Rational Design of TDP-43 Derived α-Helical Peptide Inhibitors: An In Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders.

TDP-43, an essential RNA/DNA-binding protein, is central to the pathology of neurodegenerative diseases, such as amyotrophic lateral sclerosis and frontotemporal dementia. Pathological mislocalization and aggregation of TDP-43 disrupt RNA splicing, mRNA stability, and mRNA transport, thereby impairing neuronal function and survival. The formation of amyloid-like TDP-43 filaments is largely facilitated by the destabilization of an α-helical segment within the disordered C-terminal region. In this study, we hypothesized that preventing the destabilization of the α-helical domain could potentially halt the growth of these pathological filaments. To explore this, we utilized a range of in silico techniques to design and evaluate peptide-based therapeutics that bind to pathological TDP-43 amyloid-like filament crystal structures and resist ß sheet conversion. Our computational approaches, including biophysical and secondary structure property prediction, molecular docking, 3D structure prediction, and molecular dynamics simulations, were used to assess the structure, stability, and binding affinity of these peptides in relation to pathological TDP-43 filaments. The results of our in silico analyses identified a selection of promising peptides which displayed a stable α-helical structure, exhibited an increased number of intramolecular hydrogen bonds within the helical domain, and demonstrated high binding affinities for pathological TDP-43 amyloid-like filaments. Molecular dynamics simulations provided further support for the structural and thermodynamic stability of these peptides, as they exhibited lower root-mean-square deviation and more favorable free energy landscapes over 300 ns. These findings establish α-helical propensity peptides as potential lead molecules for the development of novel therapeutics against TDP-43 aggregation. This structure-based computational approach for the rational design of peptide inhibitors opens a new direction in the search for effective interventions for ALS, FTD, and other related neurodegenerative diseases. The peptides identified as the most promising candidates in this study are currently subject to further testing and validation through both in vitro and in vivo experiments.

Rational Design of TDP-43 Derived α-Helical Peptide Inhibitors: an In-Silico Strategy to Prevent TDP-43 Aggregation in Neurodegenerative Disorders.

TDP-43, an essential RNA/DNA-binding protein, is central to the pathology of neurodegenerative diseases such as Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Pathological mislocalization and aggregation of TDP-43 disrupts RNA splicing, mRNA stability, and mRNA transport, thereby impairing neuronal function and survival. The formation of amyloid-like TDP-43 filaments is largely facilitated by the destabilization of an α-helical segment within the disordered C-terminal region. In this study, we hypothesized that preventing the destabilization of the α-helical domain could potentially halt the growth of these pathological filaments. To explore this, we utilized a range of in-silico techniques to design and evaluate peptide-based therapeutics. Various pathological TDP-43 amyloid-like filament crystal structures were selected for their potential to inhibit the binding of additional TDP-43 monomers to the growing filaments. Our computational approaches included biophysical and secondary structure property prediction, molecular docking, 3D structure prediction, and molecular dynamics simulations. Through these techniques, we were able to assess the structure, stability, and binding affinity of these peptides in relation to pathological TDP-43 filaments. The results of our in-silico analyses identified a selection of promising peptides, which displayed a stable α-helical structure, exhibited an increased number of intramolecular hydrogen bonds within the helical domain, and demonstrated high binding affinities for pathological TDP-43 amyloid-like filaments. Additionally, molecular dynamics simulations provided further support for the stability of these peptides, as they exhibited lower root mean square deviations in their helical propensity over 100ns. These findings establish α-helical propensity peptides as potential lead molecules for the development of novel therapeutics against TDP-43 aggregation. This structure-based computational approach for rational design of peptide inhibitors opens a new direction in the search for effective interventions for ALS, FTD, and other related neurodegenerative diseases. The peptides identified as the most promising candidates in this study are currently subject to further testing and validation through both in vitro and in vivo experiments.

Phase separation and pathologic transitions of RNP condensates in neurons: implications for amyotrophic lateral sclerosis, frontotemporal dementia and other neurodegenerative disorders.

Liquid-liquid phase separation results in the formation of dynamic biomolecular condensates, also known as membrane-less organelles, that allow for the assembly of functional compartments and higher order structures within cells. Multivalent, reversible interactions between RNA-binding proteins (RBPs), including FUS, TDP-43, and hnRNPA1, and/or RNA (e.g., RBP-RBP, RBP-RNA, RNA-RNA), result in the formation of ribonucleoprotein (RNP) condensates, which are critical for RNA processing, mRNA transport, stability, stress granule assembly, and translation. Stress granules, neuronal transport granules, and processing bodies are examples of cytoplasmic RNP condensates, while the nucleolus and Cajal bodies are representative nuclear RNP condensates. In neurons, RNP condensates promote long-range mRNA transport and local translation in the dendrites and axon, and are essential for spatiotemporal regulation of gene expression, axonal integrity and synaptic function. Mutations of RBPs and/or pathologic mislocalization and aggregation of RBPs are hallmarks of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease. ALS/FTD-linked mutations of RBPs alter the strength and reversibility of multivalent interactions with other RBPs and RNAs, resulting in aberrant phase transitions. These aberrant RNP condensates have detrimental functional consequences on mRNA stability, localization, and translation, and ultimately lead to compromised axonal integrity and synaptic function in disease. Pathogenic protein aggregation is dependent on various factors, and aberrant dynamically arrested RNP condensates may serve as an initial nucleation step for pathologic aggregate formation. Recent studies have focused on identifying mechanisms by which neurons resolve phase transitioned condensates to prevent the formation of pathogenic inclusions/aggregates. The present review focuses on the phase separation of neurodegenerative disease-linked RBPs, physiological functions of RNP condensates, and the pathologic role of aberrant phase transitions in neurodegenerative disease, particularly ALS/FTD. We also examine cellular mechanisms that contribute to the resolution of aberrant condensates in neurons, and potential therapeutic approaches to resolve aberrantly phase transitioned condensates at a molecular level.

An in silico approach to study the role of epitope order in the multi-epitope-based peptide (MEBP) vaccine design.

With different countries facing multiple waves, with some SARS-CoV-2 variants more deadly and virulent, the COVID-19 pandemic is becoming more dangerous by the day and the world is facing an even more dreadful extended pandemic with exponential positive cases and increasing death rates. There is an urgent need for more efficient and faster methods of vaccine development against SARS-CoV-2. Compared to experimental protocols, the opportunities to innovate are very high in immunoinformatics/in silico approaches, especially with the recent adoption of structural bioinformatics in peptide vaccine design. In recent times, multi-epitope-based peptide vaccine candidates (MEBPVCs) have shown extraordinarily high humoral and cellular responses to immunization. Most of the publications claim that respective reported MEBPVC(s) assembled using a set of in silico predicted epitopes, to be the computationally validated potent vaccine candidate(s) ready for experimental validation. However, in this article, for a given set of predicted epitopes, it is shown that the published MEBPVC is one among the many possible variants and there is high likelihood of finding more potent MEBPVCs than the published candidates. To test the same, a methodology is developed where novel MEBP variants are derived by changing the epitope order of the published MEBPVC. Further, to overcome the limitations of current qualitative methods of assessment of MEBPVC, to enable quantitative comparison and ranking for the discovery of more potent MEBPVCs, novel predictors, Percent Epitope Accessibility (PEA), Receptor specific MEBP vaccine potency (RMVP), MEBP vaccine potency (MVP) are introduced. The MEBP variants indeed showed varied MVP scores indicating varied immunogenicity. Further, the MEBP variants with IDs, SPVC_446 and SPVC_537, had the highest MVP scores indicating these variants to be more potent MEBPVCs than the published MEBPVC and hence should be preferred candidates for immediate experimental testing and validation. The method enables quicker selection and high throughput experimental validation of vaccine candidates. This study also opens the opportunity to develop new software tools for designing more potent MEBPVCs in less time.

Sequence Determinants of TDP-43 Ribonucleoprotein Condensate Formation and Axonal Transport in Neurons.

Mutations in TDP-43, a RNA-binding protein with multiple functions in RNA metabolism, cause amyotrophic lateral sclerosis (ALS), but it is uncertain how defects in RNA biology trigger motor neuron degeneration. TDP-43 is a major constituent of ribonucleoprotein (RNP) granules, phase separated biomolecular condensates that regulate RNA splicing, mRNA transport, and translation. ALS-associated TDP-43 mutations, most of which are found in the low complexity domain, promote aberrant liquid to solid phase transitions and impair the dynamic liquid-like properties and motility of RNP transport granules in neurons. Here, we perform a comparative analysis of ALS-linked mutations and TDP-43 variants in order to identify critical structural elements, aromatic and charged residues that are key determinants of TDP-43 RNP transport and condensate formation in neurons. We find that A315T and Q343R disease-linked mutations and substitutions of aromatic residues within the α-helical domain and LARKS, show the most severe defects in TDP-43 RNP granule transport and impair both anterograde and retrograde motility. F313L and F313-6L/Y substitutions of one or both phenylalanine residues in LARKS suggest the aromatic rings are important for TDP-43 RNP transport. Similarly, W334F/L substitutions of the tryptophan residue in the α-helical domain, impair TDP-43 RNP motility (W334L) or anterograde transport (W334F). We also show that R293A and R293K mutations, which disrupt the only RGG in the LCD, profoundly reduce long-range, directed transport and net velocity of TDP-43 RNP granules. In the disordered regions flanking the α-helical domain, we find that F283Y, F397Y or Y374F substitutions of conserved GF/G and SYS motifs, also impair anterograde and/or retrograde motility, possibly by altering hydrophobicity. Similarly, ALS-linked mutations in disordered regions distant from the α-helical domain also show anterograde transport deficits, consistent with previous findings, but these mutations are less severe than A315T and Q343R. Overall our findings demonstrate that the conserved α-helical domain, phenylalanine residues within LARKS and RGG motif are key determinants of TDP-43 RNP transport, suggesting they may mediate efficient recruitment of motors and adaptor proteins. These results offer a possible mechanism underlying ALS-linked TDP-43 defects in axonal transport and homeostasis.