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.

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.

Rationally designed helical peptidomimetics disrupt α-synuclein fibrillation.

Misfolding of the human protein α-synuclein results in toxic fibrils and the aggregation of Lewy bodies, which are a hallmark of Parkinson's disease in brain tissue. Here we disclose a supramolecular approach where peptidomimetics are rationally designed and pre-organised to recognize the surface of native helical α-Syn by forming complementary contacts with key patches of protein surface composed of charged and hydrophobic residues. Under lipid-catalyzed conditions the mimetics slow the rate of aggregation (thioflavin-T assay) and disrupt the misfolding pathway (electron microscopy of aggregates). This hypothesis is supported by comparison with a series of negative control compounds and with circular dichroism spectroscopy. Given the approach relies on selective recognition of both amino acid sequence and conformation (helical secondary structure) there is potential to develop these compounds as tools to unravel the currently intractable structure-function relationships of (i) missense mutation, and (ii) amyloid polymorphism with disease pathogenesis.

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.

The helical supramolecular assembly of oligopyridylamide foldamers in aqueous media can be guided by adenosine diphosphates.

A metal-free and achiral tri-pyridylamide foldamer, DM 11, containing a critical naphthalimide side chain self-assembles in a left-handed helical manner in the presence of chiral adenosine phosphates, under physiological conditions. Surprisingly, a very high degree of helicity in the foldamer assemblies was observed with ADP compared to other nucleoside phosphates, including ATP.

Protein mimetic amyloid inhibitor potently abrogates cancer-associated mutant p53 aggregation and restores tumor suppressor function.

Missense mutations in p53 are severely deleterious and occur in over 50% of all human cancers. The majority of these mutations are located in the inherently unstable DNA-binding domain (DBD), many of which destabilize the domain further and expose its aggregation-prone hydrophobic core, prompting self-assembly of mutant p53 into inactive cytosolic amyloid-like aggregates. Screening an oligopyridylamide library, previously shown to inhibit amyloid formation associated with Alzheimer's disease and type II diabetes, identified a tripyridylamide, ADH-6, that abrogates self-assembly of the aggregation-nucleating subdomain of mutant p53 DBD. Moreover, ADH-6 targets and dissociates mutant p53 aggregates in human cancer cells, which restores p53's transcriptional activity, leading to cell cycle arrest and apoptosis. Notably, ADH-6 treatment effectively shrinks xenografts harboring mutant p53, while exhibiting no toxicity to healthy tissue, thereby substantially prolonging survival. This study demonstrates the successful application of a bona fide small-molecule amyloid inhibitor as a potent anticancer agent.

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.

Antimicrobial Peptide Mimetics Based on a Diphenylacetylene Scaffold: Synthesis, Conformational Analysis, and Activity.

Mimics of natural antimicrobial peptides are promising compounds to fight the rising threat of multi-drug resistant bacteria. Here we report the design, synthesis and conformational analysis of a new class of antimicrobial peptide mimetics incorporating a diphenylacetylene scaffold. Within a small set of compounds, we observe a correlation between amphiphilicity, the efficiency of partitioning into negatively charged membranes and antibacterial activity. The most amphiphilic compound, which contains four isoleucine residues and four lysine residues, displays species-selective antibacterial activity (most active against Bacillus subtills) and low haemolytic activity. Solution-phase conformational analysis of this compound indicates that a defined structure is adopted in the presence of negatively charged phospholipid membranes and aqueous 2,2,2-trifluoroethanol but not in water. A conformation model indicates that the cationic and hydrophobic functional groups are segregated. These results may inform the development of highly selective antimicrobial peptide mimetics for therapeutic applications.

Sub-stoichiometric inhibition of IAPP aggregation: a peptidomimetic approach to anti-amyloid agents.

Membrane-catalysed misfolding of islet amyloid polypeptide is associated with the death of ß-cells in type II diabetes (T2D). Most active compounds so far reported require high doses for inhibition of membrane bound IAPP fibrillation. Here, we describe a naphthalimide-appended oligopyridylamide-based α-helical mimetic, DM 1, for targeting membrane bound IAPP. DM 1 completely inhibits the aggregation of IAPP at doses of 0.2 equivalents. DM 1 is also effective at similarly low doses for inhibition of seed-catalyzed secondary nucleation. An NMR based study demonstrates that DM 1 modulates IAPP self-assembly by stabilizing and/or perturbing the N-terminus helix conformation. DM 1 at substoichiometric doses rescues rat insulinoma cells from IAPP-mediated cytotoxicity. Most importantly, 0.2 equivalents of DM 1 disaggregate preformed oligomers and fibrils and can reverse cytotoxicity by modulating toxic preformed oligomers and fibrils of IAPP into non-toxic conformations.

Heterofunctionalized Cavitands by Macrocyclization of Sequence-Defined Foldamers.

Macrocyclic hosts have long been the workhorses of molecular recognition. Despite the widespread use of container-shaped molecules as synthetic receptors, an efficient preparation of cavitands bearing multiple functional groups has not been realized. This Letter describes a new cavitand derived from a sequence-defined oligoamide foldamer scaffold. A solid-phase synthesis approach is reported, which enables the display of multiple chemically diverse functional groups on the cavitand rim.

Dual Farnesyl and Geranylgeranyl Transferase Inhibitor Thwarts Mutant KRAS-Driven Patient-Derived Pancreatic Tumors.

PURPOSE: Mutant KRAS is a major driver of pancreatic oncogenesis and therapy resistance, yet KRAS inhibitors are lacking in the clinic. KRAS requires farnesylation for membrane localization and cancer-causing activity prompting the development of farnesyltransferase inhibitors (FTIs) as anticancer agents. However, KRAS becomes geranylgeranylated and active when cancer cells are treated with FTIs. To overcome this geranylgeranylation-dependent resistance to FTIs, we designed FGTI-2734, a RAS C-terminal mimetic dual FT and geranylgeranyltransferase-1 inhibitor (GGTI). EXPERIMENTAL DESIGN: Immunofluorescence, cellular fractionation, and gel shift assays were used to assess RAS membrane association, Western blotting to evaluate FGTI-2734 effects on signaling, and mouse models to demonstrate its antitumor activity. RESULTS: FGTI-2734, but not the selective FTI-2148 and GGTI-2418, inhibited membrane localization of KRAS in pancreatic, lung, and colon human cancer cells. FGTI-2734 induced apoptosis and inhibited the growth in mice of mutant KRAS-dependent but not mutant KRAS-independent human tumors. Importantly, FGTI-2734 inhibited the growth of xenografts derived from four patients with pancreatic cancer with mutant KRAS (2 G12D and 2 G12V) tumors. FGTI-2734 was also highly effective at inhibiting, in three-dimensional cocultures with resistance promoting pancreatic stellate cells, the viability of primary and metastatic mutant KRAS tumor cells derived from eight patients with pancreatic cancer. Finally, FGTI-2734 suppressed oncogenic pathways mediated by AKT, mTOR, and cMYC while upregulating p53 and inducing apoptosis in patient-derived xenografts in vivo. CONCLUSIONS: The development of this novel dual FGTI overcomes a major hurdle in KRAS resistance, thwarting growth of patient-derived mutant KRAS-driven xenografts from patients with pancreatic cancer, and as such it warrants further preclinical and clinical studies.

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.

Mimicry of a ß-Hairpin Turn by a Nonpeptidic Laterally Flexible Foldamer.

The design and characterization of a proteomimetic foldamer that displays lateral flexibility endowed by intramolecular bifurcated hydrogen bonds is reported. The MAMBA scaffold, derived from meta-aminomethylbenzoic acid, adopts a serpentine conformation that mimics the side chain projection of all four residues in a ß-hairpin turn.

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".

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.

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.

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.

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.

An α-helical peptidomimetic scaffold for dynamic combinatorial library formation.

A novel oligobenzamide-based α-helix mimetic was designed and synthesised with either imine or hydrazone functionalities that serve both to pre-organise the side-chain vectors to mimic the i, i + 4 and i + 7 residues of an α-helix, and to allow for the facile creation of dynamic libraries.