Recent advances in the modulation of amyloid protein aggregation using the supramolecular host-guest approaches.

Misfolding of proteins is associated with many incurable diseases in human beings. Understanding the process of aggregation from monomers to fibrils, the characterization of all intermediate species, and the origin of toxicity is very challenging. Extensive research including computational and experimental shed some light on these tricky phenomena. Non-covalent interactions between amyloidogenic domains of proteins play a major role in their self-assembly which can be disrupted by designed chemical tools. This will lead to the development of inhibitors of detrimental amyloid formations. In supramolecular host-guest chemistry approaches, different macrocycles function as hosts for encapsulating hydrophobic guests, i.e. phenylalanine residues of proteins, in their hydrophobic cavities via non-covalent interactions. In this way, they can disrupt the interactions between adjacent amyloidogenic proteins and prevent their self-aggregation. This supramolecular approach has also emerged as a prospective tool to modify the aggregation of several amyloidogenic proteins. In this review, we discussed recent supramolecular host-guest chemistry-based strategies for the inhibition of amyloid protein aggregation.

Small molecule-based fluorescent probes for the detection of α-Synuclein aggregation states.

The formation of aggregates due to protein misfolding is encountered in various neurodegenerative diseases. α-Synuclein (α-Syn) aggregation is linked to Parkinson's disease (PD). It is one of the most prevalent neurodegenerative disorders after Alzheimer's disease. Aggregation of α-Syn is associated with Lewy body formation and degeneration of the dopaminergic neurons in the brain. These are the pathological hallmarks of PD progression. α-Syn aggregates in a multi-step process. The native unstructured α-Syn monomers combine to form oligomers, followed by amyloid fibrils, and finally Lewy bodies. Recent evidence suggests that α-Syn oligomerization and fibrils formation play major roles in PD development. α-Syn oligomeric species is the main contributor to neurotoxicity. Therefore, the detection of α-Syn oligomers and fibrils has drawn significant attention for potential diagnostic and therapeutic development. In this regard, the fluorescence strategy has become the most popular approach for following the protein aggregation process. Thioflavin T (ThT) is the most frequently used probe for monitoring amyloid kinetics. Unfortunately, it suffers from several significant drawbacks including the inability to detect neurotoxic oligomers. Researchers developed several small molecule-based advanced fluorescent probes compared to ThT for the detection/monitoring of α-Syn aggregates states. These are summarized here.

Inhibition of Amyloid Protein Aggregation Using Selected Peptidomimetics.

Aberrant protein aggregation leads to the formation of amyloid fibrils. This phenomenon is linked to the development of more than 40 irremediable diseases such as Alzheimer's disease, Parkinson's disease, type 2 diabetes, and cancer. Plenty of research efforts have been given to understanding the underlying mechanism of protein aggregation, associated toxicity, and the development of amyloid inhibitors. Recently, the peptidomimetic approach has emerged as a potential tool to modulate several protein-protein interactions (PPIs). In this review, we discussed selected peptidomimetic-based approaches for the modulation of important amyloid proteins (Islet Amyloid Polypeptide, Amyloid Beta, α-synuclein, mutant p53, and insulin) aggregation. This approach holds a powerful platform for creating an essential stepping stone for the vital development of anti-amyloid therapeutic agents.

Genetic Diversity and Structure of Persian Walnut (Juglans regia L.) in Pakistan: Implications for Conservation.

Persian (Common) walnut (Juglans regia L.) is a famous fruit tree species valued for its nutritious nuts and high-quality wood. Although walnut is widely distributed and plays an important role in the economy and culture of Pakistan, the genetic diversity and structure of its populations in the country remains poorly understood. Therefore, using 31 nuclear microsatellites, we assessed the genetic diversity and population structure of 12 walnut populations sampled across Pakistan. We also implemented the geostatistical IDW technique in ArcGIS to reveal "hotspots" of genetic diversity. Generally, the studied populations registered relatively low indices of genetic diversity (NA = 3.839, HO = 0.558, UHE = 0.580), and eight populations had positive inbreeding coefficient (FIS) values. Low among-population differentiation was indicated by AMOVA, pairwise FST and DC. STRUCTURE, PCoA and neighbor joining (NJ) analysis revealed a general lack of clear clustering in the populations except that one population in Upper Dir was clearly genetically distinct from the rest. Furthermore, the Mantel test showed no correlation between the geographic and genetic distance (r = 0.14, p = 0.22), while barrier analysis suggested three statistically significant genetic barriers. Finally, the spatial interpolation results indicated that populations in Ziarat, Kashmir, Dir, Swat, Chitral, and upper Dir had high intrapopulation genetic diversity, suggesting the need to conserve populations in those areas. The results from this study will be important for future breeding improvement and conservation of walnuts in Pakistan.

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.

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.

Selected peptide-based fluorescent probes for biological applications.

To understand the molecular interactions, present in living organisms and their environments, chemists are trying to create novel chemical tools. In this regard, peptide-based fluorescence techniques have attracted immense interest. Synthetic peptide-based fluorescent probes are advantageous over protein-based sensors, since they are synthetically accessible, more stable, and can be easily modified in a site-specific manner for selective biological applications. Peptide receptors labeled with environmentally sensitive/FRET fluorophores have allowed direct detection/monitoring of biomolecules in aqueous media and in live cells. In this review, key peptide-based approaches for different biological applications are presented.

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.

A Selective Cucurbit[8]uril-Peptide Beacon Ensemble for the Ratiometric Fluorescence Detection of Peptides.

A convenient supramolecular strategy for constructing a ratiometric fluorescent chemosensing ensemble, consisting of a macrocyclic host (cucurbit[8]uril CB[8]), and a pyrene-tagged amphiphilic peptide beacon (AP 1), is reported. AP 1 unfolds upon encapsulation of the pyrene termini into the hydrophobic CB[8] cavity. This changes pyrene excimer to monomer emission. Substrates with higher affinity for the CB[8] cavity can displace AP 1 from the ensemble. The released AP 1 folds again to form a pyrene excimer, which allows for the ratiometric fluorescence monitoring of the substrate. In this report, the ensemble capacity for ratiometric fluorescence monitoring of biological substrates, such as amino acid derivatives, specific peptides, and proteins, in aqueous media is demonstrated.

Arginine mimetic appended peptide-based probes for fluorescence turn-on detection of 14-3-3 proteins.

14-3-3 proteins are adaptor elements in intracellular signaling pathways. Recently, this protein family has been identified as a relevant therapeutic target involved in many human diseases. Therefore, identification of 14-3-3 proteins in biological systems is very important. Two cationic peptide-based probes are reported for the fluorescence detection of 14-3-3 proteins at physiological pH. The design of these probes consists of two symmetric peptidic arms equipped with a guanidiniocarbonyl pyrrole moiety (an arginine mimetic aka GCP), and an environment-sensitive amino-naphthalimide fluorophore as a third arm. These peptide sequences also contain lysine and phenylalanine/tryptophan amino acids for additional charge-charge and hydrophobic interactions. Both probes show high affinity and sensitivity for the 14-3-3 family, as well as good selectivity against other relevant biological proteins and ions.

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.

Correction: A metal-free fluorescence turn-on molecular probe for detection of nucleoside triphosphates.

Correction for 'A metal-free fluorescence turn-on molecular probe for detection of nucleoside triphosphates' by Debabrata Maity et al., Chem. Commun., 2017, 53, 208-211.

Peptide-Based Probes with an Artificial Anion-Binding Motif for Direct Fluorescence "Switch-On" Detection of Nucleic Acid in Cells.

This work reports two new peptide-based fluorescence probes (1 and 2) for the detection of ds-DNA at physiological pH. Probes 1 and 2 contain a fluorophore, either amino-naphthalimide or diethyl-aminocoumarin, respectively, and two identical peptide arms each equipped with a guanidiniocarbonylpyrrole (GCP) anion-binding motif. These probes show "switch-on" fluorescence response upon binding to ds-DNA, whereby they can differentiate between various types of polynucleotides. For instance, they exhibit more pronounced fluorescence response for AT-rich polynucleotides than GC-rich polynucleotides, and both give only negligible response to ds-RNA. The fluorimetric response of 1 is proportional to the AT-basepair content in DNA, whereas the fluorescence of 2 is sensitive to the secondary structure of the polynucleotide. Fluorescence experiments, thermal melting experiments and circular dichroism studies suggest that 1 interacts with ds-DNA in a combined intercalation and minor groove binding, whereas 2 interacts mainly with the outer surface of DNA/RNA. As 1 and 2 have a very low cytotoxicity, 1 can be applied for the imaging of nuclear DNA in cells.

A metal-free fluorescence turn-on molecular probe for detection of nucleoside triphosphates.

We report a fluorescence probe 1, which contains a naphthalimide fluorophore with two symmetric peptidic arms equipped with a tailor-made anion-binding motif, the guanidiniocarbonyl pyrrole moiety, for the detection of nucleoside triphosphates. Upon binding to nucleoside triphosphates, especially ATP, 1 shows significant turn-on fluorescence response. Probe 1 can also be applied for the imaging of ATP in cells.

Fluorescent Peptide Beacons for the Selective Ratiometric Detection of Heparin.

Heparin is extensively used as an anticoagulant drug during surgery. Two fluorophore-functionalized cationic oligopeptides HS 1 and HS 2 were developed to monitor heparin ratiometrically in aqueous media. Upon binding to heparin, HS 1 and HS 2 undergo a conformational change from an open form to a folded form, which leads to a distinct change in the fluorescence properties. HS 1 switches from pyrene monomer emission to an excimer emission. For HS 2, a fluorescence resonance energy transfer (FRET) process is enabled between a naphthalene donor and a dansyl acceptor. This method is highly selective for heparin relative to other similar biological analytes such as hyaluronic acid or chondroitin sulfate. HS 1 and HS 2 could also detect heparin ratiometrically in diluted bovine serum. The strong ratiometric emission color change can also be observed by the naked eye. Addition of the polycationic protein protamine releases both HS 1 and HS 2 from their heparin complex, which simultaneously restores pyrene monomer emission for the first case and decreases the FRET process for the latter case, respectively. Dynamic light scattering (DLS) and AFM studies confirm aggregate formation of heparin with HS 1 and HS 2.

A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids.

A cationic molecular peptide beacon NAP1 functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences. NAP1 most likely binds in a folded form into the minor groove of ds-DNA, which results in a remarkable change in its fluorescence properties. As NAP1 exhibits quite low cytotoxicity, it can also be used for imaging of nuclear DNA in cells.