Philip C. Sharpe

Amyloid peptides and proteins in review

Amyloids are filamentous protein deposits ranging in size from nanometres to microns and composed of aggregated peptide beta-sheets formed from parallel or anti-parallel alignments of peptide beta-strands. Amyloid-forming proteins have attracted a great deal of recent attention because of their association with over 30 diseases, notably neurodegenerative conditions like Alzheimers, Huntingtons, Parkinsons, Creutzfeldt-Jacob and prion disorders, but also systemic diseases such as amyotrophic lateral sclerosis (Lou Gehrigs disease) and type II diabetes. These diseases are all thought to involve important conformational changes in proteins, sometimes termed misfolding, that usually produce beta-sheet structures with a strong tendency to aggregate into water-insoluble fibrous polymers. Reasons for such conformational changes in vivo are still unclear. Intermediate aggregated state(s), rather than precipitated insoluble polymeric aggregates, have recently been implicated in cellular toxicity and may be the source of aberrant pathology in amyloid diseases. Numerous in vitro studies of short and medium length peptides that form amyloids have provided some clues to amyloid formation, with an alpha-helix to beta-sheet folding transition sometimes implicated as an intermediary step leading to amyloid formation. More recently, quite a few non-pathological amyloidogenic proteins have also been identified and physiological properties have been ascribed, challenging previous implications that amyloids were always disease causing. This article summarises a great deal of current knowledge on the occurrence, structure, folding pathways, chemistry and biology associated with amyloidogenic peptides and proteins and highlights some key factors that have been found to influence amyloidogenesis.

Novel thiosemicarbazones of the ApT and DpT Series and their copper complexes: Identification of pronounced redox activity and characterization of their antitumor activity

The novel chelators 2-acetylpyridine-4,4-dimethyl-3-thiosemicarbazone (HAp44mT) and di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (HDp44mT) have been examined to elucidate the structure-activity relationships necessary to form copper (Cu) complexes with pronounced antitumor activity. Electrochemical studies demonstrated that the Cu complexes of these ligands had lower redox potentials than their iron complexes. Moreover, the Cu complexes where the ligand/metal ratio was 1:1 rather than 2:1 had significantly higher intracellular oxidative properties and antitumor efficacy. Interestingly, the 2:1 complex was shown to dissociate to give significant amounts of the 1:1 complex that could be the major cytotoxic effector. Both types of Cu complex showed significantly more antiproliferative activity than the ligand alone. We also demonstrate the importance of the inductive effects of substituents on the carbonyl group of the parent ketone, which influence the Cu(II/I) redox potentials because of their proximity to the metal center. The structure-activity relationships described are important for the design of potent thiosemicarbazone Cu complexes.

2-Acetylpyridine thiosemicarbazones are potent iron chelators and antiproliferative agents: redox activity, iron complexation and characterization of their antitumor activity.

Through systematic structure-activity studies of the 2-benzoylpyridine thiosemicarbazone (HBpT), 2-(3-nitrobenzoyl)pyridine thiosemicarbazone (HNBpT) and dipyridylketone thiosemicarbazone (HDpT) series of iron (Fe) chelators, we identified structural features necessary to form Fe complexes with potent anticancer activity (J. Med. Chem. 2007, 50, 3716-3729). In this investigation, we generated the related 2-acetylpyridine thiosemicarbazone (HApT) analogues to examine the influence of the methyl group at the imine carbon. Four of the six HApT chelators had potent antitumor activity (IC(50): 0.001-0.002 microM) and Fe chelation efficacy that was similar to the most effective HBpT and HDpT ligands. The HApT Fe complexes had the lowest Fe(III/II) redox potentials of any thiosemicarbazone series we have generated. This property, in combination with their ability to effectively chelate cellular Fe, make the HApT series one of the most potent antiproliferative agents developed by our group.

Iron chelators of the dipyridylketone thiosemicarbazone class: Precomplexation and transmetalation effects on anticancer activity

We previously reported a series of di-2-pyridylketone thiosemicarbazone (HDpT) chelators that showed marked and selective antitumor activity (Whitnall, M.; et al. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 14901-14906). To further understand their biological efficacy, we report the characterization and activity of their Mn(II), Co(III), Ni(II), Cu(II), and Zn(II) complexes. The X-ray crystal structures of four divalent (Mn, Ni, Cu, and Zn) and one trivalent (Fe) complexes are reported. Electrochemistry shows the Fe(III/II) and Cu(II/I) potentials of the complexes may be redox-active within cells. Stability constants were also determined for the Mn(II), Ni(II), Cu(II), and Zn(II) complexes. All divalent complexes underwent transmetalation upon encountering Fe(II), to form low spin ferrous complexes. Importantly, the divalent Mn(II), Ni(II), Cu(II), and Zn(II) complexes of the HDpT analogues are equally active in preventing proliferation as their ligands, suggesting the complexes act as lipophilic vehicles facilitating intracellular delivery of the free ligand upon metal dissociation.

Novel Second-Generation Di-2-Pyridylketone Thiosemicarbazones Show Synergism with Standard Chemotherapeutics and Demonstrate Potent Activity against Lung Cancer Xenografts after Oral and Intravenous Administration in Vivo

We developed a series of second-generation di-2-pyridyl ketone thiosemicarbazone (DpT) and 2-benzoylpyridine thiosemicarbazone (BpT) ligands to improve the efficacy and safety profile of these potential antitumor agents. Two novel DpT analogues, Dp4e4mT and DpC, exhibited pronounced and selective activity against human lung cancer xenografts in vivo via the intravenous and oral routes. Importantly, these analogues did not induce the cardiotoxicity observed at high nonoptimal doses of the first-generation DpT analogue, Dp44mT. The Cu(II) complexes of these ligands exhibited potent antiproliferative activity having redox potentials in a range accessible to biological reductants. The activity of the copper complexes of Dp4e4mT and DpC against lung cancer cells was synergistic in combination with gemcitabine or cisplatin. It was demonstrated by EPR spectroscopy that dimeric copper compounds of the type [CuLCl](2), identified crystallographically, dissociate in solution to give monomeric 1:1 Cu:ligand complexes. These monomers represent the biologically active form of the complex.

Novel diaroylhydrazine ligands as iron chelators: coordination chemistry and biological activity

The search for orally effective drugs for the treatment of iron overload disorders is an important goal in improving the health of patients suffering diseases such as β-thalassemia major. Herein, we report the syntheses and characterization of some new members of a series of N-aroyl-N′-picolinoyl hydrazine chelators (the H2IPH analogs). Both 1:1 and 1:2 FeIII:L complexes were isolated and the crystal structures of Fe(HPPH)Cl2, Fe(4BBPH)Cl2, Fe(HAPH)(APH) and Fe(H3BBPH)(3BBPH) were determined (H2PPH=N,N′-bis-picolinoyl hydrazine; H2APH=N-4-aminobenzoyl-N′-picolinoyl hydrazine, H23BBPH=N-3-bromobenzoyl-N′-picolinoylhydrazine and H24BBPH=N-(4-bromobenzoyl)-N′-(picolinoyl)hydrazine). In each case, a tridentate N,N,O coordination mode of each chelator with Fe was observed. The FeIII complexes of these ligands have been synthesized and their structural, spectroscopic and electrochemical characterization are reported. Five of these new chelators, namely H2BPH (N-(benzoyl)-N′-(picolinoyl)hydrazine), H2TPH (N-(2-thienyl)-N′-(picolinoyl)-hydrazine), H2PPH, H23BBPH and H24BBPH, showed high efficacy at mobilizing 59Fe from cells and inhibiting 59Fe uptake from the serum Fe transport protein, transferrin (Tf). Indeed, their activity was much greater than that found for the chelator in current clinical use, desferrioxamine (DFO), and similar to that observed for the orally active chelator, pyridoxal isonicotinoyl hydrazone (H2PIH). The ability of the chelators to inhibit 59Fe uptake could not be accounted for by direct chelation of 59Fe from 59Fe–Tf. The most effective chelators also showed low antiproliferative activity which was similar to or less than that observed with DFO, which is important in terms of their potential use as agents to treat Fe-overload disease.

Tuning the antiproliferative activity of biologically active iron chelators: characterization of the coordination chemistry and biological efficacy of 2-acetylpyridine and 2-benzoylpyridine hydrazone ligands

Abstract2-Pyridinecarbaldehyde isonicotinoyl hydrazone (HPCIH) and di-2-pyridylketone isonicotinoyl hydrazone (HPKIH) are two Fe chelators with contrasting biological behavior. HPCIH is a well-tolerated Fe chelator with limited antiproliferative activity that has potential applications in the treatment of Fe-overload disease. In contrast, the structurally related HPKIH ligand possesses significant antiproliferative activity against cancer cells. The current work has focused on understanding the mechanisms of the Fe mobilization and antiproliferative activity of these hydrazone chelators by synthesizing new analogs (based on 2-acetylpyridine and 2-benzoylpyridine) that resemble both series and examining their Fe coordination and redox chemistry. The Fe mobilization activity of these compounds is strongly dependent on the hydrophobicity and solution isomeric form of the hydrazone (E or Z). Also, the antiproliferative activity of the hydrazone ligands was shown to be influenced by the redox properties of the Fe complexes. This indicated that toxic Fenton-derived free radicals are important for the antiproliferative activity for some hydrazone chelators. In fact, we show that any substitution of the H atom present at the imine C atom of the parent HPCIH analogs leads to an increase in antiproliferative efficacy owing to an increase in redox activity. These substituents may deactivate the imine R–C=N–Fe (R is Me, Ph, pyridyl) bond relative to when a H atom is present at this position preventing nucleophilic attack of hydroxide anion, leading to a reversible redox couple. This investigation describes novel structure–activity relationships of aroylhydrazone chelators that will be useful in designing new ligands or fine-tuning the activity of others.

Methemoglobin formation by triapine, di-2-pyridylketone-4,4-dimethyl-3- thiosemicarbazone (Dp44mT), and other anticancer thiosemicarbazones: identification of novel thiosemicarbazones and therapeutics that prevent this effect

Thiosemicarbazones are a group of compounds that have received comprehensive investigation as anticancer agents. The antitumor activity of the thiosemicarbazone, 3-amino-2-pyridinecarboxaldehyde thiosemicarbazone (3-AP; triapine), has been extensively assessed in more than 20 phase I and II clinical trials. These studies have demonstrated that 3-AP induces methemoglobin (metHb) formation and hypoxia in patients, limiting its usefulness. Considering this problem, we assessed the mechanism of metHb formation by 3-AP compared with that of more recently developed thiosemicarbazones, including di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT). This was investigated using intact red blood cells (RBCs), RBC lysates, purified oxyhemoglobin, and a mouse model. The chelation of cellular labile iron with the formation of a redox-active thiosemicarbazone-iron complex was found to be crucial for oxyhemoglobin oxidation. This observation was substantiated using a thiosemicarbazone that cannot ligate iron and also by using the chelator, desferrioxamine, that forms a redox-inactive iron complex. Of significance, cellular copper chelation was not important for metHb generation in contrast to its role in preventing tumor cell proliferation. Administration of Dp44mT to mice catalyzed metHb and cardiac metmyoglobin formation. However, ascorbic acid administered together with the drug in vivo significantly decreased metHb levels, providing a potential therapeutic intervention. Moreover, we demonstrated that the structure of the thiosemicarbazone is of importance in terms of metHb generation, because the DpT analog, di-2-pyridylketone-4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), does not induce metHb generation in vivo. Hence, DpC represents a next-generation thiosemicarbazone that possesses markedly superior properties. This investigation is important for developing more effective thiosemicarbazone treatment regimens.

Head phantom for testing microwave systems for head imaging

The fabrication of a human head phantom for the use in microwave systems designed for head imaging is presented. Since most of the ongoing research activities on microwave imaging of human head use the band from 1 GHz to 4 GHz, the fabricated phantom is designed to emulate the electrical properties of human brain tissues across that band. A material that has electrical properties equal to those of the soft human brain tissues is manufactured using simple substances. A fabricated material that resembles the shape of human skull is used to contain the developed soft tissues. The measured properties of the fabricated phantom using a suitable dielectric probe are in good agreement with the properties of the real head tissues.

Amyloid Formation from an α‐Helix Peptide Bundle Is Seeded by 310‐Helix Aggregates

Transformation of proteins and peptides to fibrillar aggregates rich in β sheets underlies many diseases, but mechanistic details of these structural transitions are poorly understood. To simulate aggregation, four equivalents of a water-soluble, α-helical (65 %) amphipathic peptide (AEQLLQEAEQLLQEL) were assembled in parallel on an oxazole-containing macrocyclic scaffold. The resulting 4α-helix bundle is monomeric and even more α helical (85 %), but it is also unstable at pH 4 and undergoes concentration-dependent conversion to β-sheet aggregates and amyloid fibrils. Fibrils twist and grow with time, remaining flexible like rope (>1 μm long, 5-50 nm wide) with multiple strings (2 nm), before ageing to matted fibers. At pH 7 the fibrils revert back to soluble monomeric 4α-helix bundles. During α→β folding we were able to detect soluble 3(10) helices in solution by using 2D-NMR, CD and FTIR spectroscopy. This intermediate satisfies the need for peptide elongation, from the compressed α helix to the fully extended β strand/sheet, and is driven here by 3(10) -helix aggregation triggered in this case by template-promoted helical bundling and by hydrogen-bonding glutamic acid side chains. A mechanism involving α⇌α(4) ⇌(3(10) )(4) ⇌(3(10) )(n) ⇌(β)(n) ⇋m(β)(n) equilibria is plausible for this peptide and also for peptides lacking hydrogen-bonding side chains, with unfavourable equilibria slowing the α→β conversion.