Aggregation of Aβ Alzheimer's disease-related peptide studied by dynamic light scattering

The aggregation behavior of the major component of Alzheimer's disease-related, amyloid peptides, Aβ-(1–40) and Aβ-(1–42), was studied in solution using dynamic light scattering. With most solvents employed, we found fibrils coexisting with oligomeric Aβ species. Pronounced differences were observed in aggregation of Aβ-(1–40) and (1–42) sequences in acetonitrile-water mixtures. Cofactors such as Zn²⁺ were found to induce deaggregation of Aβ instead of aggregation. The results indicated that the initial state of the peptide immediately after synthesis is rather poorly defined. Using freezing instead of lyophilization after the final peptide synthesis step, may partially relieve these problems.     

Structure‐Activity Relationships for a Series of Compounds that Inhibit Aggregation of the Alzheimer's Peptide,Aβ42

Inhibiting aggregation of the amyloid‐beta (Aβ) peptide may be an effective strategy for combating Alzheimer's disease. As the high‐resolution structure of the toxic Aβ aggregate is unknown, rational design of small molecule inhibitors is not possible, and inhibitors are best isolated by high‐throughput screening. We applied high‐throughput screening to a collection of 65 000 compounds to identify compound D737 as an inhibitor of Aβ aggregation. D737 diminished the formation of oligomers and fibrils, and reduced Aβ42‐induced cytotoxicity. Most importantly, D737 increased the life span and locomotive ability of transgenic flies in a Drosophila melanogaster model of Alzheimer's disease (J Biol Chem, 287, 2012, 38992). To explore the chemical features that make D737 an effective inhibitor of Aβ42 aggregation and toxicity, we tested a small collection of eleven analogues of D737 . Overall, the ability of a compound to inhibit Aβ aggregation was a good predictor of its efficacy in prolonging the life span and locomotive ability of transgenic flies expressing human Aβ42 in the central nervous system. Two compounds ( D744 and D830 ) with fluorine substitutions on an aromatic ring were effective inhibitors of Aβ42 aggregation and increased the longevity of transgenic flies beyond that observed for the parent compound, D737 .     

Key mechanisms underlying netrin‐1 prevention of impaired spatial and object memory in Aβ1‐42 CA1‐injected rats

Alzheimer's disease (AD) is a neurodegenerative disorder with an incompletely defined aetiology that is associated with memory and cognitive impairment. Currently available therapeutics only provide temporary assistance with symptoms. In spite of plentiful research in the field and the generation of thousands of studies, much is still to be clarified on precise mechanisms of pathobiology, prevention modalities, disease course and cure. Netrin‐1, a laminin family protein, is said to have anti‐inflammatory and anti‐apoptotic effects and has a key role in neurogenesis and morphogenesis of neural structures. Accordingly, this study was designed to investigate protective effects of bilateral intrahippocampal fissure microinjections of netrin‐1 on memory impairment in rat model of AD. Concomitant administration of netrin‐1 with amyloid beta 1‐42 (Aβ_1‐42) improved cognitive dysfunction in novel object recognition task (NOR), ameliorated impaired spatial memory in Morris water maze (MWM) setting, increased neuronal density and reduced amyloid aggregation in rat AD model. Netrin‐1 was also seen to prevent Aβ_1‐42‐induced caspase‐3, caspase‐7 and NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) activation. Therefore, based on the data reported here, netrin‐1 may be a promising biologic therapeutic that addresses the memory and neuronal loss associated with AD.     

Effects of N‐Methylated Amyloid‐β30–40 Peptides on the Fibrillation of Amyloid‐β1–40

Alzheimer's disease is a neurodegenerative disorder associated with amyloid‐β (Aβ) fibrillation. N‐Methylated amyloid‐β peptides are potent inhibitors of amyloid‐β fibrillation. We investigated the inhibitory effect of N‐Methylated Aβ_30–40 peptides on Aβ_1–40 fibrillation. N‐Methylated Aβ_30–40 peptides affected the fibrillation, and this effect was dependent on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. N‐Methylated Aβ_30–40 peptides were co‐aggregated with Aβ_1–40. Spectroscopic technique was adopted to investigate an origin of the observed dependence. Suppression of thioflavin T (ThT) fluorescence count was correlated with the dissociation constant K_d of monomer–dimer equilibrium of each N‐Methylated Aβ_30–40 peptide. Monomeric N‐Methylated peptides decreased ThT fluorescence count during Aβ_1–40 fibrillation. Secondary structure content was not largely different between Aβ_1–40 fibrils and co‐aggregates. These results suggested that N‐Methylated Aβ_30–40 peptides disrupted the regular β‐sheet structure of Aβ_1–40 fibrils and affected the ThT fluorescence count. The monomer–dimer equilibrium of N‐Methylated peptides was (partly) responsible for the observed dependence of their inhibitory effect on the concentration of N‐Methylated peptide and the number and position of N‐Methylated groups. Our study provides a hint to design new N‐Methylated inhibitor peptides of fibrillation.     

Protective effects of β‐sheet breaker α/β‐hybrid peptide against amyloid β‐induced neuronal apoptosis in vitro

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid‐β oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid‐β (Aβ). Increased production of Aβ invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a β‐sheet breaker α/β‐hybrid peptide (BSBHp) and the underlying mechanisms against Aβ₄₀‐induced neurotoxicity in human neuroblastoma SH‐SY5Y cells. Cells were pretreated with the peptide Aβ₄₀ to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca²⁺, and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aβ₄₀‐induced toxicity by retaining cell viability, suppressing generation of ROS, Ca²⁺ levels, and effectively protects neuronal apoptosis by suppressing pro‐apoptotic protein Bax and up‐regulating antiapoptotic protein Bcl‐2. These results suggest that α/β‐hybrid peptide has neuroprotective effects against Aβ₄₀‐induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.     

The CEC behaviour of several synthetic peptides related to the activin βA–βD subunits

Abstract: The resolution of several structurally related synthetic peptides, derived from the loop 3 region of the activin β_A–β_D subunits, has been studied using capillary electrochromatography (CEC) with Hypersil n‐octadecylsilica as the sorbent. The results confirm that the CEC migration of these peptides can be varied in a charge‐state‐specific manner as the properties of the background electrolyte, such as pH, salt concentration and content of organic modifier, or temperature are systematically changed. Acidic peptides followed similar trends in retention behaviour, which was distinctly different to that shown by more basic peptides. The CEC separation of these peptides with the Hypersil n‐octadecyl‐silica involved distinguishable contributions from both electrophoretic mobility and chromatographic retention. Temperature effects were reflected as variations in both the electro‐osmotic flow and the electrophoretic mobility of the peptides. When the separation forces acting on the peptides were synergistic with the electro‐osmotic flow, as, for example, with the positively charged peptides at a particular pH and buffer electrolyte composition, their retention coefficient, κ_cec, decreased with increasing capillary temperature, whereas when the separation forces worked in opposite directions, as for example with negatively charged peptides, their κ_cec values increased slightly with increasing temperature. Moreover, when the content of organic modifier, acetonitrile, was sufficiently high, e.g. > 40% (v/v) and nonpolar interactions with the Hypersil n‐octadecyl‐silica sorbent were suppressed, mixtures of both the basic and acidic synthetic peptides could be baseline resolved under isocratic conditions by exploiting the mutual processes of electrophoretic mobility and electrostatic interaction. A linear relationship between the ln κ_cec values and the volume fractions, ψ, of the organic modifier over a limited range of ψ‐values, was established for the negatively charged peptides under these isocratic conditions. These findings thus provide useful guidelines in a more general context for the resolution and analysis of structurally related synthetic peptides using CEC methods.     

Does Aluminium Bind to Histidine? An NMR Investigation of Amyloid β12 and Amyloid β16 Fragments

Aluminium and zinc are known to be the major triggering agents for aggregation of amyloid peptides leading to plaque formation in Alzheimer's disease. While zinc binding to histidine in Aβ (amyloid β) fragments has been implicated as responsible for aggregation, not much information is available on the interaction of aluminium with histidine. In the NMR study of the N‐terminal Aβ fragments, DAEFRHDSGYEV (Aβ12) and DAEFRHDSGYEVHHQK (Aβ16) presented here, the interactions of the fragments with aluminium have been investigated. Significant chemical shifts were observed for few residues near the C‐terminus when aluminium chloride was titrated with Aβ12 and Aβ16 peptides. Surprisingly, it is nonhistidine residues which seem to be involved in aluminium binding. Based on NMR constrained structure obtained by molecular modelling, aluminium‐binding pockets in Aβ12 were around charged residues such as Asp, Glu. The results are discussed in terms of native structure propagation, and the relevance of histidine residues in the sequences for metal‐binding interactions. We expect that the study of such short amyloid peptide fragments will not only provide clues for plaque formation in aggregated conditions but also facilitate design of potential drugs for these targets.     

Thiophilic interaction chromatography of Alzheimer's β‐amyloid peptides

Abstract: Alzheimer's disease is characterized by a progressive formation of senile plaques in the brain, the major constituent of which is β‐amyloid (Aβ) peptide, a proteolytic product of the transmembrane β‐amyloid precursor protein (APP). Prior to the measurement of levels of the Aβ peptide for diagnostic purposes, this peptide must be isolated from the myriad of proteins resident in the human serum. Thiophilic interaction chromatography is an effective method for the isolation of proteins and peptides containing clusters of aromatic residues such as tryptophan, phenylalanine and tyrosine. The purpose of the present study was to develop a protocol for binding and recovery of Aβ peptides (1–38), (1–40) and (1–42) to T‐gels by varying T‐gel type and elution conditions such as the salt concentration and type of eluent. We established the minimal salt concentration necessary for the binding of the Aβ(1–40) peptide to the 3S‐gel; binding at that concentration was subsequently compared with that of model proteins, lysozyme and α‐chymotrypsin and this methodology was extended to 2S‐gels and PyS. β‐Amyloid peptide (1–40) showed a remarkably strong affinity for all three types of T‐gels in comparison to lysozyme and α‐chymotrypsin and was found to bind best to 2S‐gel.     

Structural analysis of fertilinβ cyclic peptide mimics that are ligands for α6β1 integrin

Abstract: The NMR structural analysis of two fertilinβ mimics cyclo(EC²DC¹)YNH_2, 1 , and cyclo(D²EC²D¹C¹)YNH_2, 2 is described. Both of these mimics are moderate inhibitors of sperm?egg binding with IC₅₀ values of 500 µm in a mouse in vitro fertilization assay. For peptide 1 , the optimized conformations that best match the NMR data have a pseudo‐type II′β‐turn with the linker and Glu at the i+1 and i+2 positions, respectively. The EC²D¹C¹ sequence is in a nonclassical (type IV) β‐turn. For peptide 2 , the conformation that best matches the NMR data has two turns: a pseudo‐type II′β‐turn in the D²EC²D¹ sequence followed by a nonclassical β‐turn in the EC²D¹C¹ sequence. The Cβ?Cβ distance between E and D¹ in peptide 1 is 9.1 Å, in peptide 2 , it is 7.7 Å. Thus, one possibility for the high IC₅₀ values of these cyclic peptides is that the acidic residues are not constrained to a sufficiently tight turn, and thus much entropy must still be lost upon binding to the α₆β₁ integrin. This explains why the cyclic peptides are the same as linear peptides at inhibiting sperm?egg binding.     

Metformin inhibits Aβ25‐35‐induced apoptotic cell death in SH‐SY5Y cells

Metformin, a first‐line drug for type‐2 diabetes, plays a potentially protective role in preventing Alzheimer's disease (AD), but its underlying mechanism is unclear. In this study, Aβ_25‐35‐treated SH‐SY5Y cells were used as a cell model of AD to investigate the neuroprotective effect of metformin, as well as its underlying mechanisms. We found that metformin decreased the cell apoptosis rate and death, ratio of Bcl‐2/Bax, and expression of NR2A and NR2B, and increased the expression of LC3 in Aβ_25‐35‐treated SH‐SY5Y cells. Metformin also reduced intracellular and extracellular Glu concentrations, as well as the intracellular concentration of Ca²⁺ and ROS in Aβ_25‐35‐treated SH‐SY5Y cells. These findings suggest that metformin inhibits Aβ_25‐35‐treated SH‐SY5Y cell death by inhibiting apoptosis, decreasing intracellular Ca²⁺ and ROS by reducing neurotoxicity of excitatory amino acids, and by possibly reversing autophagy disorder via regulating autophagy process.     

Inhibition of β‐amyloid toxicity by short peptides containing N‐methyl amino acids

Abstract: Single N‐methyl amino acid‐containing peptides related to the central hydrophobic region β_16–20 (Lys‐Leu‐Val‐Phe‐Phe) of the β‐amyloid protein are able to reduce the cytotoxicity of natural β_1–42 in PC12 cell cultures. N‐methyl phenylalanine analogs yield statistically significant increments in cell viability (Student's t‐test < 0.01%) and are nontoxic in the same assay. These promising results indicate that these peptide molecules could be a starting point for the development of potential therapeutic compounds for the treatment of Alzheimer's disease.     

Selectively 2H‐labeledGlu/Asp: application to pKa measurements in Aβ amyloid peptides

Abstract: Human Aβ peptides have been linked to Alzheimer’s disease, and it is hypothesized that formation of amyloid as well as neurotoxicity are important events in the etiology of the disease. Previous studies have shown that the soluble precursor to Alzheimer’s amyloid undergoes a pH‐dependent folding transition as the self‐assembly activity appears, and based upon inter‐residue proximities, it was suspected that stabilization of the soluble form might rely upon formation of an intramolecular salt‐bridge. However, pK_a studies on a model 17‐residue Aβ fragment supported an electrostatic model where a solvation imperative for charged side‐chain atoms drives the folding process. To explore this model in an active 26‐residue fragment as well as the full‐length 40‐residue Αβ peptide, pK_a measurements were performed via ¹H and ²H NMR. To overcome issues related to sensitivity and spin system degeneracy, specifically deuterated allyl protected‐Fmoc amino acids were synthesized for incorporation into a series of peptides, and a high sensitivity ²H observe NMR probe was constructed.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Conformational investigation of α,β-dehydropeptides

Conformations of three series of model peptides: homochiral Ac-Pro-L-Xaa-NHCH₃ and heterochiral Ac-Pro-D-Xaa-NHCH₃ (Xaa=Phe, Val, Leu. Abu. Ala) as ivell as α,β-dehydro Ac-Pro-ΔXaa-NHCH_s [ΔXaa = (Z)-ΔPhe, ΔVal. (Z)-ΔLeu, (Z)-ΔAbu] were investigated by CD spectroscopy in 2 % dichloromethanecyclohexane, trifluoroethanol. water. and occasionally in other solvents. The spectra of homochiral peptides show a significant solvent dependence. Folded structures are present in 2% dichloromethane-cyclohexane and unordered ones occur in water. The folded conformers are of the inverse γ-turn type for all the peptides but Ac-Pro-L-Phe-NHCH₃ for which the type-I β-turn is preferred. The changes in the spectra of the heterochiral peptides are limited. The compounds adopt the typc-II β–turn in 2% dichloromethanecyclohexane, represented by class B spectra, and retain this conformation in water as well as in fluorinated alcohols but not always to a full extent. The CD spectra of the unsaturated peptides in 2%, dichloromethanecyclohexane, although they cannot be assigned to any common spectral class, must be attributed to the βII-turn conformation as determined for these coinpounds by NMR and IR spectroscopy. The CD spectra of dehydropeptides exhibit a considerable solvent dependence and suggest unordered structures in water.     

Integrin α5β1: a new purification strategy based on immobilized peptides

Abstract: Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C‐terminally modified peptide Ac‐Gly‐Ala‐c‐(Cys^SS‐Arg‐Arg‐Glu‐Thr‐Ala‐Trp‐Ala‐Cys^SS)‐Gly‐Ala‐O(CH₂CH₂O)₂CH₂CH₂‐NH₂ allows for the affinity purification of the integrin α₅β₁. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α₅β₁. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well‐characterized affinity chromatography material with a site‐specifically immobilized peptide allows to obtain integrin α₅β₁ in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein‐ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.     

Synthesis of a non‐peptidic PET tracer designed for α5β1 integrin receptor

Arginine–glycine–aspartic acid (RGD)‐containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α₅β₁ integrin receptor have been developed with promising results. Sixty‐one antagonists were screened, and tert‐butyl (S)‐3‐(2‐((3R,5S)‐1‐(3‐(1‐(2‐fluoroethyl)‐1H‐1,2,3‐triazol‐4‐yl)propanoyl)‐5‐((pyridin‐2‐ylamino)methyl)pyrrolidin‐3‐yloxy)acetamido)‐2‐(2,4,6‐trimethylbenzamido)propanoate (FPMt) was selected for the development of a PET tracer to image the expression of α₅β₁ integrin receptors. An alkynyl precursor (PMt) was initially synthesized in six steps, and its radiolabeling was performed according to the azide–alkyne copper(II)‐catalyzed Huisgen's cycloaddition by using 1‐azido‐2‐[¹⁸F]fluoroethane ([¹⁸F]12). Different reaction conditions between PMt and [¹⁸F]12 were investigated, but all of them afforded [¹⁸F]FPMt in 15 min with similar radiochemical yields (80–83%, decay corrected). Overall, the final radiopharmaceutical ([¹⁸F]FPMt) was obtained after a synthesis time of 60–70 min in 42–44% decay‐corrected radiochemical yield. Copyright © 2014 John Wiley & Sons, Ltd.     

177Lu‐labeled monomeric,dimeric and multimeric RGD peptides for the therapy of tumors expressing α(ν)β(3) integrins

The conjugation of peptides to gold nanoparticles (AuNPs) produces biocompatible and stable multimeric systems with target‐specific molecular recognition. Peptides based on the cyclic Arg‐Gly‐Asp (RGD) sequence have been reported as high‐affinity agents for the α(ν)β(3) integrin. The aim of this research was to prepare a multimeric system of ¹⁷⁷Lu‐labeled gold nanoparticles conjugated to c(RGDfK)C (cyclo(Arg‐Gly‐Asp‐Phe‐Lys)Cys) and to compare the radiation‐absorbed dose with that of ¹⁷⁷Lu‐labeled monomeric and dimeric RGD peptides to α(ν)β(3) integrin‐positive U87MG tumors in mice. DOTA‐GGC (1,4,7,10‐tetraazacyclododecane‐N‐N′,N″,N?‐tetraacetic acid‐Gly‐Gly‐Cys) and c(RGDfK)C peptides were synthesized and conjugated to AuNPs by a spontaneous reaction of the thiol groups. Transmission electron microscopy, ultraviolet–visible, X‐ray photoelectron spectroscopy, Raman and far‐infrared spectroscopy techniques demonstrated that AuNPs were functionalized with the peptides. For the ¹⁷⁷Lu‐AuNP‐c(RGDfK)C to be obtained, the ¹⁷⁷Lu‐DOTA‐GGC radiopeptide was first prepared and added to a solution of AuNPs followed by c(RGDfK)C (25 µl, 5 µ m ) at 18 °C for 15 min. ¹⁷⁷Lu‐DOTA‐GGC, ¹⁷⁷Lu‐DOTA‐cRGDfK and ¹⁷⁷Lu‐DOTA‐E‐c(RGDfK)₂ were prepared by adding ¹⁷⁷LuCl₃ (370 MBq) to 5 µl (1 mg/ml) of the DOTA derivative diluted with 50 µl of 1 m acetate buffer pH 5. The mixture was incubated at 90 °C in a block heater for 30 min. Radiochemical purity was determined by ultrafiltration and HPLC analyses. Biokinetic studies were accomplished in athymic mice with U87MG‐induced tumors. The radiochemical purity for all ¹⁷⁷Lu‐RGD derivatives was 96 ± 2%. ¹⁷⁷Lu‐absorbed doses per injected activity delivered to U87MG tumors were 0.357 ± 0.052 Gy/MBq (multimer), 0.252 ± 0.027 Gy/MBq (dimer) and 0.102 ± 0.018 Gy/MBq (monomer). ¹⁷⁷Lu‐labeled dimeric and multimeric RGD peptides demonstrated properties suitable for targeted radionuclide therapy of tumors expressing α(ν)β(3) integrins. Copyright © 2012 John Wiley & Sons, Ltd.     

Design of high affinity cyclic pentapeptide ligands for κ‐opioid receptors

Abstract: Using results from our previously reported cyclic opioid peptide series and reliable models for μ‐, δ‐, and κ‐opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr‐c[d ‐Cys‐Phe‐Phe‐X]‐NH₂, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = d ‐ or l ‐Cys; or d ‐ or l ‐penicillamine (Pen; β,β‐dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = d ‐ or l ‐Cys display KOR affinities in the low nanomolar range, demonstrating that a ‘DPDPE‐like’ tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8 , Tyr‐c[d ‐Cys‐Phe‐Phe‐Cys]‐NH₂‐cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm ) with more than 100‐fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Structure‐based derivation of peptide inhibitors to target TGF‐β1 receptor for the suppression of hypertrophic scarring fibroblast activation

The intermolecular recognition and interaction between human transforming growth factor β‐1 (TGF‐β1) and its cognate receptor TβRII have been implicated in the pathological condition of hypertrophic scarring (HS). Here, we attempted to rationally derive peptide inhibitors from the complex interface of TGF‐β1 with TβRII to disrupt such interaction for the suppression of fibroblast activation involved in HS. A synthetic strategy that integrated computational design and fluorescence‐based assay was described to examine the structural basis and energetic property of TGF‐β1–TβRII crystal structure, from which a small peptide segment in the complex binding site was stripped artificially. Molecular dynamics simulations revealed that the linear peptide possesses a large intrinsic disorder that would incur considerable entropy penalty upon binding to TβRII; the peptide segment was then extended and cyclized by introducing a disulfide bond across its terminal residues that were premutated to cysteine. Normal mode analysis indicated that, as expected, the peptide flexibility was largely reduced upon the cyclization, and thus, the entropy penalty was minimized substantially, consequently promoting the spontaneous binding of peptide to TβRII. Fluorescence polarization assay confirmed that all linear peptides are typical non‐binders of TβRII (K_d = ND), while the designed cyclic peptides exhibit moderate or high affinity with K_d at micromolar level.     

Preparation and preliminary biological evaluation of 177Lu‐labeled GluDTPA‐cyclo(RGDfK) for integrin ανβ3 receptor‐positive tumor targeting

Integrin α_νβ₃ is a receptor and is highly expressed on activated and proliferating endothelial cells during the growth and metastasis of solid tumors but not on resting endothelial cells and normal organs. Because RGD peptide binds to integrin α_νβ₃ receptor, a variety of radiolabeled RGD peptides have been evaluated for non‐invasive imaging of integrin α_νβ₃‐positive tumors. In an attempt to develop RGD‐based radiopharmaceuticals, a novel GluDTPA‐cyclo arginine‐glycine‐aspartic acid‐d ‐phenylalanine‐lysine (GluDTPA‐cycloRGDfK) was simply synthesized and radiolabeled with ¹⁷⁷Lu. Also, tumor targeting and retention of the radiolabeled complex were evaluated in U87MG glioma‐bearing mice. The ¹⁷⁷Lu‐labeled GluDTPA‐cyclo(RGDfK) was formulated with a high radiolabeling yield (>98%) under mild condition, and the radiochemical purity was sustained in both saline and serum for over 4 days at 37°C. The radiolabeled compounds were rapidly cleared from the blood pool and non‐target tissue. Tumor‐to‐blood ratio was 12.09 at 2 h post injection and increased to 134.67 at 24 h, while tumor to liver ratio was 2.01 at 24 h similar to that of 2 h. Though it is inappropriate for targeted therapy due to its low uptake in tumor (~ 1 %ID/g), the acceptable results on radiochemistry and biodistribution propose to take a further assessment for non‐invasive imaging and detection of integrin α_νβ₃‐positive tumors by applying diagnostic radionuclides. Copyright © 2011 John Wiley & Sons, Ltd.