Molecular Dynamics Studies of Atomistically Determined Fibrillar Assemblies: Comparison of the Rippled ß-Sheet, Pleated ß-Sheet, and Herringbone Structures.

Pauling and Corey expected that a racemic mixture would result in a rippled ß-sheet, however, it has been known from experiments that the racemic mixtures of triphenylalanine lead to a herringbone structure. Because of the theoretical limitations concerning crystal structures such as rippled ß-sheet, it is inevitable to understand how the interplay of the amino acids prefers a specific structural motif. In this paper we use molecular dynamics to understand the sequence- and enantiomer-dependent structures by comparisons between rippled ß-sheet and pleated ß-sheet, solvated and anhydrous rippled ß-sheet, and rippled ß-sheet and the herringbone structure, based on thermodynamics and structures at the atomic level. The tripeptides select the favored structure that can be stabilized through aromatic or hydrogen bonding interactions between tripeptides. Furthermore, the solubility is determined by the environment of space that is created around the side chains. Our findings provide comprehensive insight into the crystallized fibril motif of the polypeptide.

Racemic Peptides from Amyloid ß and Amylin Form Rippled ß-Sheets Rather Than Pleated ß-Sheets.

The rippled ß-sheet was theorized by Pauling and Corey in 1953 as a structural motif in which mirror image peptide strands assemble into hydrogen-bonded periodic arrays with strictly alternating chirality. Structural characterization of the rippled ß-sheet was limited to biophysical methods until 2022 when atomic resolution structures of the motif were first obtained. The crystal structural foundation is restricted to four model tripeptides composed exclusively of aromatic residues. Here, we report five new rippled sheet crystal structures derived from amyloid ß and amylin, the aggregating toxic peptides of Alzheimer's disease and type II diabetes, respectively. Despite the variation in peptide sequence composition, all five structures form antiparallel rippled ß-sheets that extend, like a fibril, along the entire length of the crystalline needle. The long-range packing of the crystals, however, varies. In three of the crystals, the sheets pack face-to-face and exclude water, giving rise to cross-ß architectures grossly resembling the steric zipper motif of amyloid fibrils but differing in fundamental details. In the other two crystals, the solvent is encapsulated between the sheets, yielding fibril architectures capable of host-guest chemistry. Our study demonstrates that the formation of rippled ß-sheets from aggregating racemic peptide mixtures in three-dimensional (3D) assemblies is a general phenomenon and provides a structural basis for targeting intrinsically disordered proteins.

An Enantiomeric Fragment Pair (EFP) Approach for the Study of Cellular Uptake of Intrinsically Disordered Proteins.

The study of intrinsically disordered and amyloidogenic proteins poses a major challenge to researchers due to the propensity of the system to aggregate and to form amyloid fibrils and deposits. This intrinsic nature limits the way amyloids can be studied and increases the level of complexity of the techniques needed to study the system of interest. Recent reports suggest that cellular recognition and internalization of pre-fibrillary species of amyloidogenic peptides and proteins may initiate some of its toxic actions. Therefore, developing novels tools to facilitate the understanding and determination of the interactions between intrinsically disordered proteins and the cellular membrane is becoming increasingly valuable. Here, we present and propose an approach for the study of the interactions of intrinsically disordered proteins with the cellular surface based on the use of enantiomeric fragment pairs (EFPs). By following a stepwise methodology in which the amyloidogenic peptide or protein is fragmented into specific segments, we show how this approach can be exploited to differentiate between different types of cellular uptake, to determine the degree of receptor-mediated cellular internalization of intrinsically disordered peptides and proteins, and to pinpoint the specific regions within the amino acid sequence responsible for the cellular recognition. Adopting this approach overcomes aggregation-related challenges and offers a particularly well-suited platform for the elucidation of receptor-intermediated recognition, uptake, and toxicity.

Defining the Landscape of the Pauling-Corey Rippled Sheet: An Orphaned Motif Finding New Homes.

When peptides are mixed with their mirror images in an equimolar ratio, two-dimensional periodic structural folds can form, in which extended peptide strands are arrayed with alternating chirality. The resultant topography class, termed the rippled ß-sheet, was introduced as a theoretical concept by Pauling and Corey in 1953. Unlike other fundamental protein structural motifs identified around that time, including the α-helix and the pleated ß-sheet, it took several decades before conclusive experimental data supporting the proposed rippled ß-sheet motif were gained. Much of the key experimental evidence was provided over the course of the past decade through the concurrent efforts of our three laboratories. Studies that focused on developing new self-assembling hydrogel materials have shown that certain amphiphilic peptides form fibrils and hydrogel networks that are more rigid and have a higher thermodynamic stability when made from racemic peptide mixtures as opposed to pure enantiomers. Related interrogation of assemblies composed of mixtures of l- and d-amphiphilic peptides confirmed that the resulting fibrils were composed of alternating l/d peptides consistent with rippled ß-sheets. It was also demonstrated that mirror-image amyloid beta (Aß) could act as a molecular chaperone to promote oligomer-to-fibril conversion of the natural Aß enantiomer, which was found to reduce Aß neurotoxicity against different neuronal cell models. With a cross-disciplinary approach that combines experiment and theory, our three laboratories have demonstrated the unique biophysical, biochemical, and biological properties that arise upon mixing of peptide enantiomers, in consequence of rippled ß-sheet formation. In this Account, we give an overview of the early history of the rippled ß-sheet and provide a detailed structural description/definition of this motif relative to the pleated ß-sheet. We then summarize the key findings, obtained on three unique sets of aggregating mirror-image peptide pairs through independent efforts of our three laboratories, and use these results to delineate the landscape of the rippled ß-sheet structural motif to inspire future studies. Peptide sequence parameters that favor rippled ß-sheet assembly are described, along with the accompanying kinetic and thermodynamic properties, as well as the resulting emergent physical properties of the assemblies. The Account then concludes with a brief overview of some key unresolved challenges in this nascent field. There is much potential for future applications of this unique supramolecular motif in the realm of materials design and biomedical research. We hope this Account will stimulate much-needed discussion of this fascinating structural class to eventually produce a fully quantitative, rational framework for the molecular engineering of rippled ß-sheets in the future.

Understanding and controlling amyloid aggregation with chirality.

Amyloid aggregation and human disease are inextricably linked. Examples include Alzheimer disease, Parkinson disease, and type II diabetes. While seminal advances on the mechanistic understanding of these diseases have been made over the last decades, controlling amyloid fibril formation still represents a challenge, and it is a subject of active research. In this regard, chiral modifications have increasingly been proved to offer a particularly well-suited approach toward accessing to previously unknown aggregation pathways and to provide with novel insights on the biological mechanisms of action of amyloidogenic peptides and proteins. Here, we summarize recent advances on how the use of mirror-image peptides/proteins and d-amino acid incorporations have helped modulate amyloid aggregation, offered new mechanistic tools to study cellular interactions, and allowed us to identify key positions within the peptide/protein sequence that influence amyloid fibril growth and toxicity.

A DFT study of structure and stability of pleated and rippled cross-ß sheets with hydrophobic sidechains.

The rippled cross-ß sheet, a topography, in which mirror-image peptides are arranged with alternating chirality into a periodic two-dimensional network, is burgeoning as a new design principle for materials and biomedical applications. Experiments by the Schneider, Nilsson, and Raskatov labs have independently shown diverse racemic mixtures of aggregation-prone peptide of different sizes to favor the rippled over the pleated topography. Yet, systematic ab initio studies are lacking, and the field is yet to develop rules that would enable the design of new rippled cross-ß frameworks from first principles. Here, DFT calculations were performed on a set of model systems, designed to begin understanding the impact that bulky, hydrophobic sidechains have upon the formation of pleated and rippled cross-ß frameworks. It is hoped that this study will help stimulate the development of a predictive, general framework to enable rational design of rippled cross-ß sheets in the future.

Trapping and Characterization of Nontoxic Aß42 Aggregation Intermediates.

Amyloid ß (Aß) 42 is an aggregation-prone peptide and the believed seminal etiological agent of Alzheimer's disease (AD). Intermediates of Aß42 aggregation, commonly referred to as diffusible oligomers, are considered to be among the most toxic forms of the peptide. Here, we studied the effect of the age-related epimerization of Ser26 (i.e., S26s chiral edit) in Aß42 and discovered that this subtle molecular change led to reduced fibril formation propensity. Surprisingly, the resultant soluble aggregates were nontoxic. To gain insight into the structural changes that occurred in the peptide upon S26s substitution, the system was probed using an array of biophysical and biochemical methods. These experiments consistently pointed to the stabilization of aggregation intermediates in the Aß42-S26s system. To better understand the changes arising as a consequence of the S26s substitution, molecular level structural studies were performed. Using a combined nuclear magnetic resonance (NMR)- and density functional theory (DFT)-computational approach, we found that the S26s chiral edit induced only local structural changes in the Gly25-Ser26-Asn27 region. Interestingly, these subtle changes enabled the formation of an intramolecular Ser26-Asn27 H-bond, which disrupted the ability of Asn27 to engage in the fibrillogenic side chain-to-side chain H-bonding pattern. This reveals that intermolecular stabilizing interactions between Asn27 side chains are a key element controlling Aß42 aggregation and toxicity.

Chirality Dependence of Amyloidâ€ ß Cellular Uptake and a New Mechanistic Perspective.

Amyloidâ€…ß is an inherently disordered peptide that can form diverse neurotoxic aggregates, and its 42-amino-acid isoform is believed to be the agent responsible for Alzheimer's disease (AD). Cellular uptake of the peptide is a pivotal step for it to be able to exert many of its toxic actions. The cellular uptake process is complex, and numerous competing internalization pathways have been proposed. To date, it remains unclear which of the uptake mechanisms are particularly important for the overall process, and improvement of this understanding is needed, so that better molecular AD therapeutics can be designed. Chirality can be used as a unique tool to study this process, because some of the proposed mechanisms are expected to proceed in stereoselective fashion, whereas others are not. To shed light on this important issue, we synthesized fluorescently labeled enantiomers of amyloidâ€…ß and quantified their cellular uptake, finding that uptake occurs in stereoselective fashion, with a typical preference for the l stereoisomer of ≈5:1. This suggests that the process is predominantly receptor-mediated, with likely minor contributions of non-stereoselective mechanisms.

Suppression of Oligomer Formation and Formation of Non-Toxic Fibrils upon Addition of Mirror-Image Aß42 to the Natural l-Enantiomer.

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aß) 42 is an aggregation-prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aß42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror-image d-Aß42 should reduce the concentration of toxic oligomers formed from natural l-Aß42. We synthesized l- and D-Aß42 and found their equimolar mixing to lead to accelerated fibril formation. Confocal microscopy with fluorescently labeled analogues of the enantiomers showed their colocalization in racemic fibrils. Owing to the enhanced fibril formation propensity, racemic Aß42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l-Aß42 at concentrations up to 50 µm. The mixing of Aß42 enantiomers thus accelerates the formation of non-toxic fibrils.

Introduction of d-Glutamate at a Critical Residue of Aß42 Stabilizes a Prefibrillary Aggregate with Enhanced Toxicity.

The amyloid beta peptide 42 (Aß42) is an aggregation-prone peptide that plays a pivotal role in Alzheimer's disease. We report that a subtle perturbation to the peptide through a single chirality change at glutamate 22 leads to a pronounced delay in the ß-sheet adoption of the peptide. This was accompanied by an attenuated propensity of the peptide to form fibrils, which was correlated with changes at the level of the fibrillary architecture. Strikingly, the incorporation of d-glutamate was found to stabilize a soluble, ordered macromolecular assembly with enhanced cytotoxicity to PC12 cells, highlighting the importance of advanced prefibrillary Aß aggregates in neurotoxicity.

4-Azidobenzyl ferrocenylcarbamate as an anticancer prodrug activated under reductive conditions.

Aminoferrocene-based prodrugs are activated in the presence of cancer-specific amounts of reactive oxygen species, e.g. H2O2, with the formation of products of two types: Fe-containing complexes, which catalyze generation of HO and O2(-), and quinone methides, which alkylate glutathione and inhibit the antioxidative system of the cell. Both processes act synergistically by increasing the oxidative stress in cancer cells thereby leading to their death. However, in the activation step including the cleavage of a B-C bond one molecule of H2O2 is consumed that counteracts the desired effect of the products released from aminoferrocenes. We replaced an H2O2-sensitive trigger in original prodrugs with an azide group. This trigger is slowly reduced in the presence of glutathione with the formation of an unstable arylamine intermediate, which decomposes with the release of iron ions and iminoquinone methides. These products induce strong oxidative stress in cells as we confirmed using 2',7'-dichlorodihydrofluorescin diacetate reagent in combination with flow cytometry. In this case the activation process does not consume H2O2. Correspondingly, we observed that the azide-containing prodrug is substantially more toxic towards human promyelocytic leukemia cell line HL-60 (IC50=27±4µM) than its H2O2-responsive analogue (IC50>50µM).

An HRE-Binding Py-Im Polyamide Impairs Hypoxic Signaling in Tumors.

Hypoxic gene expression contributes to the pathogenesis of many diseases, including organ fibrosis, age-related macular degeneration, and cancer. Hypoxia-inducible factor-1 (HIF1), a transcription factor central to the hypoxic gene expression, mediates multiple processes including neovascularization, cancer metastasis, and cell survival. Pyrrole-imidazole polyamide 1: has been shown to inhibit HIF1-mediated gene expression in cell culture but its activity in vivo was unknown. This study reports activity of polyamide 1: in subcutaneous tumors capable of mounting a hypoxic response and showing neovascularization. We show that 1: distributes into subcutaneous tumor xenografts and normal tissues, reduces the expression of proangiogenic and prometastatic factors, inhibits the formation of new tumor blood vessels, and suppresses tumor growth. Tumors treated with 1: show no increase in HIF1α and have reduced ability to adapt to the hypoxic conditions, as evidenced by increased apoptosis in HIF1α-positive regions and the increased proximity of necrotic regions to vasculature. Overall, these results show that a molecule designed to block the transcriptional activity of HIF1 has potent antitumor activity in vivo, consistent with partial inhibition of the tumor hypoxic response. Mol Cancer Ther; 15(4); 608-17. ©2015 AACR.

Tumor xenograft uptake of a pyrrole-imidazole (Py-Im) polyamide varies as a function of cell line grafted.

Subcutaneous xenografts represent a popular approach to evaluate efficacy of prospective molecular therapeutics in vivo. In the present study, the C-14 labeled radioactive pyrrole-imidazole (Py-Im) polyamide 1, targeted to the 5'-WGWWCW-3' DNA sequence, was evaluated with regard to its uptake properties in subcutaneous xenografts, derived from the human tumor cell lines LNCaP (prostate), A549 (lung), and U251 (brain), respectively. Significant variation in compound tumor concentrations was seen in xenografts derived from these three cell lines. Influence of cell line grafted on systemic polyamide elimination was established. With A549, a marked variation in localization of 1 was determined between Matrigel-negative and -positive xenografts. An extensive tissue distribution analysis of 1 in wild-type animals was conducted, enabling the comparison between the xenografts and the corresponding host organs of origin.

A C-14 labeled Py-Im polyamide localizes to a subcutaneous prostate cancer tumor.

In an effort to quantitate Py-Im polyamide concentrations in vivo, we synthesized the C-14 radioactively labeled compounds 1-3, and investigated their tumor localization in a subcutaneous xenograft model of prostate cancer (LNCaP). Tumor concentrations were compared with representative host tissues, and exhibited a certain degree of preferential localization to the xenograft. Compound accumulation upon repeated administration was measured. Py-Im polyamide 1 was found to accumulate in LNCaP tumors at concentrations similar to the IC50 value for this compound in cell culture experiments.

Activity of a Py-Im polyamide targeted to the estrogen response element.

Pyrrole-imidazole (Py-Im) polyamides are a class of programmable DNA minor groove binders capable of modulating the activity of DNA-binding proteins and affecting changes in gene expression. Estrogen receptor alpha (ERα) is a ligand-activated hormone receptor that binds as a homodimer to estrogen response elements (ERE) and is a driving oncogene in a majority of breast cancers. We tested a selection of structurally similar Py-Im polyamides with differing DNA sequence specificity for activity against 17ß-estadiol (E2)-induced transcription and cytotoxicity in ERα positive, E2-stimulated T47DKBluc cells, which express luciferase under ERα control. The most active polyamide targeted the sequence 5'-WGGWCW-3' (W = A or T), which is the canonical ERE half site. Whole transcriptome analysis using RNA-Seq revealed that treatment of E2-stimulated breast cancer cells with this polyamide reduced the effects of E2 on the majority of those most strongly affected by E2 but had much less effect on the majority of E2-induced transcripts. In vivo, this polyamide circulated at detectable levels following subcutaneous injection and reduced levels of ER-driven luciferase expression in xenografted tumors in mice after subcutaneous compound administration without significant host toxicity.

Gene expression changes in a tumor xenograft by a pyrrole-imidazole polyamide.

Gene regulation by DNA binding small molecules could have important therapeutic applications. This study reports the investigation of a DNA-binding pyrrole-imidazole polyamide targeted to bind the DNA sequence 5'-WGGWWW-3' with reference to its potency in a subcutaneous xenograft tumor model. The molecule is capable of trafficking to the tumor site following subcutaneous injection and modulates transcription of select genes in vivo. An FITC-labeled analogue of this polyamide can be detected in tumor-derived cells by confocal microscopy. RNA deep sequencing (RNA-seq) of tumor tissue allowed the identification of further affected genes, a representative panel of which was interrogated by quantitative reverse transcription-PCR and correlated with cell culture expression levels.

Characterization and solubilization of pyrrole-imidazole polyamide aggregates.

To optimize the biological activity of pyrrole-imidazole polyamide DNA-binding molecules, we characterized the aggregation propensity of these compounds through dynamic light scattering and fractional solubility analysis. Nearly all studied polyamides were found to form measurable particles 50-500 nm in size under biologically relevant conditions, while HPLC-based analyses revealed solubility trends in both core sequences and peripheral substituents that did not correlate with overall ionic charge. The solubility of both hairpin and cyclic polyamides was increased upon addition of carbohydrate solubilizing agents, in particular, 2-hydroxypropyl-ß-cyclodextrin (HpßCD). In mice, the use of HpßCD allowed for improved injection conditions and subsequent investigations of the availability of polyamides in mouse plasma to human cells. The results of these studies will influence the further design of Py-Im polyamides and facilitate their study in animal models.

Modulation of NF-κB-dependent gene transcription using programmable DNA minor groove binders.

Nuclear factor κB (NF-κB) is a transcription factor that regulates various aspects of immune response, cell death, and differentiation as well as cancer. In this study we introduce the Py-Im polyamide 1 that binds preferentially to the sequences 5'-WGGWWW-3' and 5'GGGWWW-3'. The compound is capable of binding to κB sites and reducing the expression of various NF-κB-driven genes including IL6 and IL8 by qRT-PCR. Chromatin immunoprecipitation experiments demonstrate a reduction of p65 occupancy within the proximal promoters of those genes. Genome-wide expression analysis by RNA-seq compares the DNA-binding polyamide with the well-characterized NF-κB inhibitor PS1145, identifies overlaps and differences in affected gene groups, and shows that both affect comparable numbers of TNF-α-inducible genes. Inhibition of NF-κB DNA binding via direct displacement of the transcription factor is a potential alternative to the existing antagonists.

Exploring the reactions of CO2 with PCP supported nickel complexes.

The reactions of PCP supported Ni hydride, methyl and allyl species with CO(2) to generate Ni carboxylates are described. Computational studies suggest that all three reactions follow different pathways.