Allosteric Activation Dictates PRC2 Activity Independent of Its Recruitment to Chromatin.

PRC2 is a therapeutic target for several types of cancers currently undergoing clinical trials. Its activity is regulated by a positive feedback loop whereby its terminal enzymatic product, H3K27me3, is specifically recognized and bound by an aromatic cage present in its EED subunit. The ensuing allosteric activation of the complex stimulates H3K27me3 deposition on chromatin. Here we report a stepwise feedback mechanism entailing key residues within distinctive interfacing motifs of EZH2 or EED that are found to be mutated in cancers and/or Weaver syndrome. PRC2 harboring these EZH2 or EED mutants manifested little activity in vivo but, unexpectedly, exhibited similar chromatin association as wild-type PRC2, indicating an uncoupling of PRC2 activity and recruitment. With genetic and chemical tools, we demonstrated that targeting allosteric activation overrode the gain-of-function effect of EZH2Y646X oncogenic mutations. These results revealed critical implications for the regulation and biology of PRC2 and a vulnerability in tackling PRC2-addicted cancers.

Improving Structural Stability and Anticoagulant Activity of a Thrombin Binding Aptamer by Aromatic Modifications.

The thrombin binding aptamer (TBA) is a 15-mer DNA oligonucleotide (5'-GGT TGG TGT GGT TGG-3'), that can form a stable intramolecular antiparallel chair-like G-quadruplex structure. This aptamer shows anticoagulant properties by interacting with one of the two anion binding sites of thrombin, namely the fibrinogen-recognition exosite. Here, we demonstrate that terminal modification of TBA with aromatic fragments such as coumarin, pyrene and perylene diimide (PDI), improves the G-quadruplex stability. The large aromatic surface of these dyes can π-π stack to the G-quadruplex or to each other, thereby stabilizing the aptamer. With respect to the original TBA, monoPDI-functionalized TBA exhibited the most remarkable improvement in melting temperature (ΔTm ≈+18 °C) and displayed enhanced anticoagulant activity.

Cucurbit[7]uril Inhibits Islet Amyloid Polypeptide Aggregation by Targeting N Terminus Hot Segments and Attenuates Cytotoxicity.

Two "hot segments" within an islet amyloid polypeptide are responsible for its self-assembly, which in turn is linked to the decline of ß-cells in type 2 diabetes (T2D). A readily available water-soluble, macrocyclic host, cucurbit[7]uril (CB[7]), effectively inhibits islet amyloid polypeptide (IAPP) aggregation through ion-dipole and hydrophobic interactions with different residues of the monomeric peptide in its random-coil conformation. A HSQC NMR study shows that CB[7] likely modulates IAPP self-assembly by interacting with and masking major residues present in the "hot segments" at the N terminus. CB[7] also prevents the formation of toxic oligomers and inhibits seed-catalyzed fibril proliferation. Importantly, CB[7] recovers rat insulinoma cells (RIN-m) from IAPP-assembly associated cytotoxicity.

Cucurbit[7]uril Inhibits Islet Amyloid Polypeptide Aggregation by Targeting N†Terminus Hot Segments and Attenuates Cytotoxicity.

Invited for the cover of this issue is the group of Prof. Hamilton at New York University. The image depicts how cucurbit[7]uril inhibits islet amyloid polypeptide self-assembly that rescues rat insulinoma cells (a pancreatic ß-cell model) from assembly-associated cytotoxicity. Read the full text of the article at 10.1002/chem.202200456.

Peptidomimetic-Based Vesicles Inhibit Amyloid-ß Fibrillation and Attenuate Cytotoxicity.

An interruption in Aß homeostasis leads to the deposit of neurotoxic amyloid plaques and is associated with Alzheimer's disease. A supramolecular strategy based on the assembly of peptidomimetic agents into functional vesicles has been conceived for the simultaneous inhibition of Aß42 fibrillation and expedited clearance of Aß42 aggregates. Tris-pyrrolamide peptidomimetic, ADH-353, contains one hydrophobic N-butyl and two hydrophilic N-propylamine side chains and readily forms vesicles under physiological conditions. These vesicles completely rescue both mouse neuroblastoma N2a and human neuroblastoma SH-SY5Y cells from the cytotoxicity that follows from Aß42 misfolding likely in mitochondria. Biophysical studies, including confocal imaging, demonstrate the biocompatibility and selectivity of the approach toward this aberrant protein assembly in cellular milieu.

α-Helix-Mimetic Foldamers for Targeting HIV-1 TAR RNA.

An oligopyridylamide-based foldamer approach has been employed to target HIV TAR RNA-TAT assembly as a model system to study RNA-protein interactions. The oligopyridylamide scaffold adopts a constrained conformation which presents surface functionalities at distinct spatial locations and mimic the chemical features of the secondary structure of proteins. We have designed a library of oligopyridylamides containing diverse surface functionalities which mimic the side chain residues of the TAT protein domain. The interaction of TAR RNA and TAT plays a pivotal role in facilitating HIV replication. The library was screened using various fluorescent based assays to identify antagonists of the TAR RNA-TAT complex. A tricationic oligopyridylamide ADH-19, possessed the highest affinity towards TAR and efficiently inhibited the TAR RNA-TAT interaction with apparent Kd of 4.1±1.0 µm. Spectroscopic studies demonstrated that ADH-19 interacts with the bulge and the lower bulge regions of TAR RNA, the domains important for TAT interaction. ADH-19 demonstrated appreciable in vivo efficacy (IC50 =25±1 µm) by rescuing TZM-bl cells infected with the pseudovirus HIV-1HXB-2.

Peptidomimetic-Based Multidomain Targeting Offers Critical Evaluation of Aß Structure and Toxic Function.

The prevailing hypothesis stipulates that the preamyloid oligomers of Aß are the main culprits associated with the onset and progression of Alzheimer's disease (AD), which has prompted efforts to search for therapeutic agents with the ability to inhibit Aß oligomerization and amyloidogenesis. However, clinical progress is impeded by the limited structural information about the neurotoxic oligomers. To address this issue, we have adopted a synthetic approach, where a library of oligopyridylamide-based small molecules was tested against various microscopic events implicated in the self-assembly of Aß. Two oligopyridylamides bind to different domains of Aß and affect distinct microscopic events in Aß self-assembly. The study lays the foundations for a dual recognition strategy to simultaneously target different domains of Aß for further improvement in antiamyloidogenic activity. The data demonstrate that one of the most effective oligopyridylamides forms a high affinity complex with Aß, which sustains the compound's activity in cellular milieu. The oligopyridylamide was able to rescue cells when introduced 24 h after the incubation of Aß. The rescue of Aß toxicity is potentially a consequence of the colocalization of the oligopyridylamide with Aß. The synthetic tools utilized here provide a straightforward strategic framework to identify a range of potent antagonists of Aß-mediated toxic functions. This approach could be a powerful route to the design of candidate drugs for various amyloid diseases that have so far proven to be "untargetable".

Teaching an old scaffold new recognition tricks: oligopyrrolamide antagonists of IAPP aggregation.

A library of N-substituted oligopyrrolamides was designed to modulate the aggregation kinetics of islet amyloid polypeptide (IAPP). IAPP is a hormonal peptide, co-secreted with insulin in the pancreatic ß-cells. IAPP samples a variety of conformations, starting from a native random coil to membrane-associated α-helical intermediates and eventually terminates in the amyloid plaques rich in ß-sheet structures. A growing body of evidence suggests that membrane bound α-helical intermediates are the key cytotoxic species that impair the functionality and viability of ß-cells and contribute to the onset of type 2 diabetes mellitus (DM2). The N-substituted oligopyrrolamides were screened against the aggregation of IAPP using amyloid kinetic assays. A tripyrrole, ADH-101, was the most effective antagonist of IAPP fibrillation in a physiologically relevant lipid membrane system as well as under de novo conditions. ADH-101 induces/stabilizes a secondary structure in IAPP which potentially affects its downstream functions. ADH-101 efficiently affects IAPP-mediated liposome leakage and cell toxicity in insulin secreting cells. ADH-101 inhibits the elongation process potentially binding to the monomeric IAPP and attenuating its access to the preformed fibers. More importantly, oligopyrrolamides are better inhibitors of IAPP aggregation than analogous oligopyridylamides and have more desirable biological properties reflected by their partition coefficients. In essence, an oligopyrrolamide scaffold has been designed which modulates the membrane bound helical intermediates of IAPP and affects their downstream functions such as oligomerization, membrane poration, and cytotoxicity.

α-Helix Mimetics as Modulators of Aß Self-Assembly.

A key molecular species in Alzheimer's disease (AD) is the Aß42 alloform of Aß peptide, which is dominant in the amyloid plaques deposited in the brains of AD patients. Recent studies have decisively demonstrated that the prefibrillar soluble oligomers are the neurotoxic culprits and are associated with the pathology of AD. Nascent Aß42 is predominantly disordered but samples α-helical conformations covering residues 15-24 and 29-35 in the presence of micelles and structure-inducing solvents. In this report, a focused library of oligopyridylamide based α-helical mimetics was designed to target the central α-helix subdomain of Aß (Aß13-26). A tripyridylamide, ADH-41, was identified as one of the most potent antagonists of Aß fibrillation. Amyloid-assembly kinetics, transmission electron microscopy (TEM), and atomic force microscopy (AFM) show that ADH-41 wholly suppresses the aggregation of Aß at a substoichiometric dose. Dot blot and ELISA assays demonstrate the inhibition of the putative neurotoxic Aß oligomers. ADH-41 targets Aß in a sequence and structure-specific manner, as it did not have any effect on the aggregation of islet amyloid polypeptide (IAPP), a peptide which shares sequence similarity with Aß. Spectroscopic studies using NMR and CD confirm induction of α-helicity in Aß mediated by ADH-41. Calorimetric and fluorescence titrations yielded binding affinity in the low micromolar range. ADH-41 was also effective at inhibiting the seed-catalyzed aggregation of Aß probably by modulating the Aß conformation into a fiber incompetent structure. Overall, we speculate that ADH-41 directs Aß into off-pathway structures, and thereby alters various solution based functions of Aß. Cell-based assays to assess the effect of ADH-41 on Aß are underway and will be presented in due course.

Foldamer-Mediated Structural Rearrangement Attenuates Aß Oligomerization and Cytotoxicity.

The conversion of the native random coil amyloid beta (Aß) into amyloid fibers is thought to be a key event in the progression of Alzheimer's disease (AD). A significant body of evidence suggests that the highly dynamic Aß oligomers are the main causal agent associated with the onset of AD. Among many potential therapeutic approaches, one is the modulation of Aß conformation into off-pathway structures to avoid the formation of the putative neurotoxic Aß oligomers. A library of oligoquinolines was screened to identify antagonists of Aß oligomerization, amyloid formation, and cytotoxicity. A dianionic tetraquinoline, denoted as 5, was one of the most potent antagonists of Aß fibrillation. Biophysical assays including amyloid kinetics, dot blot, ELISA, and TEM show that 5 effectively inhibits both Aß oligomerization and fibrillation. The antagonist activity of 5 toward Aß aggregation diminishes with sequence and positional changes in the surface functionalities. 5 binds to the central discordant α-helical region and induces a unique α-helical conformation in Aß. Interestingly, 5 adjusts its conformation to optimize the antagonist activity against Aß. 5 effectively rescues neuroblastoma cells from Aß-mediated cytotoxicity and antagonizes fibrillation and cytotoxicity pathways of secondary nucleation induced by seeding. 5 is also equally effective in inhibiting preformed oligomer-mediated processes. Collectively, 5 induces strong secondary structure in Aß and inhibits its functions including oligomerization, fibrillation, and cytotoxicity.

Unpicking the determinants of amide NHO[double bond, length as m-dash]C hydrogen bond strength with diphenylacetylene molecular balances.

Hydrogen bonding plays an essential part in dictating the properties of natural and synthetic materials. Secondary amides are well suited to cross-strand interactions through the display of both hydrogen bond donors and acceptors and are prevalent in polymers such as proteins, nylon, and Kevlar™. In attempting to measure hydrogen bond strength and to delineate the stereoelectronic components of the interaction, context frequently becomes vitally important. This makes molecular balances - systems in which direct comparison of two groups is possible - an appealing bottom up approach that allows the complexity of larger systems to be stripped away. We have previously reported a family of single molecule conformational switches that are responsive to diverse stimuli including Brønsted and Lewis acids, anions, and redox gradients. In this work we assess the ability of the scaffold, based on a 2,6-disubstituted diphenylacetylene, to measure accurately the difference in hydrogen bond strength between variously functionalised amides. In all of the examples investigated hydrogen bond strength closely correlate to measures of Brønstead acidity suggesting that the scaffold is well-suited as a platform for the accurate determination of bond strength in variously substituted systems.

Hybrid Diphenylalkyne-Dipeptide Oligomers Induce Multistrand ß-Sheet Formation.

Functionalized diphenylalkynes provide a template for the presentation of protein-like surfaces composed of multistrand ß-sheets. The conformational properties of three-, four-, and seven-stranded systems have been investigated in the solid- and solution-state. This class of molecule may be suitable for the mediation of therapeutically relevant protein-protein interactions.

A Modular Synthesis of Conformationally Preorganised Extended ß-Strand Peptidomimetics.

Invited for the cover of this issue is the group of Andrew D. Hamilton and Sam Thompson at the University of Oxford (UK). The image depicts a new class of conformationally constrained ß-strand mimetics mediating the interaction between two subunits of a protein that controls transcription. Read the full text of the article at 10.1002/chem.201501366.

A Modular Synthesis of Conformationally Preorganised Extended ß-Strand Peptidomimetics.

A promising strategy for mediating protein-protein interactions is the use of non-peptidic mimics of secondary structural protein elements, such as the α-helix. Recent work has expanded the scope of this approach by providing proof-of-principle scaffolds that are conformationally biased to mimic the projection of side-chains from one face of another common secondary structural element-the ß-strand. Herein, we present a synthetic route that has key advantages over previous work: monomers bearing an amino acid side-chain were pre-formed before rapid assembly to peptidomimetics through a modular, iterative strategy. The resultant oligomers of alternating pyridyl and six-membered cyclic ureas accurately reproduce a recognition domain of several amino acid residues of a ß-strand, demonstrated herein by mimicry of the i, i+2, i+4 and i+6 residues.

ß-Strand mimetic foldamers rigidified through dipolar repulsion.

Many therapeutically relevant protein-protein interactions contain hot-spot regions on secondary structural elements, which contribute disproportionately to binding enthalpy. Mimicry of such α-helical regions has met with considerable success, however the analogous approach for the ß-strand has received less attention. Presented herein is a foldamer for strand mimicry in which dipolar repulsion is a central determinant of conformation. Computation as well as solution- and solid-phase data are consistent with an ensemble weighted almost exclusively in favor of the desired conformation.

α-Helix mimetics: outwards and upwards.

α-Helices are common secondary structural elements forming key parts of the large, generally featureless interfacial regions of many therapeutically-relevant protein-protein interactions (PPIs). The rational design of helix mimetics is an appealing small-molecule strategy for the mediation of aberrant PPIs, however the first generation of scaffolds presented a relatively small number of residues on a single recognition surface. Increasingly, helices involved in PPIs are found to have more complex binding modes, utilizing two or three recognition surfaces, or binding with extended points of contact. To address these unmet needs the design and synthesis of new generations of multi-sided, extended, and supersecondary structures are underway.

Remote conformational control of a molecular switch via methylation and deprotonation.

Exacting control over conformation in response to an external stimulus is the central focus of molecular switching. Here we describe the synthesis of a series of diphenylacetylene-based molecular switches, and examine their response to covalent modification and deprotonation at remote phenolic positions. A complex interplay between multiple intramolecular hydrogen bond donors and acceptors determines the global conformation.

A Lewis acid-mediated conformational switch.

Molecules that change conformation in response to a stimulus have numerous uses, such as artificial chemoreceptors, novel drug delivery strategies and liquid crystal technology. Here we describe the design, synthesis and conformational behaviour of an isonicotinamide-substituted diphenylacetylene upon recognition of Lewis acids, including metalloporphyrins. Binding of these at a remote site - the pyridyl nitrogen - increases hydrogen-bond donor ability of the proximal amide NH, causing an increased preference for the alkyne rotamer in which this hydrogen bond is maintained.

Redox-dependent conformational switching of diphenylacetylenes.

Herein we describe the design and synthesis of a redox-dependent single-molecule switch. Appending a ferrocene unit to a diphenylacetylene scaffold gives a redox-sensitive handle, which undergoes reversible one-electron oxidation, as demonstrated by cyclic voltammetry analysis. (1)H-NMR spectroscopy of the partially oxidized switch and control compounds suggests that oxidation to the ferrocenium cation induces a change in hydrogen bonding interactions that results in a conformational switch.

Diphenylacetylene-linked peptide strands induce bidirectional ß-sheet formation.

In the search for synthetic mimics of protein secondary structures relevant to the mediation of protein-protein interactions, we have synthesized a series of tetrasubstituted diphenylacetylenes that display ß-sheet structures in two directions. Extensive X-ray crystallographic and NMR solution phase studies are consistent with these proteomimetics adopting sheet structures, displaying both hydrophobic and hydrophilic amino acid side chains.