De novo design of peptides that bind specific conformers of α-synuclein.

Insoluble amyloids rich in cross-ß fibrils are observed in a number of neurodegenerative diseases. Depending on the clinicopathology, the amyloids can adopt distinct supramolecular assemblies, termed conformational strains. However, rapid methods to study amyloids in a conformationally specific manner are lacking. We introduce a novel computational method for de novo design of peptides that tile the surface of α-synuclein fibrils in a conformationally specific manner. Our method begins by identifying surfaces that are unique to the conformational strain of interest, which becomes a "target backbone" for the design of a peptide binder. Next, we interrogate structures in the PDB with high geometric complementarity to the target. Then, we identify secondary structural motifs that interact with this target backbone in a favorable, highly occurring geometry. This method produces monomeric helical motifs with a favorable geometry for interaction with the strands of the underlying amyloid. Each motif is then symmetrically replicated to form a monolayer that tiles the amyloid surface. Finally, amino acid sequences of the peptide binders are computed to provide a sequence with high geometric and physicochemical complementarity to the target amyloid. This method was applied to a conformational strain of α-synuclein fibrils, resulting in a peptide with high specificity for the target relative to other amyloids formed by α-synuclein, tau, or Aß40. This designed peptide also markedly slowed the formation of α-synuclein amyloids. Overall, this method offers a new tool for examining conformational strains of amyloid proteins.

Cryo-EM structures reveal tau filaments from Down syndrome adopt Alzheimer's disease fold.

Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among their complex clinical features, including musculoskeletal, neurological, and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early-onset Alzheimer's disease (AD). This dementia is attributed to the increased gene dosage of the amyloid-ß (Aß) precursor protein gene, the formation of self-propagating Aß and tau prion conformers, and the deposition of neurotoxic Aß plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here, we report the characterization of brain samples from four DS cases spanning 36-63 years of age by spectral confocal imaging with conformation-specific dyes and cryo-electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures revealed paired helical filament (PHF) and straight filament (SF) conformations of tau that were identical to those determined from AD cases. The PHFs and SFs are made of two C-shaped protofilaments, each containing a cross-ß/ß-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (approximately 20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene oxide surface derivatized with anti-tau antibodies. This method improved isolation and revealed that primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II-like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.

De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from <5 nM to low micromolar. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free energy calculations performed directly on the designed models were in excellent agreement with the experimentally measured affinities. We conclude that de novo design of high-affinity small molecule-binding proteins with tuned interaction energies is feasible entirely from computation.

Cryo-EM Structures Reveal Tau Filaments from Down Syndrome Adopt Alzheimer's Disease Fold.

Down syndrome (DS) is a common genetic condition caused by trisomy of chromosome 21. Among the complex clinical features including musculoskeletal, neurological and cardiovascular disabilities, individuals with DS have an increased risk of developing progressive dementia and early onset Alzheimer's Disease (AD). This is attributed to the increased gene dosage of amyloid-ß (Aß) precursor protein gene, the formation of self-propagating Aß and tau prion conformers, and the deposition of neurotoxic Aß plaques and tau neurofibrillary tangles. Tau amyloid fibrils have previously been established to adopt many distinct conformations across different neurodegenerative conditions. Here we report the characterization of brain samples from four DS cases spanning 36 to 63 years of age by spectral confocal imaging with conformation-specific dyes and cryo-electron microscopy (cryo-EM) to determine structures of isolated tau fibrils. High-resolution structures reveal paired helical filament (PHF) and straight filament (SF) conformations of tau that are identical to those determined from AD. The PHFs and SFs are made of two C-shaped protofilaments with a cross-ß/ß-helix motif. Similar to filaments from AD cases, most filaments from the DS cases adopted the PHF form, while a minority (~20%) formed SFs. Samples from the youngest individual with no documented dementia had sparse tau deposits. To isolate tau for cryo-EM from this challenging sample we used a novel affinity-grid method involving a graphene-oxide surface derivatized with anti-tau antibodies. This improved isolation and revealed primarily tau PHFs and a minor population of chronic traumatic encephalopathy type II-like filaments were present in this youngest case. These findings expand the similarities between AD and DS to the molecular level, providing insight into their related pathologies and the potential for targeting common tau filament folds by small-molecule therapeutics and diagnostics.

Supramolecular Interactions of Teixobactin Analogues in the Crystal State.

This Note presents the X-ray crystallographic structure of the N-methylated teixobactin analogue N-Me-d-Gln4,Lys10-teixobactin (1). Eight peptide molecules comprise the asymmetric unit, with each peptide molecule binding a chloride anion through hydrogen bonding with the amide NH group of residues 7, 8, 10, and 11. The peptide molecules form hydrogen-bonded antiparallel ß-sheet dimers in the crystal lattice, with residues 1-3 comprising the dimerization interface. The dimers further assemble end-to-end in the crystal lattice.

De novo Design of Peptides that Bind Specific Conformers of α-Synuclein.

Insoluble amyloids rich in cross-ß fibrils are observed in a number of neurodegenerative diseases. Depending on the clinicopathology, the amyloids can adopt distinct supramolecular assemblies, termed conformational strains. However, rapid methods to study amyloid in a conformationally specific manner are lacking. We introduce a novel computational method for de novo design of peptides that tile the surface of α-synuclein fibrils in a conformationally specific manner. Our method begins by identifying surfaces that are unique to the conformational strain of interest, which becomes a "target backbone" for the design of a peptide binder. Next, we interrogate structures in the PDB database with high geometric complementarity to the target. Then, we identify secondary structural motifs that interact with this target backbone in a favorable, highly occurring geometry. This method produces monomeric helical motifs with a favorable geometry for interaction with the strands of the underlying amyloid. Each motif is then symmetrically replicated to form a monolayer that tiles the amyloid surface. Finally, amino acid sequences of the peptide binders are computed to provide a sequence with high geometric and physicochemical complementarity to the target amyloid. This method was applied to a conformational strain of α-synuclein fibrils, resulting in a peptide with high specificity for the target relative to other amyloids formed by α-synuclein, tau, or Aß40. This designed peptide also markedly slowed the formation of α-synuclein amyloids. Overall, this method offers a new tool for examining conformational strains of amyloid proteins.

EMBER multidimensional spectral microscopy enables quantitative determination of disease- and cell-specific amyloid strains.

In neurodegenerative diseases, proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformations in situ. Here, we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid-binding dyes to identify six distinct different conformational strains in vitro, as well as amyloid-ß (Aß) deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aß and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. EMBER has in situ identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains.

EMBER multi-dimensional spectral microscopy enables quantitative determination of disease- and cell-specific amyloid strains.

In neurodegenerative diseases proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. However, there is a need to rapidly identify amyloid conformations in situ . Here we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid binding dyes to identify six distinct different conformational strains in vitro , as well as Aß deposits in different transgenic mouse models. Our EMBER (excitation multiplexed bright emission recording) imaging method rapidly identifies conformational differences in Aß and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. EMBER has in situ identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. In future studies, EMBER should enable high-throughput measurements of the fidelity of strain transmission in cellular and animal neurodegenerative diseases models, time course of amyloid strain propagation, and identification of pathogenic versus benign strains. Significance: In neurodegenerative diseases proteins fold into amyloid structures with distinct conformations (strains) that are characteristic of different diseases. There is a need to rapidly identify these amyloid conformations in situ . Here we use machine learning on the full information available in fluorescent excitation/emission spectra of amyloid binding dyes to identify six distinct different conformational strains in vitro , as well as Aß deposits in different transgenic mouse models. Our imaging method rapidly identifies conformational differences in Aß and tau deposits from Down syndrome, sporadic and familial Alzheimer's disease human brain slices. We also identified distinct conformational strains of tau inclusions in astrocytes, oligodendrocytes, and neurons from Pick's disease. These findings will facilitate the identification of pathogenic protein aggregates to guide research and treatment of protein misfolding diseases.

De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small-molecule-binding proteins has seen exciting recent progress; however, the ability to achieve exquisite affinity for binding small molecules while tuning specificity has not yet been demonstrated directly from computation. Here, we develop a computational procedure that results in the highest affinity binders to date with predetermined relative affinities, targeting a series of PARP1 inhibitors. Two of four designed proteins bound with affinities ranging from < 5 nM to low µM, in a predictable manner. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free-energy calculations performed directly on the designed models are in excellent agreement with the experimentally measured affinities, suggesting that the de novo design of small-molecule-binding proteins with tuned interaction energies is now feasible entirely from computation. We expect these methods to open many opportunities in biomedicine, including rapid sensor development, antidote design, and drug delivery vehicles.

Covalent labeling of a chromatin reader domain using proximity-reactive cyclic peptides.

Chemical probes for chromatin reader proteins are valuable tools for investigating epigenetic regulatory mechanisms and evaluating whether the target of interest holds therapeutic potential. Developing potent inhibitors for the plant homeodomain (PHD) family of methylation readers remains a difficult task due to the charged, shallow and extended nature of the histone binding site that precludes effective engagement of conventional small molecules. Herein, we describe the development of novel proximity-reactive cyclopeptide inhibitors for PHD3-a trimethyllysine reader domain of histone demethylase KDM5A. Guided by the PHD3-histone co-crystal structure, we designed a sidechain-to-sidechain linking strategy to improve peptide proteolytic stability whilst maintaining binding affinity. We have developed an operationally simple solid-phase macrocyclization pathway, capitalizing on the inherent reactivity of the dimethyllysine ε-amino group to generate scaffolds bearing charged tetraalkylammonium functionalities that effectively engage the shallow aromatic 'groove' of PHD3. Leveraging a surface-exposed lysine residue on PHD3 adjacent to the ligand binding site, cyclic peptides were rendered covalent through installation of an arylsulfonyl fluoride warhead. The resulting lysine-reactive cyclic peptides demonstrated rapid and efficient labeling of the PHD3 domain in HEK293T lysates, showcasing the feasibility of employing proximity-induced reactivity for covalent labeling of this challenging family of reader domains.

Microbowls with Controlled Concavity for Accurate Microscale Mass Spectrometry.

Patterned surfaces can enhance the sensitivity of laser desorption ionization mass spectrometry by segregating and concentrating analytes, but their fabrication can be challenging. Here, a simple method to fabricate substrates patterned with micrometer-scale wells that yield more accurate and sensitive mass spectrometry measurements compared to flat surfaces is described. The wells can also concentrate and localize cells and beads for cell-based assays.

Exploration of α/ß/γ-peptidomimetics design for BH3 helical domains.

Systematic incorporation of ring-constrained ß- and γ-amino acid residues into α-helix mimetics engenders stable helical secondary structures. In this paper, functional α/ß/γ-helical peptidomimetics were explored for mimicry of BH3 helical domains, Bim as a pioneering study. The Bim-based α/ß/γ-peptides in an αγααßα-hexad repeat with five helical turns inhibited the interaction between Bak and Bcl-xL with excellent resistance towards proteolytic digestion. Further optimization of the α/ß/γ-backbone strategy will considerably expand the utility of functional α/ß/γ-peptidomimetics, in particular due to its prominent stability against proteolysis.

Dual antagonists of α5ß1/αvß1 integrin for airway hyperresponsiveness.

Inhibition of integrin α5ß1 emerges as a novel therapeutic option to block transmission of contractile forces during asthma attack. We designed and synthesized novel inhibitors of integrin α5ß1 by backbone replacement of known αvß1 integrin inhibitors. These integrin α5ß1 inhibitors also retain the nanomolar potency against αvß1 integrin, which shows promise for developing dual integrin α5ß1/αvß1 inhibitor. Introduction of hydrophobic adamantane group significantly boosted the potency as well as selectivity over integrin αvß3. We also demonstrated one of the inhibitors (11) reduced airway hyperresponsiveness in ex vivo mouse tracheal ring assay. Results from this study will help guide further development of integrin α5ß1 inhibitors as potential novel asthma therapeutics.

Design, Synthesis, and Study of Lactam and Ring-Expanded Analogues of Teixobactin.

This paper describes the chemical synthesis, X-ray crystallographic structure, and antibiotic activity assay of lactam analogues of teixobactin and explores ring-expanded analogues of teixobactin with ß3-homo amino acids. Lactam analogues of teixobactin containing all four stereoisomers of aza-threonine at position 8 were synthesized on a solid support from commercially available stereoisomeric threonine derivatives. The threonine stereoisomers are converted to the diastereomeric aza-threonines by mesylation, azide displacement, and reduction during the synthesis. d-Aza-Thr8,Arg10-teixobactin exhibits 2-8-fold greater antibiotic activity than the corresponding macrolactone Arg10-teixobactin. Azateixobactin analogues containing other stereoisomers of aza-threonine are inactive. A dramatic 16-128-fold increase in the activity of teixobactin and teixobactin analogues is observed with the inclusion of 0.002% of the mild detergent polysorbate 80 in the MIC assay. The X-ray crystallographic structure of N-Me-d-Gln4,d-aza-Thr8,Arg10-teixobactin reveals an amphipathic hydrogen-bonded antiparallel ß-sheet dimer that binds chloride anions. In the binding site, the macrolactam amide NH groups of residues 8, 10, and 11, as well as the extra amide NH group of the lactam ring, hydrogen bond to the chloride anion. The teixobactin pharmacophore tolerates ring expansion of the 13-membered ring to 14-,15-, and 16-membered rings containing ß3-homo amino acids with retention of partial or full antibiotic activity.

X-ray Crystallographic Structure of a Teixobactin Derivative Reveals Amyloid-like Assembly.

This paper describes the X-ray crystallographic structure of a derivative of the antibiotic teixobactin and shows that its supramolecular assembly through the formation of antiparallel ß-sheets creates binding sites for oxyanions. An active derivative of teixobactin containing lysine in place of allo-enduracididine assembles to form amyloid-like fibrils, which are observed through a thioflavin T fluorescence assay and by transmission electron microscopy. A homologue, bearing an N-methyl substituent, to attenuate fibril formation, and an iodine atom, to facilitate X-ray crystallographic phase determination, crystallizes as double helices of ß-sheets that bind sulfate anions. ß-Sheet dimers are key subunits of these assemblies, with the N-terminal methylammonium group of one monomer and the C-terminal macrocycle of the other monomer binding each anion. These observations suggest a working model for the mechanism of action of teixobactin, in which the antibiotic assembles and the assemblies bind lipid II and related bacterial cell wall precursors on the surface of Gram-positive bacteria.

Elucidation of the Teixobactin Pharmacophore.

This paper elucidates the teixobactin pharmacophore by comparing the arginine analogue of teixobactin Arg10-teixobactin to seven homologues with varying structure and stereochemistry. The roles of the guanidinium group at position 10, the stereochemistry of the macrolactone ring, and the "tail" comprising residues 1-5 are investigated. The guanidinium group is not necessary for activity; Lys10-teixobactin is more active than Arg10-teixobactin against Gram-positive bacteria in minimum inhibitory concentration (MIC) assays. The relative stereochemistry of the macrolactone ring is important. Diastereomer l-Thr8,Arg10-teixobactin is inactive, and diastereomer d-allo-Ile11,Arg10-teixobactin is less active. The macrolactone ring is critical; seco-Arg10-teixobactin is inactive. The absolute stereochemistry is not important; the enantiomer ent-Arg10-teixobactin is comparable in activity. The hydrophobic N-terminal tail is important. Truncation of residues 1-5 results in loss of activity, and replacement of residues 1-5 with a dodecanoyl group partially restores activity. These findings pave the way for developing simpler homologues of teixobactin with enhanced pharmacological properties.