Peptide-cleaving agents for human islet amyloid polypeptide containing substrate recognition site based on quinoxaline: cleavage efficiency enhanced by lowering substrate concentration

Oligomers of human islet amyloid polypeptide (h-IAPP) are believed to be the pathogenic species for type 2 diabetes mellitus. Peptide-cleaving agents selective for oligomers of h-IAPP were synthesized by using quinoxaline derivatives as recognition sites attached to the Co(III) complex of cyclen in this study. When the initial concentration of h-IAPP was lowered from 4.0 to 0.20 μM, cleavage yield of the new agents was enhanced by 3 times reaching 16-22 mol%. This shows that the agents would have significant activities at subnano molar concentrations if the concentration of h-IAPP is lowered to the in vivo values. This further indicates that the peptide-cleaving agents prepared previously in this laboratory possess sufficiently high activity for application as a new therapeutic option for Alzheimer's disease, type 2 diabetes mellitus, and Parkinson's disease.     

A Co(III) complex cleaving soluble oligomers of h-IAPP in the presence of polymeric aggregates of h-IAPP

Soluble oligomers of human islet amyloid polypeptide (h-IAPP) are believed to be the pathogenic species for type 2 diabetes mellitus. In search of the peptide-cleavage agent cleaving oligomers of h-IAPP with low affinity for polymeric aggregates of h-IAPP, a chemical library was constructed by using the Ugi condensation. From the library, a Co(III) complex was discovered to cleave soluble oligomers of h-IAPP in the presence of polymeric aggregates of h-IAPP without being captured by the aggregates considerably. The peptide-cleavage agent inhibited apoptosis of INS-1 cell by h-IAPP even in the presence of preformed polymeric aggregates of h-IAPP. This suggests that target-selective peptide-cleavage agents may be applied clinically not only to diabetes but also to various other amyloid diseases.     

Cell-penetration by Co(III)cyclen-based peptide-cleaving catalysts selective for pathogenic proteins of amyloidoses

Derivatives of the Co(III) complex of 1,4,7,10-tetraazacyclododecane (cyclen) with various organic pendants have been reported as target-selective peptide-cleaving catalysts, which can be exploited as catalytic drugs. In order to provide a firm basis for the catalytic drugs based on Co(III)cyclen, the ability of the Co(III)cyclen-containing peptide-cleaving catalysts to penetrate animal cells such as mouse fibroblast NIH-3T 3 or human embryonic kidney (HEK) 293 cells is demonstrated in the present study. Since the catalysts destroy pathogenic proteins for amyloidoses, results of the present study are expected to initiate extensive efforts to obtain therapeutically safe catalytic drugs for amyloidoses such as Alzheimer’s disease, type 2 diabetes mellitus, Parkinson’s disease, Huntington’s disease, mad cow disease, and so on.     

Angiotensin-cleaving catalysts: conversion of N-terminal aspartate to pyruvate through oxidative decarboxylation catalyzed by Co(III)cyclen

To provide a firm basis for the new paradigm of drug discovery based on peptide-cleaving catalysts, oligopeptide-cleaving catalysts were searched for by using human angiotensin I (Ang-I) and angiotensin II (Ang-II) as the substrates. Catalyst candidates containing the Co(III) complex of cyclen as the catalytic center were prepared by multicomponent condensation reactions. From two types of chemical libraries containing about 3,600 catalyst candidates, two compounds [SS-Co(III)X and S-Co(III)Y] were selected as the most active catalysts. On incubation with SS-Co(III)X and S-Co(III)Y, both Ang-I and Ang-II were cleaved by oxidative decarboxylation instead of peptide hydrolysis: the N-terminal Asp residues of Ang-I and Ang-II were converted to pyruvate residues. Catalysts for oxidative decarboxylation of the N-terminal Asp residue contained in an oligopeptide are unprecedented in both biological and chemical systems. Detailed kinetics analysis suggested that Ang-I and Ang-II can be cleaved with half-lives much less than 1 h if the structures of the chelating ligands of the catalysts are further improved. The results indicated that the concept of the peptide-cleaving catalysts can be expanded to include oligopeptides as the targets and nonhydrolytic reactions as the means for cleavage.     

Cleavage agents for soluble oligomers of human islet amyloid polypeptide

Soluble oligomers of human islet amyloid polypeptide (h-IAPP) are implicated in the initiation of beta-cell apoptosis leading to type 2 diabetes mellitus (T2DM). Cleavage of the h-IAPP included in an oligomer may provide a novel method for reducing the level of h-IAPP oligomers, offering a new therapeutic option for T2DM. From the combinatorial library of triazine derivatives prepared by exploiting the Co(III) complex of cyclen as the cleavage center for peptide bonds, eight compounds were selected as cleavage agents for oligomers of h-IAPP. After reaction with cleavage agents for 36 h at 37 degrees C and pH 7.50, up to 20 mol% of h-IAPP (initial concentration: 4.0 microM) was cleaved, although the target oligomers existed as transient species. Considerable activity was manifested at agent concentrations as low as 100 nM.     

Toward protein-cleaving catalytic drugs: artificial protease selective for myoglobin

A protein-cleaving catalyst highly selective for a disease-related protein can be used as a catalytic drug. As the first protein-cleaving catalyst selective for a protein substrate, a catalyst for myoglobin (Mb) was designed by attaching the Cu(II) or Co(III) complex of cyclen to a binding site searched by a combinatorial method using peptide nucleic acid monomers as building units. Various linkers were inserted between the catalytic Co(III) center and the binding site of the Mb-cleaving catalyst. Kinetic data revealed catalytic turnover of the Mb cleavage by the Cu(II) or Co(III) complex. MALDI-TOF MS revealed cleavage of the polypeptide backbone of Mb at selected positions. N-Terminal sequencing of the cleavage products identified the cleavage site and provided evidence for the hydrolytic nature of the Mb cleavage. Various chelating ligands were tested as the ligand for the Co(III) center of the Mb-cleaving catalyst. Among the nine chelating ligands examined, only cyclen and its triaza-monooxo analogue manifested catalytic activity.     

Proteolytic activity of Co(III) complex of 1-oxa-4,7,10-triazacyclododecane: a new catalytic center for peptide-cleavage agents

Catalytic drugs based on target-selective artificial proteases have been proposed as a new paradigm in drug design. Peptide-cleavage agents selective for pathogenic proteins of Alzheimer’s disease, type 2 diabetes mellitus or Parkinson’s disease have been prepared using the Co(III) aqua complex (Co(III)cyclen) of 1,4,7,10-tetraazacyclododecane as the catalytic center. In the present study, the Co(III) aqua complex (Co(III)oxacyclen) of 1-oxa-4,7,10-triazacyclododecane was examined in search of an improved catalytic center for peptide-cleavage agents. An X-ray crystallographic study of [Co(oxacyclen)(CO₃)](ClO₄), titration of Co(III)oxacyclen, and kinetic studies on the cleavage of albumin, γ-globulin, lysozyme, and myoglobin by Co(III)oxacyclen were carried out. Considerably higher proteolytic activity was observed for Co(III)oxacyclen in comparison with Co(III)cyclen, indicating that better target-selective artificial metalloproteases would be obtained using Co(III)oxacyclen as the catalytic center. The improved proteolytic activity was attributed to either steric effects or the increased Lewis acidity of the Co(III) center. The kinetic data also predicted that side effects due to the cleavage of nontarget proteins by a catalytic drug based on Co(III)oxacyclen would be insignificant.     

Cu(II) cyclen cleavage agent for human islet amyloid peptide

Type 2 diabetes mellitus (T2DM) is characterized by a substantial reduction in β-cell mass and the amyloid fibrils which are formed by the aggregation of the human islet amyloid polypeptide (h-IAPP) in the islet of Langerhans. Cleavage agents with Co(III) cyclen as the catalytic group have been studied as a novel therapeutic option for T2DM patients. However, recent research has suggested that the cytotoxicity of h-IAPP might be mediated by interactions with Cu(II); furthermore, it has been shown in vitro that Cu(II) prevents h-IAPP from forming the β-sheet conformers. Therefore, we synthesized a cleavage agent using Cu(II) cyclen. The resulting cleaved fragments and estimated cleavage yield (8.3 mol %) were evaluated after incubation with h-IAPP.     

Oligonucleotide conjugates of Eu(III) tetraazamacrocycles with pendent alcohol and amide groups promote sequence-specific RNA cleavage

Eu(III) complexes of two neutral bifunctional tetraaaza macrocyclic ligands {1-[1-carboxamido-3-(4-nitrophenyl)propyl]-4,7,10-tris(2-hydroxyethyl)-1,4,7,10-tetraazacyclododecane and 2-(4-nitrobenzyl)-1,4,7,10-tetrakis(2-hydroxyethyl)-1,4,7,10-tetraazacyclododecane} are prepared. Eu(III) complexes of the isothiocyanate derivatives of these macrocycles are treated with oligonucleotides containing 2′-O-propylamine linkers to form conjugates. Hydrolytic cleavage of an oligoribonucleotide is promoted by Eu(III) macrocyclic oligonucleotide conjugates containing complementary (antisense) sequences. Cleavage is not observed in the presence of Eu(III) conjugates containing scrambled sequences nor by free complex. Despite the fact that one of the free macrocyclic complexes is more reactive than the other, the extent of cleavage observed is similar for conjugates containing either Eu(III) macrocyclic complex.     

Peptide-cleaving catalyst selective for melanin-concentrating hormone: oxidative decarboxylation of N-terminal aspartate catalyzed by Co(III)cyclen

To provide a firm basis for the new paradigm of drug discovery based on catalysts for oxidative cleavage of N-terminal aspartate (Asp) residues of oligopeptides, oligopeptide-cleaving catalysts were searched by using melanin-concentrating hormone (MCH) as the substrate. MCH is a target for designing drugs to reduce obesity. Catalyst candidates containing the Co(III) complex of cyclen as the catalytic center were prepared by multicomponent condensation reactions. From three kinds of chemical libraries containing about 19,000 catalyst candidates, one compound was identified as the MCH-cleaving catalyst. On incubation with the catalyst, the N-terminal Asp residue of MCH was converted to the pyruvate residue by oxidative decarboxylation. Detailed kinetics analysis revealed the catalytic nature of the action of the catalyst. In addition, the kinetics data indicated that MCH can be cleaved with half-lives of 3 h or less with submicromolar catalyst concentrations if the structure of the catalyst is further improved.     

The superoxide dismutase activities of two higher valent manganese complexes, MnIV desferrioxamine and MnIII-cyclam.

A green manganese desferrioxamine complex is rapidly formed at room temperature upon stirring freshly precipitated manganese dioxide in a solution of the ligand. Spectral studies and low-temperature ESR indicate that this compound, which has been previously described as a manganese(III) complex, is better characterized as containing tetravalent manganese. The complex appears to form oligomers in solution. The extinction coefficient at 635 nm is 137 +/- 6 M-1 cm-1 (per manganese) at pH 7.8 and 88 +/- 4 M-1 s-1 at pH 6.6 after purification by chromatography. The superoxide dismutase activity was measured and compared to that of mononuclear manganese(III) 1,4,8,11-tetraazacyclodecane (cyclam). The catalytic rate constants for superoxide dismutase activity are 1.7 x 10(6) M-1 s-1 and 2.9 x 10(6) M-1 s-1 for the desferrioxamine and the cyclam complexes, respectively.     

Synthesis of a novel 'smart' bifunctional chelating agent 1-(2-[beta,D-galactopyranosyloxy]ethyl)-7-(1-carboxy-3-[4-aminophenyl]propyl)-4,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (Gal-PA-DO3A-NH2) and its Gd(III) complex

A new synthetic pathway to 1-(2-[beta,D-galactopyranosyloxy]ethyl)-7-(1-carboxy-3-[4-aminophenyl]propyl)-4,10-bis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (Gal-PA-DO3A-NH2) and 1-(2-[beta,D-galactopyranosyloxy]ethyl)-4,7,10-tris(carboxymethyl)-1, 4,7,10-tetraazacyclododecane (Gal-DO3A) chelating agents was developed involving full hydroxyl- and carboxyl-group protection in precursors to product. Two sequences of cyclen-N-functionalisation were subsequently investigated, one successfully, towards synthesis of the novel 'smart' bifunctional Gal-PA-DO3A-NH2 chelate. The longitudinal proton relaxivities of the neutral [Gd-(Gal-PA-DO3A-NH2)] and [Gd-(Gal-DO3A)] complexes were increased by 28% and 37% in the presence of beta-galactosidase, respectively.     

Islet amyloid polypeptide (IAPP) transgenic rodents as models for type 2 diabetes

Blood glucose concentrations are maintained by insulin secreted from beta-cells located in the islets of Langerhans. There are approximately 2000 beta-cells per islet, and approximately one million islets of Langerhans scattered throughout the pancreas. The islet in type 2 diabetes mellitus (T2D) has deficient beta-cell mass due to increased beta-cell apoptosis and islet amyloid derived from islet amyloid polypeptide (IAPP). Accumulating evidence implicates toxic IAPP oligomers in the mediation of beta-cell apoptosis in T2D. Humans, monkeys, and cats express an amyloidogenic toxic form of IAPP and spontaneously develop diabetes characterized by islet amyloid deposits. However, longitudinal studies of islet pathology in humans are impossible, and studies in nonhuman primates and cats are costly and impractical. Rodent IAPP is not amyloidogenic, thus commonly used rodent models of diabetes do not recapitulate islet pathology in humans. To investigate the diabetogenic role of human IAPP (h-IAPP), several mouse models and, more recently, a rat model transgenic for h-IAPP have been developed. Studies in these models have revealed that the toxic effect of h-IAPP on beta-cell apoptosis demonstrates a threshold-dependent effect. Specifically, increasing h-IAPP transgene expression by breeding or induction of insulin resistance leads to increased beta-cell apoptosis and diabetes. These transgenic rodent models for h-IAPP provide an opportunity to elucidate the mechanisms responsible for h-IAPP-induced beta-cell apoptosis further and to test novel approaches to the prevention and treatment of T2D.     

Chemical cleavage of plasmid DNA by Cu(II), Ni(II) and Co(III) desferal complexes.

It is demonstrated that the Cu(II), Co(III) and Ni(II) complexes of a siderophore chelating drug desferal cleave DNA, in contrast to the corresponding Fe(II) complex which does not bring about DNA scission. Hydroxy radical scavengers inhibit the cleavage reaction.     

Ester hydrolysis with 2,6-di(pyrazol-3-yl)pyridines and their CoII complexes in homogeneous and micellar media

The 1:1 complex of Co(ClO4)2 with the H2O-insoluble tridentate 2,6-di(1H-4,5,6,7-tetrahydroindazol-3-yl)pyridine (H21) was found to be an excellent catalyst for the hydrolysis of para-nitrophenyl acetate in aqueous buffers over the pH 7.05-7.90 range, with an estimated second-order rate constant of 0.50 M(-1) s(-1). The Co2+ complexes of the N,N'-di-1-dodecyl analogue in micellar media and the N,N'-di-(4-carboxyphenyl) analogue in aqueous media were much poorer catalysts, poorer than the free ligands. In all cases, the pH-rate profiles indicated that free base, deprotonated or hydroxo forms were the active species. The greater success with Co(H(2)1)2+ indicated a catalytic role for N-H deprotonation.     

The search for new hydrogenation catalyst motifs based on N-heterocyclic carbene ligands

A series of new iridium complexes containing N-heterocyclic carbene ligands (NHCs) has been prepared and tested for hydrogenation of primary (1-octene), secondary (cyclohexene), tertiary (1-methylcyclohexene) and quartenary (2,3-dimethyl-2-butene) alkenes. Taking inspiration from Crabtree’s catalyst, [Ir(COD)(PCy₃)(py)]PF₆, the labile pyridine ligand was exchanged with an NHC ligand, and was found to produce catalysts with similar activities and rates. Further refinement of the ligands produced the optimal version of the iridium phosphine/NHC combination: Using a smaller phosphine, tri-n-butylphosphine, a saturated NHC and the non-coordinating anion, tetrakis(3,5-bis(trifluoromethylphenyl))borate (BARF), a highly active catalyst that has substantially better longevity and stability, but similar activity, to the parent Crabtree’s catalyst was prepared in three steps. The effect of the BARF counteranion was explored, and was shown to improve even Crabtree’s catalyst, and is thus a general phenomenon for iridium catalysts in non-polar solvents. Mechanistic studies suggest that the catalyst resting state is a dimeric complex, presumably hydride-bridged, that dissociates into catalytically active species under catalytic conditions. Thus, the catalyst dead-end for Crabtree’s catalyst, hydride-bridged dimers and trimers, may in fact be a reversible resting state for the iridium complexes described here.     

Comparison of yttrium and indium complexes of DOTA-BA and DOTA-MBA: models for (90)Y- and (111)In-labeled DOTA-biomolecule conjugates

Yttrium and indium complexes of 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecylacetylbenzylamine (DOTA-BA) and 1,4,7,10-tetraaza-4,7,10-tris(carboxymethyl)-1-cyclododecylacetyl-R-(+)-alpha-methylbenzylamine (DOTA-MBA) were prepared in order to study solution structures of (90)Y- and (111)In-labeled DOTA-biomolecule conjugates. (90)Y and (111)In complexes M(L) (M = (90)Y and (111)In; L = DOTA-BA and DOTA-MBA) were prepared from the reaction of MCl(3) with DOTA-BA and DOTA-MBA, respectively, in ammonium acetate buffer. A reverse phase HPLC method revealed that both (90)Y and (111)In complexes show only one radiometric peak in their radio-HPLC chromatograms. It was also found that (111)In(DOTA-BA) and (111)In(DOTA-MBA) are more hydrophilic than their corresponding (90)Y analogues, suggesting different coordination spheres in (111)In and (90)Y complexes of the same DOTA conjugate. Complexes M(L) (M = Y and In; L = DOTA-BA and DOTA-MBA) were prepared and characterized by HPLC, LC-MS, and NMR ((1)H and (13)C) methods. The HPLC concordance experiments for (90)Y(DOTA-MBA)/Y(DOTA-MBA) and (111)In(DOTA-MBA)/In(DOTA-MBA) show that the same complex is prepared at both tracer and macroscopic levels. The NMR data ((1)H and (13)C) clearly demonstrates that Y(DOTA-BA) and Y(DOTA-MBA) exist in solution as one predominant isomer. VT NMR data ((1)H and (13)C) show that In(DOTA-BA) and In(DOTA-MBA) are fluxional at room temperature while Y(DOTA-BA) and Y(DOTA-MBA) become fluxional only at elevated temperatures. The fluxionality of these complexes is due to rapid rotation of acetate/acetamide chelating arms and inversion of ethylenic groups of the macrocyclic ring.     

Preparation and preliminary biological evaluation of a 177Lu labeled sanazole derivative for possible use in targeting tumor hypoxia

The preparation of a polyazamacrocyclic-nitrotriazole conjugate for radiolabeling with the therapeutic radioisotope viz. (177)Lu is described. The nitroimidazole used for the present study is [N-2'(carboxyethyl)-2-(3'-nitro-1'-triazolyl)acetamide], the carboxylic acid derivative of sanazole, which possesses an optimal combination of desired properties such as, selective toxicity for hypoxic cells, lowered lipophilicity resulting in lowered neurotoxicity. The bifunctional chelating agent is a DOTA derivative viz. 1,4,7,10-tetraaza-1-(4'-aminobenzylacetamido)-cyclododecane-4,7,10- triacetic acid (p-amino-DOTA-anilide). (177)Lu was produced in adequate specific activity (110TBq/g) and high radionuclidic purity (approximately 100%) by irradiating enriched (60.6% (176)Lu) Lu(2)O(3) target and used for radiolabeling of the sanazole-BFCA conjugate. approximately 98% Complexation yield was achieved under optimized conditions. The complex has been characterized by paper chromatography and HPLC studies. Bioevaluation studies in Swiss mice bearing fibrosarcoma tumors revealed moderate tumor uptake (0.88%/g at 1h post-injection) with favorable tumor to blood (4.00 at 1h post-injection) and tumor to muscle (4.63 at 1h post-injection) ratios.     

A chelate complex‐enhanced luminol system for selective determination of Co(II), Fe(II) and Cr(III)

A determination method for Co(II), Fe(II) and Cr(III) ions by luminol‐H₂O₂ system using chelating reagents is presented. A metal ion‐chelating ligand complex with a Co(II) ion and a chelating reagent like ethylenediaminetetraacetic acid (EDTA) produced highly enhanced chemiluminescence (CL) intensity as well as longer lifetime in the luminol‐H₂O₂ system compared to metals that exist as free ions. Whereas free Cu(II) and Pb(II) ions had a strong catalytic effect on the luminol‐H₂O₂ system, significantly, the complexes of Cu(II) and Pb(II) with chelating reagents lost their catalytic activity due to the chelating reagents acting as masking agents. Based on the observed phenomenon, it was possible to determine Co(II), Fe(II) and Cr(III) ions with enhanced sensitivity and selectivity using the chelating reagents of the luminol‐H₂O₂ system. The effects of ligand, H₂O₂ concentration, pH, buffer solution and concentrations of chelating reagents on CL intensity of the luminol‐H₂O₂ system were investigated and optimized for the determination of Co(II), Fe(II) and Cr(III) ions. Under optimized conditions, the calibration curve of metal ions was linear over the range of 2.0 × 10^‐8 to 2.0 × 10^‐5 M for Co(II), 1.0 × 10^‐7 to 2.0 × 10^‐5 M for Fe (II) and 2.0 × 10^‐7 to 1.0 × 10^‐4 M for Cr(III). Limits of detection (3σ/s) were 1.2 × 10^‐8, 4.0 × 10^‐8 and 1.2 × 10^‐7 M for Co(II), Fe(II) and Cr(III), respectively. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and characterization of folic acid modified water-soluble chitosan derivatives for folate-receptor-mediated targeting

Three chemical modification methods of carboxymethylation, quaternization and hydroxypropylation were used to synthesize water-soluble chitosan derivatives. In order to study the feasibility of these chitosan derivatives as backbones of nuclear imaging agents, folic acid (FA) and Technetium-99m were introduced onto the water-soluble chitosan chains. The bifunctional chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to the folate grafted chitosan derivatives for chelating with radionuclides such as (64)Cu and (68)Ga. The structures of these new ligands were characterized with multiple methods. The solubility and stability of the (99m)Tc-complexes were both favorable. Further study of their radiochemical and biological properties will be performed to evaluate the usefulness of these water-soluble chitosan derivatives for nuclear imaging agent design.