K. Rajasekhar

Rationally Designed Peptidomimetic Modulators of Aβ Toxicity in Alzheimer's Disease

Alzheimers disease is one of the devastating illnesses mankind is facing in the 21st century. The main pathogenic event in Alzheimers disease is believed to be the aggregation of the β-amyloid (Aβ) peptides into toxic aggregates. Molecules that interfere with this process may act as therapeutic agents for the treatment of the disease. Use of recognition unit based peptidomimetics as inhibitors are a promising approach, as they exhibit greater protease stability compared to natural peptides. Here, we present peptidomimetic inhibitors of Aβ aggregation designed based on the KLVFF (P1) sequence that is known to bind Aβ aggregates. We improved inhibition efficiency of P1 by introducing multiple hydrogen bond donor-acceptor moieties (thymine/barbiturate) at the N-terminal (P2 and P3), and blood serum stability by modifying the backbone by incorporating sarcosine (N-methylglycine) units at alternate positions (P4 and P5). The peptidomimetics showed moderate to good activity in both inhibition and dissolution of Aβ aggregates as depicted by thioflavin assay, circular dichroism (CD) measurements and microscopy (TEM). The activity of P4 and P5 were studied in a yeast cell model showing Aβ toxicity. P4 and P5 could rescue yeast cells from Aβ toxicity and Aβ aggregates were cleared by the process of autophagy.

Natural Tripeptide-Based Inhibitor of Multifaceted Amyloid β Toxicity

Accumulation of amyloid beta (Aβ) peptide and its aggregates in the human brain is considered as one of the hallmarks of Alzheimers disease (AD). The polymorphic oligomers and fully grown fibrillar aggregates of Aβ exhibit different levels of neuronal toxicity. Moreover, aggregation of Aβ in the presence of redox-active metal ions like Cu(2+) is responsible for the additional trait of cellular toxicity induced by the generation of reactive oxygen species (ROS). Herein, a multifunctional peptidomimetic inhibitor (P6) has been presented, based on a naturally occurring metal chelating tripeptide (GHK) and the inhibitor of Aβ aggregation. It was shown by employing various biophysical studies that P6 interact with Aβ and prevent the formation of toxic Aβ forms like oligomeric species and fibrillar aggregates. Further, P6 successfully sequestered Cu(2+) from the Aβ-Cu(2+) complex and maintained it in a redox-dormant state to prevent the generation of ROS. P6 inhibited membrane disruption by Aβ oligomers and efficiently prevented DNA damage caused by the Aβ-Cu(2+) complex. PC12 cells were rescued from multifaceted Aβ toxicity when treated with P6, and the amount of ROS generated in cells was reduced. These attributes make P6 a potential therapeutic candidate to ameliorate the multifaceted Aβ toxicity in AD.

A High Affinity Red Fluorescence and Colorimetric Probe for Amyloid β Aggregates.

A major challenge in the Alzheimer’s disease (AD) is its timely diagnosis. Amyloid β (Aβ) aggregates have been proposed as the most viable biomarker for the diagnosis of AD. Here, we demonstrate hemicyanine-based benzothiazole-coumarin (TC) as a potential probe for the detection of highly toxic Aβ42 aggregates through switch-on, enhanced (~30 fold) red fluorescence (Emax = 654 nm) and characteristic colorimetric (light red to purple) optical outputs. Interestingly, TC exhibits selectivity towards Aβ42 fibrils compared to other abnormal protein aggregates. TC probe show nanomolar binding affinity (Ka = 1.72 × 107 M−1) towards Aβ42 aggregates and also displace ThT bound to Aβ42 fibrils due to its high binding affinity. The Aβ42 fibril-specific red-shift in the absorption spectra of TC responsible for the observed colorimetric optical output has been attributed to micro-environment change around the probe from hydrophilic-like to hydrophobic-like nature. The binding site, binding energy and changes in optical properties observed for TC upon interaction with Aβ42 fibrils have been further validated by molecular docking and time dependent density functional theory studies.

Aβ plaque-selective NIR fluorescence probe to differentiate Alzheimer's disease from tauopathies

Selective detection and staining of toxic amyloid plaques, a potential biomarker present in the Alzheimers disease (AD) brain is crucial for both clinical diagnosis and monitoring AD disease progression. Herein, we report a coumarin-quinoline (CQ) conjugate-based turn-on near-infrared (NIR) fluorescence probe for specific detection of β-amyloid (Aβ) aggregates. CQ probe is highly sensitive and exhibits ~100-fold fluorescence enhancement in vitro upon binding Aβ aggregates with enhanced quantum yield. Furthermore, the probe has ~10-fold higher binding affinity towards Aβ aggregates (86nM) compared to commonly used Thioflavin T. Most importantly, CQ probe displays unambiguous selectivity towards Aβ aggregates compared to other toxic protein aggregates such as tau, α-synuclein (α-Syn) and islet amyloid polypeptide (IAPP). In addition, CQ is nontoxic to neuronal cells and shows significant blood brain barrier permeability. Remarkably, CQ stains Aβ plaques in human brain tissue over co-existing tau aggregates and neurofibrillary tangles (NFTs), which are associated in AD and tauopathies. This is a highly desirable attribute to distinguish AD from tau pathology and mixed dementia.

Hybrid Multifunctional Modulators Inhibit Multifaceted Aβ Toxicity and Prevent Mitochondrial Damage

Amyloid beta (Aβ) aggregation is the key trait responsible for the pathological devastation caused by Alzheimers disease (AD). Among the various pathways of multifaceted toxicity exhibited by Aβ aggregates in neuronal cells, generation of reactive oxygen species (ROS) by Aβ-CuII complex and mitochondrial damage are prominent. Aβ interferes with mitochondrial transport channels, causing mitochondrial dysfunction. Herein, we present nontoxic hybrid multifunctional modulators (HMMs, TGR86-88) developed by integrating the structural and functional features of the metal chelating aggregation modulator, clioquinol (Clq), and the antioxidant epigallocatechin gallate (EGCG). Detailed biophysical and docking studies show that TGR86 interacts with Aβ and efficiently modulates both metal-dependent and metal-independent Aβ aggregation. TGR86 complexes with CuII, arrests its redox cycle, and thereby prevents the generation of ROS. The antioxidant nature of the HMMs effectively prevents DNA damage and protein oxidation. TGR86 rescued PC12 cells from Aβ-induced neurotoxicity by preventing the generation of ROS and foiling the interaction of toxic Aβ species with mitochondria, thereby averting its damage. These key attributes make TGR86 a potential candidate to develop therapeutics for the multifactorial Aβ toxicity in Alzheimers disease.

Current progress, challenges and future prospects of diagnostic and therapeutic interventions in Alzheimer's disease

Alzheimers disease (AD) is the most prevalent, progressive and multifaceted neurodegenerative disorder associated with cognition, memory and behavioural impairments. There is no approved diagnosis or cure for AD, and it affects both developed and developing countries and causes a significant social and economic burden. Extracellular senile plaques of amyloid beta (Aβ) and intracellular neurofibrillary tangles of phosphorylated Tau (pTau) in the brain are considered to be the pathophysiological hallmarks of AD. In an attempt to explain the complexity and multifactorial nature of AD, various hypotheses (Aβ aggregation, Tau aggregation, metal dyshomeostasis, oxidative stress, cholinergic dysfunction, inflammation and downregulation of autophagy) based on pathophysiological changes that occur during the onset and progression of AD have been proposed. However, none of the hypotheses is capable of independently explaining the pathological conditions observed in AD. The complex and multifaceted pathophysiological nature of AD has hampered the identification and validation of effective biomarkers for early diagnosis and the development of disease-modifying therapies. Nevertheless, the amyloid hypothesis is the most widely accepted and is closely correlated with disease symptoms of AD that encompass all the disease hypotheses. Therefore, amyloid plaques are ideal biomarkers for the development of an early diagnosis of AD. Similarly, the formation of amyloid plaques can also serve as a target for the design of therapeutic tools via an inclusive approach that considers multiple disease pathways involved in AD. Our review article briefly introduces pathophysiological factors involved in AD using interdependent but diverse hypotheses. Recent advances in the development of effective molecular tools and techniques for diagnostic and therapeutic interventions in AD, especially those in the advanced stages (clinical trials) of development, are given special consideration. In addition, contributions from our laboratory to the development of selective molecular tools for diagnostic and therapeutic interventions that target multifaceted toxicity in AD are also covered. In summary, we discuss diverse aspects of molecular mechanisms that underlie the pathogenesis of multifactorial AD, current progress and possible bottlenecks that have hampered the development of early diagnostic tools and effective drugs. Challenges and future prospects include the integration of various disease pathways for the successful development of an early diagnosis and effective drugs for the treatment of AD.