Early events during the aggregation of Aß16-22-derived switch-peptides tracked using Protein Charge Transfer Spectra.

BACKGROUND: Understanding Aß aggregation and inhibiting it at early stages is of utmost importance in treating Alzheimer's and other related amyloidogenic diseases. However, majority of the techniques to study Aß aggregation mainly target the late stages; while those used to monitor early stages are either expensive, use extrinsic dyes, or do not provide information on molecular level interactions. Here, we investigate the early events of Aß16-22(KLVFFAE) aggregation using Aß16-22 derived switch-peptides (SwPs) through a novel label-free approach employing Protein Charge Transfer Spectra (ProCharTS). RESULTS: When pH is increased from 2 to 7.2, the Aß-derived switch peptides undergo controlled self-assembly, where the initial random coil peptides convert into ß-sheet. We leveraged the intrinsic absorbance/luminescence arising from ProCharTS among growing peptide oligomers to observe the aggregation kinetics in real-time. In comparison to monomer, the lysine and glutamate headgroups in the peptide oligomer are expected to come in proximity enhancing ProCharTS intensity due to photoinduced electron transfer. With a combination of Aß-derived switch-peptides and ProCharTS, we obtained structural insights on the early stages of Aß-derived SwP aggregation in four unique peptides. Increase in scatter corrected ProCharTS absorbance (250-500 nm) and luminescence (320-720 nm) along with decreased mean luminescence lifetime (2.3-0.8 ns) characterize the initial stages of aggregation monitored for 1-96 h depending on the peptide. We correlated the results with Circular Dichroism (CD), 8-anilino-1-naphthalenesulfonic acid (ANS) and Thioflavin T (ThT) measurements. SIGNIFICANCE: We demonstrate ProCharTS as an intrinsic analytical probe with following advantages over other conventional methods to track aggregation: it is a label-free probe; it's intensity can be measured using a UV-Vis spectrophotometer; it is more sensitive in detecting the early molecular events in aggregation compared to ANS and ThT; and it can provide information on specific contacts made between charged headgroups of Lysine/Glutamate in the oligomer.

Copper chelating cyclic peptidomimetic inhibits Aß fibrillogenesis.

Misfolding of the amyloid-ß peptide (Aß) and its subsequent aggregation into toxic oligomers is one of the leading causes of Alzheimer's disease (AD). As a therapeutic approach, cyclic peptides have been modified in many ways and developed as a potential class of amyloid aggregation inhibitors. Head-to-tail cyclic peptides with alternating d, l amino acids inhibit amyloid aggregation significantly. On the other hand, excess deposition of copper, iron, and zinc enhances amyloid aggregation. Dysregulation of these metal ions in the brain triggers aggregation by binding to the Aß peptide. Therefore, specific metal chelators have been developed for disrupting the Aß-metal complex, thereby reducing toxicity and restoring metal ion homeostasis. Herein, we report the development of a head-to-tail cyclic peptidomimetic with a copper chelating ligand attached. The designed peptidomimetic inhibits amyloid aggregation significantly in a two-fold molar ratio to the Aß peptide, as confirmed by the thioflavin T (ThT) fluorescence assay, dynamic light scattering (DLS), transmission electron microscopy (TEM), and Congo-red stained birefringence studies. The chelating ligand attached to the cyclic peptide binds efficiently to Cu2+ but weakly to Zn2+ and Fe2+, thereby exhibiting profound selectivity, probed using UV-visible spectroscopy, thioflavin T (ThT) fluorescence assay, tyrosine (TYR10) fluorescence assay, isothermal titration calorimetry (ITC) and transmission electron microscopy (TEM). The non-toxicity of the designed peptidomimetics and their ability to reduce aggregating Aß-fragment induced cytotoxicity was confirmed by the MTT assay on the mouse neuronal cell line. Further, the molecular interaction between the peptidomimetics and the Aß-fragment was confirmed by Förster resonance energy transfer (FRET) studies using fluorescently labeled analogs. Cytotoxicity and cell internalization were also confirmed. A preliminary mechanistic investigation indicates that the peptidomimetic works by a synergistic effect of conformational restriction and metal sequestration. Such peptidomimetics can shed light on the mechanism of aggregation and a novel therapeutic approach.

Site-specific single point mutation by anthranilic acid in hIAPP8-37 enhances anti-amyloidogenic activity.

Amylin or hIAPP, together with insulin, plays a significant role in glucose metabolism. However, it undergoes ß-sheet rich amyloid formation associated with pancreatic ß-cell dysfunction leading to type-2 diabetes (T2D). Recent studies suggest that restricting ß-sheet formation in it may halt amyloid formation, which may limit the risk for the disease. Several peptide-based inhibitors have been reported to prevent aggregation. However, most of them have limitations, including low binding efficiency, active only at higher doses, poor solubility, and proteolytic degradation. Insertion of non-coded amino acids renders proteolytically stable peptides. We incorporated a structurally rigid ß-amino acid, Anthranilic acid (Ant), at different sites within the central hydrophobic region of hIAPP and developed two singly mutated hIAPP8-37 peptidomimetics. These peptidomimetics inhibited the amyloid formation of hIAPP substantially even at low concentration, as evident from in vitro ThT, CD, FT-IR, TEM, and Congo red staining birefringence results. These peptidomimetics also disrupted the preformed aggregates formed by hIAPP into non-toxic species. These ß-amino acid-based peptidomimetics can be an attractive scaffold for therapeutic design towards T2D or other protein misfolding diseases.

Peptidomimetics prepared by tail-to-side chain one component peptide stapling inhibit Alzheimer's amyloid-ß fibrillogenesis.

Alzheimer's disease (AD) is the most common form of dementia affecting the elderly population worldwide. Despite enormous efforts and considerable advancement in research, no therapeutic agents have come to light to date. However, many peptide-based and small molecule inhibitors interact efficiently with the amyloid-ß (Aß) peptide and alter its aggregation pathway. On the other hand, stapled peptides have been developed mainly to stabilize α-helix conformations and study protein-protein interactions. ß-Sheet stabilization or destabilization by stapled peptides has not been explored enough. Herein, we describe the generation of a library of "tail-to-side chain" stapled peptides via lactamization and their application for the first time as modulators of Aß1-40 self-association and fibrillogenesis. They also disrupt the preformed fibrillar aggregates into nontoxic species. Their stability in the presence of proteolytic enzymes is increased due to stapling. Therefore, the stapled peptides thus formed can be useful as potent amyloid aggregation inhibitors and pave a therapeutic pathway for combating amyloid-related diseases. Also, they may help in gaining insight into the process of aggregation.

An explicitly designed paratope of amyloid-ß prevents neuronal apoptosis in vitro and hippocampal damage in rat brain.

Synthetic antibodies hold great promise in combating diseases, diagnosis, and a wide range of biomedical applications. However, designing a therapeutically amenable, synthetic antibody that can arrest the aggregation of amyloid-ß (Aß) remains challenging. Here, we report a flexible, hairpin-like synthetic paratope (SP1, ∼2 kDa), which prevents the aggregation of Aß monomers and reverses the preformed amyloid fibril to a non-toxic species. Structural and biophysical studies further allowed dissecting the mode and affinity of molecular recognition events between SP1 and Aß. Subsequently, SP1 reduces Aß-induced neurotoxicity, neuronal apoptosis, and ROS-mediated oxidative damage in human neuroblastoma cells (SH-SY5Y). The non-toxic nature of SP1 and its ability to ameliorate hippocampal neurodegeneration in a rat model of AD demonstrate its therapeutic potential. This paratope engineering module could readily implement discoveries of cost-effective molecular probes to nurture the basic principles of protein misfolding, thus combating related diseases.

Sequence specificity of amylin-insulin interaction: a fragment-based insulin fibrillation inhibition study.

Subcutaneous insulin delivery serves as the major treatment for the ever-increasing spread of type II diabetes worldwide. However, long-term exposure to insulin results in local aggregates at the site of injection. This therapeutic concern accentuates the need to develop newer effective excipients to stabilize the insulin in pharmaceutical formulations. The fact that in normal physiological conditions, insulin interacts with the amylin hormone co-secreted from the pancreas, we targeted a peptide-mimetic approach based on the amylin sequence. The amylin-fibrillating core (NL6- N22FGAIL27 from the human Islet Amyloid Poly-Peptide) and its derivative NFGAXL (NL6X, X = 2-aminobenzoic acid) were used as potential inhibitory peptides against insulin amyloidogenesis. The fibrillation kinetics in the presence of the inhibitors was studied using an array of biophysical and microscopic techniques. High-resolution NMR spectroscopy enabled probing of the inhibitory interaction at an atomic resolution. Our results highlight the potential of using the naturally evolved NL6 peptide as an effective inhibitor against insulin fibrillation.

A Peptide Based Pro-Drug Ameliorates Amyloid-ß Induced Neuronal Apoptosis in In Vitro SH-SY5Y Cells.

BACKGROUND: Alzheimer's disease (AD), a common protein misfolding progressive neurodegenerative disorder, is one of the most common forms of dementia. Amyloid precursor protein (APP) derived amyloid-ß (Aß) protein accumulate into interneuronal spaces and plays a crucial role in the disease progression and its pathology. The aggregated Aß exerts its neurotoxic effects by inducing apoptosis and oxidative damage in neuronal cells. OBJECTIVES: We have investigated the effects of a synthesized Pro-Drug peptide (PDp) on Aß1-40 induced cytotoxicity in human neuroblastoma SH-SY5Y cells, represents one of the most effective strategies in combating human AD. METHODS: Cells were treated with Aß1-40 to induce cytotoxicity in the experimental model of AD to screen the inhibitory effect of PDp. Assays for cell viability, reactive oxygen species (ROS) generation, levels of intracellular free Ca2+ and expression of key apoptotic proteins were assessed by Western Blotting. RESULTS: Our results showed that Aß1-40 induces for 24h caused reduce cell viability, imbalance in Ca2+ homeostasis and increase in neuronal apoptosis in vitro. Treatment with PDp could effectively ameliorated Aß1-40 induced neurotoxicity and attenuates ROS generation that mediates apoptotic signaling through Bcl-2, Bax, Caspase-3 activity and cytochrome c in the cells. CONCLUSION: These findings suggested that PDp has potential role as a neuroprotective and therapeutic agent for combating human AD.

Disaggregation of Amylin Aggregate by Novel Conformationally Restricted Aminobenzoic Acid containing α/ß and α/γ Hybrid Peptidomimetics.

Diabetes has emerged as a threat to the current world. More than ninety five per cent of all the diabetic population has type 2 diabetes mellitus (T2DM). Aggregates of Amylin hormone, which is co-secreted with insulin from the pancreatic ß-cells, inhibit the activities of insulin and glucagon and cause T2DM. Importance of the conformationally restricted peptides for drug design against T2DM has been invigorated by recent FDA approval of Symlin, which is a large conformationally restricted peptide. However, Symlin still has some issues including solubility, oral bioavailability and cost of preparation. Herein, we introduced a novel strategy for conformationally restricted peptide design adopting a minimalistic approach for cost reduction. We have demonstrated efficient inhibition of amyloid formation of Amylin and its disruption by a novel class of conformationally restricted ß-sheet breaker hybrid peptidomimetics (BSBHps). We have inserted ß, γ and δ -aminobenzoic acid separately into an amyloidogenic peptide sequence, synthesized α/ß, α/γ and α/δ hybrid peptidomimetics, respectively. Interestingly, we observed the aggregation inhibitory efficacy of α/ß and α/γ BSBHps, but not of α/δ analogues. They also disrupt existing amyloids into non-toxic forms. Results may be useful for newer drug design against T2DM as well as other amyloidoses and understanding amyloidogenesis.

Protective effects of ß-sheet breaker α/ß-hybrid peptide against amyloid ß-induced neuronal apoptosis in vitro.

Alzheimer's disease is most common neurodegenerative disorder and is characterized by increased production of soluble amyloid-ß oligomers, the main toxic species predominantly formed from aggregation of monomeric amyloid-ß (Aß). Increased production of Aß invokes a cascade of oxidative damages to neurons and eventually leads to neuronal death. This study was aimed to investigate the neuroprotective effects of a ß-sheet breaker α/ß-hybrid peptide (BSBHp) and the underlying mechanisms against Aß40 -induced neurotoxicity in human neuroblastoma SH-SY5Y cells. Cells were pretreated with the peptide Aß40 to induce neurotoxicity. Assays for cell viability, cell membrane damage, cellular apoptosis, generation of reactive oxygen species (ROS), intracellular free Ca2+ , and key apoptotic protein levels were performed in vitro. Our results showed that pretreatment with BSBHp significantly attenuates Aß40 -induced toxicity by retaining cell viability, suppressing generation of ROS, Ca2+ levels, and effectively protects neuronal apoptosis by suppressing pro-apoptotic protein Bax and up-regulating antiapoptotic protein Bcl-2. These results suggest that α/ß-hybrid peptide has neuroprotective effects against Aß40 -induced oxidative stress, which might be a potential therapeutic agent for treating or preventing neurodegenerative diseases.

Phenolic ester mediated oligopeptide synthesis promoted by HOBt.

Although substituted phenolic ester mediated peptide synthesis is an efficient and well established method, the same via totally unsubstituted phenyl ester is not preferred due to the extremely slow rate of aminolysis. We have investigated the scope of the unsubstituted phenyl ester as an intermediate in peptide bond formation and found that it may be useful for the design of chemoselective peptide ligation when HOBt is used as an acyl transfer catalyst. The scope of HOBt catalyzed, oxo ester mediated ligation is explored for the synthesis of oligopeptides containing a cysteine, serine and threonine at the N-terminus of the ligating peptide.