potential affinity labels for delta opioid receptors

As part of an effort to develop peptide-based affinity labels for opioid receptors, [Leu(5)]enkephalin (LeuEnk) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), potent agonists for delta receptors, were selected as the parent peptides for further modification. The affinity label derivatives were prepared using standard Fmoc solid-phase peptide synthesis in conjunction with Fmoc-Phe(p-NHAlloc) (Fmoc: 9-flourenylmethoxycarbonyl;) and selective modification of the p-amino group on this residue. The electrophilic isothiocyanate and bromoacetamide groups were introduced into the para position of Phe(4); the corresponding free amine-containing peptides were also prepared for comparison. The pure peptides were evaluated in radioligand binding assays using Chinese hamster ovary (CHO) cells expressing delta and micro opioid receptors. Modification of Phe(4) in LeuEnk and DTLET significantly decreased delta-receptor binding affinity (40 to >2,000-fold). Among the synthesized analogues, [Phe(p-NH(2))(4)]DTLET showed the highest delta-receptor binding affinity (IC(50) = 39 nM) and enhanced selectivity for delta receptors compared to DTLET while other derivatives exhibited much lower delta receptor affinity. The differences in affinities between the two series of analogues and between the derivatives of LeuEnk and N,N-dibenzyl[Leu(5)]Enk reported previously suggest subtle differences in interactions of Phe(4) with delta receptors depending on other modifications in the sequences.     

13-Demethyl-13-substituted-13,14-dihydroretinols as potential affinity labels of retinol-binding proteins: syntheses and stability studies

13-Demethyl-13-substituted-13,14-dihydroretinols were synthesized and their stability under various conditions was measured in order to evaluate whether they would be useful as affinity labels of retinol binding proteins and retinol metabolizing enzymes. The 13-chloro analog could not be isolated because it eliminated HCl under the Wittig reaction conditions of its preparation. The trans- and cis-13,14-epoxy analogs are stable in non-protic organic solvents, but undergo an elimination reaction under various chromatographic conditions and in mixtures of organic solvents with water or alcohol. The 13-hydroxy and 13-methoxy analogs are stable in aqueous solutions and are therefore suitable for biological studies.     

Epoxysuccinyl peptide-derived affinity labels for cathepsin B

Extracellular cysteine proteases, in particular cathepsin B, have been implicated in a variety of pathological processes. Selectively targeting labels of this enzyme are important tools to gain more detailed understanding of its specific roles. Starting from our recently developed irreversible epoxysuccinyl-based inhibitor (R-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH, R=OMe), we have synthesized two affinity labels, R=NH-(CH(2))(6)-NH-rhodamine B and R=NH-(CH(2))(6)-NH-biotin. Using MCF-7 cells, the labeled inhibitors were shown to be virtually non-cell-permeant. Moreover, affinity blot analysis with the biotinylated inhibitor allowed a highly sensitive and selective non-radioactive detection of active cathepsin B.     

Synthesis and evaluation of potential affinity labels derived from endomorphin-2.

In an attempt to identify potential peptide-based affinity labels for opioid receptors, endomorphin-2 (Tyr-Pro-Phe-PheNH2), a potent and selective endogenous ligand for mu-opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc-solid phase peptide synthesis in conjunction with incorporation of Fmoc-Phe(p-NHAlloc) and modification of the p-amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe3 or Phe4; the corresponding free amine-containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand-binding assay using Chinese hamster ovary (CHO) cells expressing mu- and delta-opioid receptors. Modification on Phe4 was better tolerated than on Phe3 for mu-receptor binding. Among the analogs tested, [Phe(p-NH2)4]endomorphin-2 showed the highest affinity (IC50 = 37 nm) for mu-receptors. The Phe(p-NHCOCH2Br)4 analog displayed the highest mu-receptor affinity (IC50 = 158 nm) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for delta-receptors, similar to the parent peptide.     

Azidophenantridinium compounds as photoaffinity labels of cholinergic proteins

The synthesis of diazidopropidium and diazidoethidium is described. The applicability of these compounds as photoaffinity labels for cholinergic proteins has been investigated: diazidopropidium inhibits neuromuscular transmission. This inhibition is reversible if the compound is applied in the dark but becomes irreversible after irradiation with white light. Inhibition is accompanied by a disappearance of miniature endplate potentials. Electrophysiological analysis of this effect indicates that diazidopropidium acts postsynaptically by blocking the acetylcholine receptors. At the molecular level the action of diazidopropidium and diazidoethidium on acetylcholinesterase has been investigated: both compounds appear to bind to a peripheral acetylcholine binding site of this enzyme. Binding of ¹²⁵I-labeled α-neurotoxin from Naja naja siamensis to purified membranes from Torpedo californica electric tissue rich in acetylcholine receptors is diminished after incubation and irradiation with diazidopropidium. About half of the toxin binding sites appear to be blocked by the photoaffinity label.     

Azidophenantridinium compounds as photoaffinity labels of cholinergic proteins.

The synthesis of diazidopropidium and diazidoethidium is described. The applicability of these compounds as photoaffinity labels for cholinergic proteins has been investigated: diazidopropidium inhibits neuromuscular transmission. This inhibition is reversible if the compound is applied in the dark but becomes irreversible after irradiation with white light. Inhibition is accompanied by a disappearance of miniature endplate potentials. Electrophysiological analysis of this effect indicates that diazidopropidium acts postsynaptically by blocking the acetylcholine receptors. At the molecular level the action of diazidopropidium and diazidoethidium on acetylcholinesterase has been investigated: both compounds appear to bind to a peripheral acetylcholine binding site of this enzyme. Binding of 125I-labeled alpha-neurotoxin from Naja naja siamensis to purified membranes from Torpedo californica electric tissue rich in acetylcholine receptors is diminished after incubation and irradiation with diazidopropidium. About half of the toxin binding sites appear to be blocked by the photoaffinity label.     

Iodination of peptidyl chloromethyl ketones for protease affinity labels

The specificity of peptidyl chloromethyl ketones has been used to label proteases in complex biological systems by incorporating tyrosine into the structure for eventual radioiodination. Contrary to results with iodination of proteins, a mild reagent, that is, one which iodinates at neutrality, was unsuitable, giving complex mixtures with poor reproducibility, apparently because of side reactions at the chloromethyl ketone group. On the other hand, iodine monochloride in acetic acid provided clean products. In the cases examined where a tyrosine residue was not appropriate for the specificity of the target protease, this residue was located well displaced from the primary specificity site. The resultant diiodotyrosine-containing derivatives were generally highly active as protease inhibitors. The p-aminobenzoyl group was used as an alternative to tyrosine as an iodinatable component.     

The synthesis of diazo, halo, and sulfoxy bile acid derivatives: potential affinity labels.

Bile acid derivatives, with and without C-3 sulfate groups, and having either the diazo- or halomethylketone moieties, have been synthesized in good yield and purity. The synthetic sequence, COOH leads to COC1 leads to COCHN2 leads to COCH2X, was used with deoxycholic and cholic acids, which requires carefully controlled quench, work-up, and purification procedures, especially for the 3-sulfate esters (made from deoxycholic acid derivatives only). The pure title compounds are anticipated to be useful chemical probes (affinity labels), especially the completely water soluble sulfates, toward our studies of ileal active transport of bile salts. A new use for Sephadex LH-20 as a sulfate ester protecting group is reported. Also developed were the use of acetamide hydrochloride complex as a mild hydrochlorination reagent and a neutral desalting method for sulfate esters of deoxycholic acid derivatives.     

Gold nanomaterials and their potential use as cryo-electron tomography labels

Rapid advances in cryo-electron tomography (cryo-ET) are driving a revolution in cellular structural biology. However, unambiguous identification of specific biomolecules within cellular tomograms remains challenging. Overcoming this obstacle and reliably identifying targets in the crowded cellular environment is of major importance for the understanding of cellular function and is a pre-requisite for high-resolution structural analysis. The use of highly-specific, readily visualised and adjustable labels would help mitigate this issue, improving both data quality and sample throughput. While progress has been made in cryo-CLEM and in the development of cloneable high-density tags, technical issues persist and a robust ‘cryo-GFP’ remains elusive. Readily-synthesized gold nanomaterials conjugated to small ‘affinity modules’ may represent a solution. The synthesis of materials including gold nanoclusters (AuNCs) and gold nanoparticles (AuNPs) is increasingly well understood and is now within the capabilities of non-specialist laboratories. The remarkable chemical and photophysical properties of <3nm diameter nanomaterials and their emergence as tools with widespread biomedical application presents significant opportunities to the cryo-microscopy community. In this review, we will outline developments in the synthesis, functionalisation and labelling uses of both AuNPs and AuNCs in cryo-ET, while discussing their potential as multi-modal probes for cryo-CLEM.     

Use of fluorescently labeled caspase inhibitors as affinity labels to detect activated caspases

Activation of caspases is the key event of apoptosis and different approaches were developed to assay it. To detect their activation in situ, we applied fluorochrome labeled inhibitors of caspases (FLICA) as affinity labels of active centers of these enzymes. The FLICA ligands are fluorescein or sulforhodamine conjugated peptide-fluoromethyl ketones that covalently bind to enzymatic centers of caspases with 1:1 stoichiometry. The specificity of FLICA towards individual caspases is provided by the peptide sequence of amino acids. Exposure of live cells to FLICA results in uptake of these ligands and their binding to activated caspases; unbound FLICA is removed by cell rinse. Cells labeled with FLICA can be examined by fluorescence microscopy or subjected to quantitative analysis by cytometry. Intracellular binding sites of FLICA are consistent with known localization of caspases. Covalent binding of FLICA allowed us to identify the labeled proteins by immunoblotting: the proteins that bound individual FLICAs had molecular weight between 17 and 22 kDa, which corresponds to large subunits of the caspases. Detection of caspases activation by FLICA can be combined with other markers of apoptosis or cell cycle for multiparametric analysis. Because FLICA are caspase inhibitors they arrest the process of apoptosis preventing cell disintegration. The stathmo-apoptotic method was developed, therefore, that allows one to assay cumulative apoptotic index over long period of time and estimate the rate of cell entry into apoptosis for large cell populations. FLICA offers a rapid and convenient assay of caspases activation and can also be used to accurately estimate the incidence of apoptosis.     

Lys3]Didemnins as potential affinity ligands

The synthesis and biological activity of N(epsilon)-Z-[Lys3]didemnin B are reported. This novel analogue retains antiproliferative, cytotoxic, and protein biosynthesis inhibition activities, but at reduced levels. This result suggests the use of [Lys3]didemnin derivatives as potential affinity probes for studying the molecular target(s) of the didemnin class of natural products.     

Brain-penetrant microtubule-stabilizing compounds as potential therapeutic agents for tauopathies

Neurons within the brains of those with AD (Alzheimer's disease) and related neurodegenerative disorders, collectively termed 'tauopathies', contain fibrillar inclusions composed of hyperphosphorylated tau protein. Tau is normally enriched in axons, where it binds and stabilizes MTs (microtubules). Tau hyperphosphorylation and aggregation probably result in reduced MT binding that could affect axonal transport and neuronal function. A possible therapeutic strategy to overcome a loss of tau function in tauopathies is administration of MT-stabilizing agents, such as those used in the treatment of cancer. However, these drugs elicit severe side effects, and most existing MT-stabilizing compounds have poor BBB (blood-brain barrier) permeability, which renders them unsuitable for tauopathy treatment. We identified EpoD (epothilone D) as a brain-penetrant MT-stabilizing agent with preferred pharmacokinetic and pharmacodynamic properties. EpoD was evaluated for its ability to compensate for tau loss-of-function in an established Tg (transgenic) mouse model, using both preventative and interventional dosing paradigms. EpoD at doses much lower than previously used in human cancer patients caused improved axonal MT density and decreased axonal dystrophy in the tau Tg mice, leading to an alleviation of cognitive deficits. Moreover, EpoD reduced the extent of tau pathology in aged tau Tg mice. Importantly, no adverse side effects were observed in the EpoD-treated mice. These results suggest that EpoD might be a viable drug candidate for the treatment of AD and related tauopathies.     

Polysaccharides as potential antioxidative compounds for extended-release matrix tablets

The objective of this study was to identify polysaccharides with antioxidant properties for use as potential antioxidative compounds for extended-release matrix tablets. The antioxidant properties of five different polysaccharides, high molecular weight alginate (H-ALG), low molecular weight alginate (L-ALG), high molecular weight chitosan (H-chitosan), low molecular weight chitosan (L-chitosan), and pectic acid (PA) were examined using N-centered radicals from 1,1′-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and reducing power, based on their ability to reduce Cu²⁺. L-chitosan and PA had acceptable scavenging abilities and were good radical scavengers, with good reducing power, but the H-chitosan and alginate derivatives were much less effective. The results suggest that L-chitosan and PA could be useful in combating oxidative stress. A PA and L-chitosan interpolymer complex (IPC) tablet was prepared and evaluated as an extended-release tablet matrix using theophylline (TPH) as a model drug. The release of TPH from the matrix tablet (TPH/PA/L-chitosan = 200 mg:150 mg:50 mg) was slower than that from PA only (TPH/PA/chitosans = 200 mg:200 mg:0 mg) or L-chitosan only (TPH/PA/L-chitosan = 200 mg:0 mg:200 mg) tablet. Turbidity measurements also indicated the optimum complexation ratio for IPC between PA/L-chitosan to be 1/3, indicating an acceptable relationship between the turbidity of the complex and the release ratio of TPH. These results suggest that an L-chitosan/PA complex would be potentially useful in an extended-release IPC tablet with high antioxidant activity.     

Neurotensin analogs indications for use as potential antipsychotic compounds

This review will be an update, focusing on the central nervous system (CNS) roles of the neurotransmitter, neurotensin. We will provide a summary of current knowledge about neurotensin, why it is an important peptide to study, and where the field is heading. Special emphasis is placed on the development of neurotensin analogs, which has been a major effort of our group, the potential role of neurotensin in Parkinson's disease, and the interaction of neurotensin with other neurotransmitters as evidenced by microdialysis studies.     

The use of base pair specific DNA binding agents as affinity labels for the study of mammalian chromosomes

The fluorochromes Hoechst 33258 and olivomycin are base pair specific DNA binding agents. The fluorescence enhancement of Hoechst 33258 and olivomycin in the presence of DNA can be directly related to the A-T and G-C content of the interacting DNA respectively. Cytological observations of metaphase chromosomes treated with these two compounds suggest that the fluorescent banding patterns produced are the reverse of one another. —Non-fluorescent base pair specific DNA binding agents have been used as counterstains in chromosome preparations to enhance the contrast of the banding patterns produced by the base specific fluorochromes. The non-fluorescent G-C specific antibiotic actinomycin-D enhanced the resolution of fluorescent bands produced by the A-T specific fluorochrome Hoechst 33258. Similarly the non-fluorescent A-T specific antibiotic netropsin was found to enhance resolution of the bands produced by the G-C specific fluorochrome olivomycin. Netropsin was also found to increase the differential fluorescent enhancement of complexes of olivomycin with DNAs of various base composition in solution. These findings suggest that counterstaining agents act through a base sequence dependent inhibition of subsequent binding by base pair specific fluorochromes.—The base specific DNA binding agents have been used to differentiate different types of constitutive heterochromatin in mammalian species, and to facilitate chromosome identification in somatic cell hybrids.     

High affinity dopamine reuptake inhibitors as potential cocaine antagonists: a strategy for drug development

The addictive and euphorogenic effects of cocaine are thought to result primarily from inhibition of dopamine reuptake. Although the potency of cocaine-like drugs as inhibitors of DA reuptake is highly correlated with their potency as reinforcers in animals, several potent DA reuptake blockers (bupropion, nomifensine, benztropine, and mazindol) have not been reported to produce addiction or euphoria in humans. Based on these observations in humans, DA reuptake inhibitors are classified into two groups; type 1 blockers, which produce addiction and euphoria, and type 2 blockers, which do not. Given that type 1 and type 2 blockers act at the same site (the DA transporter), the author suggests that type 2 agents may antagonize the effects of cocaine, and might prove useful in the treatment of cocaine addiction.     

8-Azidoflavins as photoaffinity labels for flavoproteins

8-Azidoflavins have been synthesized and their potential as photoaffinity labels for flavoproteins has been explored. They are very photolabile, and in aqueous media they react with solvent to yield 8-aminoflavins and 8-hydroxlaminoflavins as the main products. They fulfill the criteria expected of a good photoaffinity label, since they bind stoichiometrically at the flavin-binding site of flavoproteins, thus minimizing problems of nonspecific labeling. Second, they absorb strongly in the visible, so that the reactive nitrene can be generated without short wavelength light, minimizing the possibility of light-induced damage of the protein. Third, in the absence of light, 8-N3-flavins are stable, permitting a study of their binding to apoproteins. 8-Azidoflavins have been bound to hen egg white riboflavin-binding protein, Megasphera elsdenii flavodoxin, yeast Old Yellow Enzyme, Aspergillus niger, glucose oxidase, and pig kidney D-amino acid oxidase, and the effect of exposure to visible light has been determined. Only small extents of covalent attachment of the flavin to the protein were found with flavodoxin, D-amino acid oxidase, and Old Yellow Enzyme; much more extensive labeling was obtained with glucose oxidase and riboflavin-binding protein. In addition to their photoreactivity, 8-azidoflavins have been found to be converted to 8-aminoflavins by reaction with sulfite or upon reduction. Similar reactions occur with 8-hydroxylamino-, 8-(O-methyl)hydroxylamino-, and 8-hydrazinoflavins, which serve as models for possible flavin-protein covalent linkages which could be formed in the photolabeling procedure. Some of the properties of these flavins, which were obtained by reaction of 8-F-flavin with the corresponding nucleophiles, are also described.     

3-Methoxy-4-(2-nitrovinyl)phenyl glycosides as potential chromogenic substrates for the assay of glycosidases

Selective glycosidation of 2,4-dihydroxybenzaldehyde with either 2,3,4, 6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide, 2-acetamido-3,4,6-tri-O-acetyl-alpha-D-glucopyranosyl chloride, or 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide afforded the corresponding 4-O-glycosyl derivatives. Subsequent O-methylation, O-deacetylation, and condensation with nitromethane afforded the appropriate beta-glycoside of 3-methoxy-4-(2-nitrovinyl)phenol. The phenol is highly coloured at alkaline pH so that these glycosides may be suitable as chromogenic substrates for the assay of glycosidases.