Amylin uncovered: a review on the polypeptide responsible for type II diabetes.

Amylin is primarily responsible for classifying type II diabetes as an amyloid (protein misfolding) disease as it has great potential to aggregate into toxic nanoparticles, thereby resulting in loss of pancreatic ß-cells. Although type II diabetes is on the increase each year, possibly due to bad eating habits of modern society, research on the culprit for this disease is still in its early days. In addition, unlike the culprit for Alzheimer's disease, amyloid ß-peptide, amylin has failed to receive attention worthy of being featured in an abundance of review articles. Thus, the aim of this paper is to shine the spotlight on amylin in an attempt to put it onto the top of researchers' to-do list since the secondary complications of type II diabetes have far-reaching and severe consequences on public health both in developing and fully developed countries alike. This paper will cover characteristics of the amylin aggregates, mechanisms of toxicity, and a particular focus on inhibitors of toxicity and techniques used to assess these inhibitors.

A direct fluorescence-based technique for cellular localization of amylin.

Amylin has been implicated in type II diabetes because of its inherent property to misfold into toxic aggregates. Although it has been shown that amylin interacts with cell membranes, no study to date has monitored the association process using a direct approach. The present study uses confocal microscopy to identify the localization of carboxyfluorescein-labeled amylin in RIN-5F cells. In addition, the size of the aggregates that are formed was evaluated using nanoparticle tracking analysis. In support of previous findings, amylin was observed to interact with and remain associated with the cell membrane. The cell membrane-associated aggregates spanned a size range of 130-800 nm.

Synthesis, 2D-NMR and molecular modelling studies of pentacycloundecane lactam-peptides and peptoids as potential HIV-1 wild type C-SA protease inhibitors.

In this study, eight non-natural peptides and peptoids incorporating the pentacycloundecane (PCU) lactam were designed and synthesized as potential inhibitors of the wild type C-SA HIV-protease. Five of these inhibitors gave IC(50) values ranging from 0.5 up to 0.75 µM against the resistance-prone wild type C-South African HIV-protease. NMR EASY-ROESY studies enabled us to describe the secondary structure of three of these compounds in solution. The 3D structures of the selected cage peptides were also modelled in solution using QM/MM/MD simulations. Satisfactory agreement between the NMR observations and the low energy calculated structures exists. Only one of these inhibitors (11 peptoid), which showed the best IC(50)(0.5 µM), exhibited a definable 3-D structure in solution. Autodock4 and AutodockVina were used to model the potential interaction between these inhibitors and the HIV-PR. It appears that the docking results are too crude to be correlated with the relative narrow range of experimental IC(50) values (0.5-10 µM). The PCU-peptides and peptoides were several orders less toxic (145 µM for 11 and 102 µM for 11 peptoid) to human MT-4 cells than lopinavir (0.025 µM). This is the first example of a polycyclic cage framework to be employed as an HIV-PR transition state analogue inhibitor and can potentially be utilized for other diseases related proteases. [Figure: see text].

Pentacycloundecane derived hydroxy acid peptides: a new class of irreversible non-scissile ether bridged type isoster as potential HIV-1 wild type C-SA protease inhibitors.

Novel peptides incorporating the PCU derived hydroxy acid (5-hydroxy-4-oxahexacyclo[5.4.1.0(2,6).0(3,10).0(5,9).0(8,11)]dodecane) were synthesized and their activity against the resistance-prone wild type C-South African (C-SA) HIV-protease is reported. The attachment of peptides and peptoids to the PCU derived hydroxy acid resulted in a series of structurally diverse promising HIV-1 protease inhibitors. Amongst the nine novel compounds, 16, 17, 20 and 23 gave IC(50) values ranging from 0.6 to 5.0 µM against the wild type C-SA HIV-1 protease enzyme. Docking studies and molecular dynamic (MD) simulations have been carried out in order to understand the binding mode of the PCU moiety at the active site of the HIV protease enzyme. A conserved hydrogen bonding pattern between the PCU derived hydroxy ether and the active site residues, ASP25/ASP25', was observed in all active compounds.

Enzymatic activation of a peptide functionalised gold nanoparticle system for prodrug delivery.

Gold nanoparticles (GNPs) with a monolayer of peptides were synthesized as a potential tumour activated cancer drug delivery system. The prodrug system was achieved by the attachment of two varying lengths of peptides to GNPs: An 18 amino acid peptide sequence encompassing a shorter fluorescent labelled (coumarin) six amino acid peptide sequence. The longer peptide chain included the sequence D-AFK that is selectively cleavable by the over-expression of proteases in the vicinity of cancer cells. The protease-mediated exposure of the coumarin was demonstrated by the incubation of peptide capped GNPs with adenocarcinomic human alveolar basal epithelial A549 cells and madin-darby bovine kidney epithelial cells. Confocal laser scanning microscopy studies revealed enhanced fluorescence emission intensities in the cancer cell line as compared to the intensity exhibited by the healthy cell line. This work suggests that GNPs functionalised with a cytotoxic agent or fluorophore encapsulated by longer peptide strands may find useful applications for development of GNPs with therapeutic or diagnostic studies.

Pentacycloundecane-based inhibitors of wild-type C-South African HIV-protease.

In this study, we present the first account of pentacycloundecane (PCU) peptide based HIV-protease inhibitors. The inhibitor exhibiting the highest activity made use of a natural HIV-protease substrate peptide sequence, that is, attached to the cage (PCU-EAIS). This compound showed nanomolar IC(50) activity against the resistance-prone wild type C-South African HIV-protease (C-SA) catalytic site via a norstatine type functional group of the PCU hydroxy lactam. NMR was employed to determine a logical correlation between the inhibitory concentration (IC(50)) results and the 3D structure of the corresponding inhibitors in solution. NMR investigations indicated that the activity is related to the chirality of the PCU moiety and its ability to induce conformations of the coupled peptide side chain. The results from docking experiments coincided with the experimental observed activities. These findings open up useful applications for this family of cage peptide inhibitors, considering the vast number of alternative disease related proteases that exist.

Design and study of peptide-based inhibitors of amylin cytotoxicity.

The incidence of type II diabetes is on the increase each year and the World Health Organisation (WHO) predicts there to be over 360 million diabetic patients worldwide by the year 2030. Deposits consisting mainly of a small protein, called islet amyloid polypeptide (amylin), which aggregates into oligo-/polymeric beta sheet structures is responsible for cytotoxicity to the pancreatic beta-cells, thus inhibition of this process has been explored as a potential prevention or treatment. N-Methylated and non N-methylated peptides spanning the length of amylin(1-37) were synthesised and evaluated for their inhibition of full length amylin mediated cytotoxicity to RIN-5F cells. The non N-methylated peptides were very effective in inhibiting the cytotoxicity while the N-methylated peptides were not. Both the N-methylated and non N-methylated versions of the 29-34 region were equally effective.

Microwave assisted SPPS of amylin and its toxicity of the pure product to RIN-5F cells.

The 37-amino acid polypeptide islet amyloid polypeptide (IAPP), or amylin, is found as amyloid aggregates in the islets of Langerhans in patients with type II diabetes. Herein, we report an efficient microwave assisted solid phase peptide synthesis of amylin (IAPP). The most efficient synthesis used double and triple couplings and 10 equiv. of amino acids. Double couplings were used for most amino acids, whereas triple couplings were utilized for amino acids in selected regions. The most effective method for formation of the disulfide bond in amylin was found to be iodine oxidation. The highest purity amylin was obtained when the crude peptide was purified with HPLC before formation of the disulfide bond. The cytotoxicity of the synthesized amylin product to RIN-5F cells was determined. The synthesized amylin exhibits an exponential increase of cytotoxicity at concentrations >35 microM. Transmission electron microscope studies of a sample of amylin shows that insoluble amyloid fibrils spontaneously formed when 45 microM solution of synthesized amylin was incubated in a suitable buffer for 6 h.