Publications by authors named "Jevgenij A Raskatov"

40 Publications

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

Acc Chem Res 2021 05 26;54(10):2488-2501. Epub 2021 Apr 26.

Department of Chemistry, University of Rochester, Rochester, New York 14627-0216, United States.

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 β-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 β-sheet, it took several decades before conclusive experimental data supporting the proposed rippled β-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 β-sheets. It was also demonstrated that mirror-image amyloid beta (Aβ) could act as a molecular chaperone to promote oligomer-to-fibril conversion of the natural Aβ enantiomer, which was found to reduce Aβ 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 β-sheet formation. In this Account, we give an overview of the early history of the rippled β-sheet and provide a detailed structural description/definition of this motif relative to the pleated β-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 β-sheet structural motif to inspire future studies. Peptide sequence parameters that favor rippled β-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 β-sheets in the future.
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http://dx.doi.org/10.1021/acs.accounts.1c00084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8154201PMC
May 2021

Understanding and controlling amyloid aggregation with chirality.

Curr Opin Chem Biol 2021 Feb 17;64:1-9. Epub 2021 Feb 17.

Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, CA, 95064, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.cbpa.2021.01.003DOI Listing
February 2021

Evidence for aggregation-independent, PrP-mediated Aβ cellular internalization.

Proc Natl Acad Sci U S A 2020 11 2;117(46):28625-28631. Epub 2020 Nov 2.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064

Evidence linking amyloid beta (Aβ) cellular uptake and toxicity has burgeoned, and mechanisms underlying this association are subjects of active research. Two major, interconnected questions are whether Aβ uptake is aggregation-dependent and whether it is sequence-specific. We recently reported that the neuronal uptake of Aβ depends significantly on peptide chirality, suggesting that the process is predominantly receptor-mediated. Over the past decade, the cellular prion protein (PrP) has emerged as an important mediator of Aβ-induced toxicity and of neuronal Aβ internalization. Here, we report that the soluble, nonfibrillizing Aβ (1-30) peptide recapitulates full-length Aβ stereoselective cellular uptake, allowing us to decouple aggregation from cellular, receptor-mediated internalization. Moreover, we found that Aβ (1-30) uptake is also dependent on PrP expression. NMR-based molecular-level characterization identified the docking site on PrP that underlies the stereoselective binding of Aβ (1-30). Our findings therefore identify a specific sequence within Aβ that is responsible for the recognition of the peptide by PrP, as well as PrP-dependent cellular uptake. Further uptake stereodifferentiation in PrP-free cells points toward additional receptor-mediated interactions as likely contributors for Aβ cellular internalization. Taken together, our results highlight the potential of targeting cellular surface receptors to inhibit Aβ cellular uptake as an alternative route for future therapeutic development for Alzheimer's disease.
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http://dx.doi.org/10.1073/pnas.2009238117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682355PMC
November 2020

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

Biopolymers 2021 Jan 1;112(1):e23391. Epub 2020 Aug 1.

Department of Chemistry and Biochemistry, University of California Santa Cruz, Santa Cruz, California, USA.

The rippled cross-β 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-β 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-β frameworks. It is hoped that this study will help stimulate the development of a predictive, general framework to enable rational design of rippled cross-β sheets in the future.
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http://dx.doi.org/10.1002/bip.23391DOI Listing
January 2021

Conformational Selection as the Driving Force of Amyloid β Chiral Inactivation.

Chembiochem 2020 10 25;21(20):2945-2949. Epub 2020 Jun 25.

Department of Chemistry and Biochemistry, University of California Santa Cruz Physical Science Building 356, 1156 High Street, Santa Cruz, CA 95064, USA.

We recently introduced amyloid β chiral inactivation (Aβ-CI) as a molecular approach that uses mirror-image peptides to chaperone the natural Aβ stereoisomer into a less toxic state. The oligomer-to-fibril conversion mechanism remains the subject of active research. Perhaps the most striking feature of Aβ-CI is the virtual obliteration of the incubation/induction phase that is so characteristic of Aβ fibril formation kinetics. This qualitative change is indicative of the distinct mechanistic pathway Aβ-CI operates through. The current working model of Aβ-CI invokes the formation of "rippled" cross-β sheets, in which alternating l- and d-peptide strands form periodic networks. However, the assumption of rippled cross-β sheets does not per se explain the dramatic changes in reaction kinetics upon mixing of Aβ enantiomers. Herein, it is shown by DFT computational methods that the individual peptide strands in rippled cross-β networks are less conformationally strained than their pleated counterparts. This means that the adoption of fibril-seeding conformations is more probable for rippled cross-β. Conformational selection is thus suggested as the mechanistic rationale for the acceleration of fibril formation upon Aβ-CI.
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http://dx.doi.org/10.1002/cbic.202000237DOI Listing
October 2020

Alzheimer's Disease "Non-amyloidogenic" p3 Peptide Revisited: A Case for Amyloid-α.

ACS Chem Neurosci 2020 06 22;11(11):1539-1544. Epub 2020 May 22.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, Santa Cruz, California 95064, United States.

Amyloid-β (Aβ) is an intrinsically disordered peptide thought to play an important role in Alzheimer's disease (AD). It has been the target of most AD therapeutic efforts, which have repeatedly failed in clinical trials. A more predominant peptidic fragment, formed through alternative processing of the amyloid precursor protein, is the p3 peptide. p3 has received little attention, which is possibly due to the prevailing view in the AD field that it is "non-amyloidogenic." By probing the self-assembly of this peptide, we found that p3 aggregates to form oligomers and fibrils and, when compared with Aβ, displays enhanced aggregation rates. Our findings highlight the solubilizing effect of the N-terminus of Aβ and the favorable formation of structures formed through C-terminal hydrophobic peptide interfaces. Based on our findings, we suggest a reevaluation of the current therapeutic approaches targeting only the β-secretase pathway of AD, given that the α- secretase pathway is also amyloidogenic.
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http://dx.doi.org/10.1021/acschemneuro.0c00160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7443049PMC
June 2020

Assessing Reproducibility in Amyloid β Research: Impact of Aβ Sources on Experimental Outcomes.

Chembiochem 2020 09 5;21(17):2425-2430. Epub 2020 May 5.

Department of Chemistry and Biochemistry, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA, 95064, USA.

The difficulty of synthesizing and purifying the amyloid β (Aβ) peptide, combined with its high aggregation propensity and low solubility under physiological conditions, leads to a wide variety of experimental results from kinetic assays to biological activity. Thus, it becomes challenging to reproduce outcomes, and this limits our ability to rely on reported results as the foundation for new research. This article examines variability of the Aβ peptide from different sources, comparing purity, and oligomer and fibril formation propensity side by side. The results highlight the importance of performing rigorous controls so that meaningful biophysical, biochemical, and neurobiological results can be obtained to improve our understanding on Aβ.
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http://dx.doi.org/10.1002/cbic.202000125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647053PMC
September 2020

A Focused Chiral Mutant Library of the Amyloid β 42 Central Electrostatic Cluster as a Tool To Stabilize Aggregation Intermediates.

J Org Chem 2020 02 3;85(3):1385-1391. Epub 2020 Jan 3.

Department of Chemistry and Biochemistry , University of California Santa Cruz , Santa Cruz , California 95064 , United States.

Amyloidogenic peptides and proteins aggregate into fibrillary structures that are usually deposited in tissues and organs and are often involved in the development of diseases. In contrast to native structured proteins, amyloids do not follow a defined energy landscape toward the fibrillary state and often generate a vast population of aggregation intermediates that are transient and exceedingly difficult to study. Here, we employ as a tool to study the aggregation mechanism of the Amyloid β (Aβ) 42 peptide, whose aggregation intermediates are thought to be one of the main driving forces in Alzheimer's disease (AD). Through the design of a focused chiral mutant library (FCML) of 16 chiral Aβ42 variants, we identified several point D-substitutions that allowed us to modulate the aggregation propensity and the biological activity of the peptide. Surprisingly, the reduced propensity toward aggregation and the stabilization of oligomeric intermediates did not always correlate with an increase in toxicity. In the present study, we show how chiral editing can be a powerful tool to trap and stabilize Aβ42 conformers that might otherwise be too transient and dynamic to study, and we identify sites within the Aβ42 sequence that could be potential targets for therapeutic intervention.
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http://dx.doi.org/10.1021/acs.joc.9b02312DOI Listing
February 2020

A Facile Method for the Separation of Methionine Sulfoxide Diastereomers, Structural Assignment, and DFT Analysis.

Chemistry 2020 Apr 6;26(20):4467-4470. Epub 2020 Feb 6.

Dept. of Chemistry and Biochemistry, UCSC, 1156 High Street, Santa Cruz, California, USA.

Methionine (Met) oxidation is an important biological redox node, with hundreds if not thousands of protein targets. The process yields methionine oxide (MetO). It renders the sulfur chiral, producing two distinct, diastereomerically related products. Despite the biological significance of Met oxidation, a reliable protocol to separate the resultant MetO diastereomers is currently lacking. This hampers our ability to make peptides and proteins that contain stereochemically defined MetO to then study their structural and functional properties. We have developed a facile method that uses supercritical CO chromatography and allows obtaining both diastereomers in purities exceeding 99 %. H NMR spectra were correlated with X-ray structural information. The stereochemical interconversion barrier at sulfur was calculated as 45.2 kcal mol , highlighting the remarkable stereochemical stability of MetO sulfur chirality. Our protocol should open the road to synthesis and study of a wide variety of stereochemically defined MetO-containing proteins and peptides.
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http://dx.doi.org/10.1002/chem.201904848DOI Listing
April 2020

Using mirror-image peptides to enhance robustness and reproducibility in studying the amyloid β-protein.

Prog Mol Biol Transl Sci 2019 18;168:57-67. Epub 2019 Jun 18.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, United States. Electronic address:

Alzheimer's disease, the most common form of dementia, is a devastating disease that affects over 44 million people worldwide. One etiological agent of Alzheimer's, the amyloid β-protein (Aβ), is an aggregation-prone, intrinsically disordered peptide that can form a wide variety of aggregates. The pathways by which Aβ aggregates in order to exert its toxicity, referred to as the Amyloid Cascade, remains largely elusive despite substantial deconvolution efforts. Preparing high-quality material that exhibits reproducible biophysical characteristics has proven challenging. Herein, we propose that mirror-image peptides can be used to rigorously control Aβ preparation quality.
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http://dx.doi.org/10.1016/bs.pmbts.2019.05.010DOI Listing
July 2020

Relative Rates of Metal-Free Azide-Alkyne Cycloadditions: Tunability over 3 Orders of Magnitude.

J Org Chem 2019 11 8;84(21):13615-13623. Epub 2019 Oct 8.

Department Chemistry and Biochemistry , University of California, Santa Cruz , 1156 High Street , Santa Cruz , California 95064 , United States.

The thermal (3 + 2) dipolar azide-alkyne cycloaddition, proceeding without copper or strained alkynes, is an underutilized ligation with potential applications in materials, bioorganic, and synthetic chemistry. Herein, we investigate the effects of alkyne substitution on the rate of this reaction, both experimentally and computationally. Electron-withdrawing groups accelerate the reaction, providing a range of relative rates from 1.0 to 2100 between the slowest and fastest alkynes studied. Unexpectedly, aryl groups conjugated to the alkyne significantly retard the reaction rate. In contrast, a sulfonyl, ester-substituted alkyne is reactive enough that it couples with an azide at room temperature in a few hours. This reactivity scale should provide a guide to those who wish to use this ligation under mild conditions.
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http://dx.doi.org/10.1021/acs.joc.9b01887DOI Listing
November 2019

Trapping and Characterization of Nontoxic Aβ42 Aggregation Intermediates.

ACS Chem Neurosci 2019 08 1;10(8):3880-3887. Epub 2019 Aug 1.

Department of Chemistry and Biochemistry , University of California Santa Cruz , Santa Cruz , California 95064 , United States.

Amyloid β (Aβ) 42 is an aggregation-prone peptide and the believed seminal etiological agent of Alzheimer's disease (AD). Intermediates of Aβ42 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 Aβ42 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 Aβ42-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 Aβ42 aggregation and toxicity.
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http://dx.doi.org/10.1021/acschemneuro.9b00340DOI Listing
August 2019

What Is the "Relevant" Amyloid β42 Concentration?

Chembiochem 2019 07 17;20(13):1725-1726. Epub 2019 May 17.

Department of Chemistry and Biochemistry, University of California Santa Cruz, Physical Science Building 356, 1156 High Street, Santa Cruz, CA, 95064, USA.

Alzheimer's amyloid beta can perform a wide variety of actions that are highly concentration dependent. This viewpoint aims to provide a framework for basic considerations on what might be considered brain-relevant concentrations of the peptide. Some implications for the therapeutic implementation of the recently emerged oligomer-to-fibril strategy are discussed.
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http://dx.doi.org/10.1002/cbic.201900097DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6711386PMC
July 2019

A DFT-Assisted Topological Analysis of Four Polymorphic, S-Shaped Aβ42 Fibril Structures.

Chembiochem 2019 07 21;20(13):1722-1724. Epub 2019 May 21.

Department of Chemistry and Biochemistry, University of California Santa Cruz, Physical Science Building 356, 1156 High Street, Santa Cruz, CA, 95064, USA.

Amyloid β 42 (Aβ42) is an inherently disordered peptide, whose toxic actions are believed to play important roles in the etiology of Alzheimer's disease. Four fibril structures of the peptide that display broadly similar characteristics were recently published, but a systematic comparison of these structures is lacking. In this paper, a topological framework was created to enable such understanding and produced new insights into subtle structural elements that underlie the overall structural diversity. A DFT-based analysis illuminated some of the energetic differences that arise as a consequence.
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http://dx.doi.org/10.1002/cbic.201900036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713286PMC
July 2019

Chirality Dependence of Amyloid β Cellular Uptake and a New Mechanistic Perspective.

Chembiochem 2019 04 27;20(8):1023-1026. Epub 2019 Feb 27.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA, 95064, USA.

Amyloid β 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 amyloid β 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.
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http://dx.doi.org/10.1002/cbic.201800708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6517241PMC
April 2019

Using chirality to probe the conformational dynamics and assembly of intrinsically disordered amyloid proteins.

Sci Rep 2017 10 2;7(1):12433. Epub 2017 Oct 2.

Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA, 90095, USA.

Intrinsically disordered protein (IDP) conformers occupy large regions of conformational space and display relatively flat energy surfaces. Amyloid-forming IDPs, unlike natively folded proteins, have folding trajectories that frequently involve movements up shallow energy gradients prior to the "downhill" folding leading to fibril formation. We suggest that structural perturbations caused by chiral inversions of amino acid side-chains may be especially valuable in elucidating these pathways of IDP folding. Chiral inversions are subtle in that they do not change side-chain size, flexibility, hydropathy, charge, or polarizability. They allow focus to be placed solely on the question of how changes in amino acid side-chain orientation, and the resultant alterations in peptide backbone structure, affect a peptide's conformational landscape (Ramachandran space). If specific inversions affect folding and assembly, then the sites involved likely are important in mediating these processes. We suggest here a "focused chiral mutant library" approach for the unbiased study of amyloid-forming IDPs.
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http://dx.doi.org/10.1038/s41598-017-10525-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5624888PMC
October 2017

Chiral Inactivation: An Old Phenomenon with a New Twist.

Chemistry 2017 Dec 23;23(67):16920-16923. Epub 2017 Oct 23.

Department of Chemistry and Biochemistry, Physical Science Building 356, University of California Santa Cruz, 1156 High Street, Santa Cruz, CA, 95064, USA.

Mixing of enantiomers of chiral molecules can have remarkable effects on the properties of their higher order assemblies. This, in turn, may have profound impact on both structural and functional outcomes, and has been noted across a wide range of contexts. This account presents key examples from organic, organometallic and bioorganic molecular sciences and showcases that, in disciplines as distinct as asymmetric catalysis and racemic protein crystallography, there is a fundamental way in which enantiomers differ from racemates.
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http://dx.doi.org/10.1002/chem.201703869DOI Listing
December 2017

Suppression of Oligomer Formation and Formation of Non-Toxic Fibrils upon Addition of Mirror-Image Aβ42 to the Natural l-Enantiomer.

Angew Chem Int Ed Engl 2017 09 19;56(38):11506-11510. Epub 2017 Jul 19.

Dept. of Chemistry and Biochemistry, UCSC, 1156 High Street, Santa Cruz, CA, USA.

Racemates often have lower solubility than enantiopure compounds, and the mixing of enantiomers can enhance the aggregation propensity of peptides. Amyloid beta (Aβ) 42 is an aggregation-prone peptide that is believed to play a key role in Alzheimer's disease. Soluble Aβ42 aggregation intermediates (oligomers) have emerged as being particularly neurotoxic. We hypothesized that the addition of mirror-image d-Aβ42 should reduce the concentration of toxic oligomers formed from natural l-Aβ42. We synthesized l- and D-Aβ42 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 Aβ42 was less prone to form soluble oligomers. This resulted in the protection of cells from the toxicity of l-Aβ42 at concentrations up to 50 μm. The mixing of Aβ42 enantiomers thus accelerates the formation of non-toxic fibrils.
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http://dx.doi.org/10.1002/anie.201706279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5623316PMC
September 2017

A Tailored HPLC Purification Protocol That Yields High-purity Amyloid Beta 42 and Amyloid Beta 40 Peptides, Capable of Oligomer Formation.

J Vis Exp 2017 03 27(121). Epub 2017 Mar 27.

Department of Chemistry, University of California, Santa Cruz;

Amyloidogenic peptides such as the Alzheimer's disease-implicated Amyloid beta (Aβ), can present a significant challenge when trying to obtain high purity material. Here we present a tailored HPLC purification protocol to produce high-purity amyloid beta 42 (Aβ42) and amyloid beta 40 (Aβ40) peptides. We have found that the combination of commercially available hydrophobic poly(styrene/divinylbenzene) stationary phase, polymer laboratory reverse phase - styrenedivinylbenzene (PLRP-S) under high pH conditions, enables the attainment of high purity (>95%) Aβ42 in a single chromatographic run. The purification is highly reproducible and can be amended to both semi-preparative and analytical conditions depending upon the amount of material wished to be purified. The protocol can also be applied to the Aβ40 peptide with identical success and without the need to alter the method.
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http://dx.doi.org/10.3791/55482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5564433PMC
March 2017

Using chiral peptide substitutions to probe the structure function relationship of a key residue of Aβ42.

Chirality 2017 01 8;29(1):5-9. Epub 2016 Dec 8.

Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA, USA.

Amyloid beta-protein 42 plays an important role in the onset and progression of Alzheimer's disease. Familial mutations have identified the glutamate residue 22 as a hotspot with regard to peptide neurotoxicity. We introduce an approach to study the influence of systematic sidechain modification at this residue, employing chirality as a structural probe. Circular dichroism experiments reveal that charge-preserving alterations of the amino acid sidechain attenuate the characteristic random coil to β-sheet transition associated with the wildtype peptide. Removal of the negative charge from residue 22, a trait observed with all known familial mutations at this residue, gives rise to a peptide with limited random coil propensity and high β-sheet characteristics. Our approach can be extended to other residues of Aβ, as well as further amyloidogenic peptides.
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http://dx.doi.org/10.1002/chir.22667DOI Listing
January 2017

Introduction of d-Glutamate at a Critical Residue of Aβ42 Stabilizes a Prefibrillary Aggregate with Enhanced Toxicity.

Chemistry 2016 Aug 30;22(34):11967-70. Epub 2016 Jun 30.

Department of Chemistry and Biochemistry, Physical Science Building, University of California, 1156 High Street, Santa Cruz, USA.

The amyloid beta peptide 42 (Aβ42) 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 β-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 Aβ aggregates in neurotoxicity.
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http://dx.doi.org/10.1002/chem.201601763DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5096037PMC
August 2016

Strict Regiospecificity of Human Epithelial 15-Lipoxygenase-2 Delineates Its Transcellular Synthesis Potential.

Biochemistry 2016 05 13;55(20):2832-40. Epub 2016 May 13.

Department Chemistry and Biochemistry, University of California at Santa Cruz , 1156 High Street, Santa Cruz, California 95064, United States.

Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (kcat/Km) of this reaction was 31% of the kcat/Km of AA. The only major product of h15-LOX-2's reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA4, indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.
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http://dx.doi.org/10.1021/acs.biochem.5b01339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5657383PMC
May 2016

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

J Inorg Biochem 2016 07 2;160:218-24. Epub 2016 Mar 2.

Friedrich-Alexander-University of Erlangen-Nürnberg, Department of Chemistry and Pharmacy, Organic Chemistry II, Henkestr. 42, 91054 Erlangen, Germany. Electronic address:

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).
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http://dx.doi.org/10.1016/j.jinorgbio.2016.02.023DOI Listing
July 2016

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

Mol Cancer Ther 2016 04 30;15(4):608-17. Epub 2015 Dec 30.

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California.

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.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-0719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873353PMC
April 2016

Genome-Directed Lead Discovery: Biosynthesis, Structure Elucidation, and Biological Evaluation of Two Families of Polyene Macrolactams against Trypanosoma brucei.

ACS Chem Biol 2015 Oct 13;10(10):2373-81. Epub 2015 Aug 13.

Department of Chemistry and Biochemistry, University of California Santa Cruz , Santa Cruz, California 95064, United States.

Marine natural products are an important source of lead compounds against many pathogenic targets. Herein, we report the discovery of lobosamides A-C from a marine actinobacterium, Micromonospora sp., representing three new members of a small but growing family of bacterially produced polyene macrolactams. The lobosamides display growth inhibitory activity against the protozoan parasite Trypanosoma brucei (lobosamide A IC50 = 0.8 μM), the causative agent of human African trypanosomiasis (HAT). The biosynthetic gene cluster of the lobosamides was sequenced and suggests a conserved cluster organization among the 26-membered macrolactams. While determination of the relative and absolute configurations of many members of this family is lacking, the absolute configurations of the lobosamides were deduced using a combination of chemical modification, detailed spectroscopic analysis, and bioinformatics. We implemented a "molecules-to-genes-to-molecules" approach to determine the prevalence of similar clusters in other bacteria, which led to the discovery of two additional macrolactams, mirilactams A and B from Actinosynnema mirum. These additional analogs have allowed us to identify specific structure-activity relationships that contribute to the antitrypanosomal activity of this class. This approach illustrates the power of combining chemical analysis and genomics in the discovery and characterization of natural products as new lead compounds for neglected disease targets.
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http://dx.doi.org/10.1021/acschembio.5b00308DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7505085PMC
October 2015

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

J Med Chem 2014 Oct 1;57(20):8471-6. Epub 2014 Oct 1.

Division of Chemistry and Chemical Engineering, California Institute of Technology , Pasadena, California 91125, United States.

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.
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http://dx.doi.org/10.1021/jm500964cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4207538PMC
October 2014

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

Bioorg Med Chem 2014 Aug 16;22(16):4371-5. Epub 2014 Apr 16.

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bmc.2014.04.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134956PMC
August 2014

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

Mol Cancer Ther 2013 May 26;12(5):675-84. Epub 2013 Feb 26.

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

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 17β-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.
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http://dx.doi.org/10.1158/1535-7163.MCT-12-1040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651785PMC
May 2013

Iridium-catalyzed allylic substitutions with cyclometalated phosphoramidite complexes bearing a dibenzocyclooctatetraene ligand: preparation of (π-allyl)Ir complexes and computational and NMR spectroscopic studies.

Chemistry 2012 Nov 27;18(45):14314-28. Epub 2012 Sep 27.

Organisch-Chemisches Institut, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 270, 69120 Heidelberg, Germany.

(π-Allyl)Ir complexes derived from dibenzocyclooctatetraene and phosphoramidites by cyclometalation are effective catalysts for allylic substitution reactions of linear monosubstituted allylic carbonates. These catalysts provide exceptionally high degrees of regioselectivity and allow the reactions to be run under aerobic conditions. A series of (π-allyl)Ir complexes were prepared and characterized by X-ray crystal structure analyses. An allylic amination with aniline displayed different resting states depending on the presence of a strong base. DFT calculations were carried out on the mechanistic aspects of these reactions. The results suggest that for the (π-allyl)Ir complexes, the formation and reactions with nucleophiles proceed with comparable rates.
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http://dx.doi.org/10.1002/chem.201201772DOI Listing
November 2012

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

Proc Natl Acad Sci U S A 2012 Oct 17;109(40):16041-5. Epub 2012 Sep 17.

Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA.

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.
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http://dx.doi.org/10.1073/pnas.1214267109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479560PMC
October 2012