Protein aggregation and neurodegenerative disease: Structural outlook for the novel therapeutics

Abstract

Before the controversial approval of humanized monoclonal antibody lecanemab, which binds to the soluble amyloid‐β protofibrils, all the treatments available earlier, for Alzheimer's disease (AD) were symptomatic. The researchers are still struggling to find a breakthrough in AD therapeutic medicine, which is partially attributable to lack in understanding of the structural information associated with the intrinsically disordered proteins and amyloids. One of the major challenges in this area of research is to understand the structural diversity of intrinsically disordered proteins under in vitro conditions. Therefore, in this review, we have summarized the in vitro applications of biophysical methods, which are aimed to shed some light on the heterogeneity, pathogenicity, structures and mechanisms of the intrinsically disordered protein aggregates associated with proteinopathies including AD. This review will also rationalize some of the strategies in modulating disease‐relevant pathogenic protein entities by small molecules using structural biology approaches and biophysical characterization. We have also highlighted tools and techniques to simulate the in vivo conditions for native and cytotoxic tau/amyloids assemblies, urge new chemical approaches to replicate tau/amyloids assemblies similar to those in vivo conditions, in addition to designing novel potential drugs.

1. INTRODUCTION

Alzheimer's disease (AD) is considered as the most prevalent neurodegenerative disorder throughout the world. This progressive disease leads to dementia and cognitive impairment. It is the sixth leading cause of death in the United States. ¹ , ² AD is particularly prevalent in the elderly and an approximately over 50 million people suffer from this disease worldwide. ³ , ⁴ , ⁵ The late‐stage progression of AD worsens the patient's health and often leads to fatal consequences even when coupled with symptomatic treatment. ⁶ , ⁷ , ⁸ In α‐synucleinopathies, neuronal death and synaptic plasticity were characterized by abnormal accumulation of α‐synuclein aggregates. ⁹ Inflammation and synaptic dysfunction mechanistically interconnected in Parkinson's disease (PD), and supports the identification of reliable biomarkers for early diagnostics and development of therapies for PD. ⁹ , ¹⁰ , ¹¹ This acts as a substantial burden on the economy, healthcare, and social systems. ¹ , ¹² The Food and Drugs Administration (FDA) approved AD drugs give symptomatic relief to the patients but they do not cure the underlying cause. ⁴ , ¹³ Most of these drugs were developed based on the cholinergic ¹⁴ and glutamatergic hypothesis, where they function to balance the disturbances in the brain neurotransmitters release and help to alleviate cognitive decline. ³ , ⁸ , ¹⁴ , ¹⁵ , ¹⁶ Additionally, monoclonal antibodies against different amyloid‐β (Aβ), based on the amyloid hypothesis, moderately slows down the cognitive (mild) decline and reduces Aβ plaques, in patients with early stage of AD. ¹⁷ , ¹⁸ Therefore, there is a surge in demand for developing potential cost‐effective drugs and therapeutic approaches for the treatment of AD. These drugs should be able to interfere with or stop the pathways of heterogeneous pathological toxic oligomer formation and bottom‐line mechanisms and kinetics of AD or other proteinopathies based on the amyloid. ¹⁹ Development in the field of structural biology techniques provide detailed knowledge about the exact tau structures and conformations, populations, sizes, toxicity pathways, and posttranslational modifications. ²⁰ , ²¹ , ²² , ²³ , ²⁴ Furthermore, knowledge of protein–protein interactions, protein binding sites, kinetics of folding and aggregation, transient and native secondary, tertiary and quaternary structures, Aβ conformations found in vivo or in vitro is demanding. ²⁵ , ²⁶ , ²⁷ These goals can be achieved by the application of biophysical methods and imaging techniques particularly nuclear magnetic resonance (NMR), ²⁸ , ²⁹ , ³⁰ , ³¹ , ³² , ³³ dynamic light scattering, ³⁴ fluorescence resonance energy transfer (FRET) biosensors, ³⁵ , ³⁶ , ³⁷ cryogenic electron microscopy (cryo‐EM), ³⁸ , ³⁹ , ⁴⁰ , ⁴¹ , ⁴² x‐ray crystallography, ⁴³ , ⁴⁴ , ⁴⁵ transmission EM (TEM), ⁴⁶ and atomic force microscopy (AFM) ⁴⁷ , ⁴⁸ all of which will be addressed in this review (Figure 1). This review will also summarize the updates for small molecule inhibitors for amyloids associated with AD, PD ⁴⁹ , ⁵⁰ and will correlate the use of the essential methods for studying protein–protein interactions, and binding affinity of protein‐drugs ⁵¹ or ligands such as surface plasmon resonance (SPR), ⁵² bio‐layer interferometry (BLI), ⁵¹ , ⁵³ isothermal titration calorimetry (ITC), ⁵⁴ , ⁵⁵ and supported by molecular docking ⁵⁶ , ⁵⁷ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ (Table 1). Finally, all these techniques discussed here will provide the awaited structural information about the heterogeneous toxic protein aggregates in AD, ⁶² allow to mimic cytotoxic species in vivo or patient‐derived material, ²³ , ⁶³ accelerating the designing of small molecules and production of more effective disease‐modifying drugs and therapeutics.

FIGURE 1.

Schematic diagram showing the fate of amyloid β plaque and tau monomers surrounded by—a ring of structural and biophysical techniques used for the characterization of monomers, oligomers, and fibrils (first circle). Second layer of techniques (in square) are applied for high throughput screening of chemical library and measurement of affinity with target proteins. Outermost layer depicts structural characterization and affinity measurements outcomes leading to toxic clearance via immunotherapy, and gene therapy.

TABLE 1.

Structural characterization of proteins and ligands using common structural and biophysical techniques.

Methods	Techniques	Properties	References
In vitro	Fluorescence probes—ThT Congo red and ANS	Quantification of amyloids	93, 94, 95, 96
	CD and FTIR	Secondary structure	97, 98, 99, 100
	ITC	Binding affinity and stoichiometry	101, 102, 103
	NMR	Structures, interactions, and dynamics	104, 105, 106
	X‐ray crystallography	Atomic structures	107, 108, 109, 110
	Cryo‐EM	Atomic structures	109, 111, 112, 113, 114, 115, 116
	SAXS	Structure	117
	TEM, AFM	Quaternary structure and surface feature	118
In vivo	Fluorescent reporter assay	Extent of folding in cell	119

Abbreviations: AFM, atomic force microscopy; cryo‐EM, cryogenic electron microscopy; ITC, isothermal titration calorimetry; NMR, nuclear magnetic resonance; TEM, transmission electron microscopy; ThT, thioflavin T; SAXS, small‐angle x‐ray scattering.

2. PROTEIN AGGREGATION AND NEURODEGENERATIVE DISEASE

Aβ is produced by the proteolytic action of secretases (β and γ) on amyloid precursor protein (APP). ⁶⁴ , ⁶⁵ Aβ accumulation particularly Aβ42 is the predominant protein species found in the senile plaque of AD patients' brains. ⁶⁶ , ⁶⁷ , ⁶⁸ , ⁶⁹ Several kinetic interrogations have revealed that once Aβ42 monomers concentration achieves critical point, it leads to the assembly of soluble oligomers. ⁷⁰ , ⁷¹ , ⁷² Aβ42 oligomers are neurotoxic ⁷³ as well as they stimulate tau pathogenicity. ⁷⁴ Therefore, development of therapeutic strategies focuses on reducing the toxic form of Aβ42, either by targeting secretase, amyloid clearance, or stabilizing the monomeric form. ⁷³ , ⁷⁵ , ⁷⁶ , ⁷⁷ , ⁷⁸ Aβ lacks well defined structure (intrinsically disordered) and exists as heterogeneous assembly of distinct conformations. ⁷⁹ Targeting intrinsically disordered proteins (IDP; e.g., Aβ, tau, α‐syn) will lead in part to the failure of conventional drugs development which follow lock and key interaction hypothesis. ⁸⁰ , ⁸¹ Search for novel therapeutics against AD and other neurodegenerative disorders require information about interaction and mechanism of action of therapeutics to alter or clear the toxic forms of tau/Aβ. ⁷⁹ , ⁸¹ For the screening and characterization of the therapeutics, various biophysical, computational, and mathematical modeling techniques, are frequently used. ⁸² Researchers working in this particular aspect are keenly interested to find drug candidates which specifically act by binding and stabilizing the IDP monomer ⁸³ , ⁸⁴ and thus significantly reduce the nucleation pathways. Successful stabilization of the monomeric IDP (tau/Aβ) can be achieved by interaction with the small molecules that act as aggregation inhibitors. The small molecule aggregation inhibitors can be screened and developed on the basis of the critical domains responsible for oligomerization of these IDPs. ⁸² , ⁸⁵

Tau is a microtubule (MT)‐associated protein that is distributed in the axonal region of the neurons of healthy people but is sorted in the somatodendritic compartment in tauopathy patients including AD. ⁸⁶ , ⁸⁷ Tau plays a role in regulating enzymatic activity of kinases and phosphatases through localization and signal transduction mechanisms. ⁸⁸ , ⁸⁹ Mutations in tau protein are associated with conformational changes, changes in isoform composition, changed affinity to the MTs, altered posttranslational modifications and increased aggregation propensity. ⁹⁰ AD is closely associated with pathologic tau modification such as phosphorylation of specific residues and oligomerization. ⁹¹ Although tau toxicity is mainly enhanced by posttranslational modification and the mechanism behind this still remains unclear. ⁹²

3. STRUCTURAL CHARACTERIZATION OF DISORDERED PROTEINS AND SMALL MOLECULES AS DRUG

3.1. X‐ray crystallography

Atomic level structure information of drug targets, designing, and optimization of lead molecules makes structural biology a powerful tool for drug discovery in human healthcare. ¹⁰⁷ , ¹⁰⁸ Structure determination of proteins and molecular complexes are limited to size of around 10–150 kDa as bulkier proteins, including membrane proteins, therefore it becomes difficult to crystallize and hence only crystal structures of few large size (greater than 150 kD) proteins are available. ¹⁰⁹ , ¹¹⁰ Complete structures of protein (with high resolution) are required for structure‐based drug design. X‐ray crystallography provides for most of the protein structures needed for drug target except complex and membrane proteins which are difficult to crystallize. This drawback of x‐ray crystallography is now compensated by advances in cryo‐EM technique. ¹¹³ , ¹²⁰ , ¹²¹

Proteins such as Aβ, tau, and α‐synuclein are the common IDPs; therefore, crystallization and structural determination of the monomeric forms of these proteins is extremely challenging. ²¹ , ⁶⁴ , ¹²² Three‐dimensional structures determined by x‐ray crystallography provide more detailed information on the structure of Aβ. Recent updates of x‐ray crystallographic structural investigations of amyloids are showing that β‐sheet dimers of a β‐Hairpin derived from Aβ16–36 reorganize to tetrameric forms, which could be used as in vitro and in vivo models for full length brain derived Aβ for further investigations including drug discovery. ¹²³ , ¹²⁴ , ¹²⁵ These crystal structures are aiding search for better small molecules in the form of novel chemical compounds, monoclonal antibodies and peptidomimetic construct that target amyloid aggregates. ¹²⁵

3.2. Cryo‐EM

Presently, structural information about large macromolecules (>100 kD) and amyloids are primarily determined by cryo‐EM up to 2 Å resolution. ¹²⁶ This can be achieved even with small molecules due to the advancements in cryo‐EM technology. ¹⁰⁹ The advantages of simple and time resolved cryo‐EM over x‐ray diffraction technique are time saving for crystallization, smaller sample requirement, can cope with noncrystalline, higher size limit and ability to capture multiple highly dynamic protein structural conformations in solution. ¹¹¹ , ¹¹² These advantages of cryo‐EM have facilitated the development of drug targeting large and membrane protein family which is not possible through x‐ray crystallography. ¹¹³ , ¹¹⁴ , ¹¹⁵ , ¹¹⁶ Cryo‐EM is also contributing to the fragment‐based drug development as demonstrated by Saur et al., by his pilot study of β‐galactosidase and pyruvate kinase isozyme M2. ¹²⁷

Nanoparticle labeled amyloid fibrils can be used for direct detection of amyloid fibrils from ex vivo sample and rapid profiling of polymorphism using cryo‐EM. ¹²⁸ Cryo‐EM structure of brain derived Aβ fibrils are found to be right‐handed twisted polymorphic form but similar to protofilaments. ¹²⁹ The mechanism of amyloid filament formation is disclosed and explained by the available high‐throughput Cryo‐EM structure of 76 different filaments. ²³ Disordered proteins self‐assemble to form diverse higher order aggregates as well as amyloid fibrils. ¹¹⁹ Initially, large insoluble fibrils were known to be the primary factors associated for neurotoxicity in disease such as AD. ¹³⁰ Recently, by the development of advance in vitro and in vivo techniques, it has demonstrated that smaller prefibrillar aggregates are the major toxic species in many neurodegenerative diseases. ¹³¹ , ¹³² , ¹³³ , ¹³⁴ Cryo‐EM structure of paired helical filament (PHF) and straight filament associated with AD, ⁶³ , ¹³⁵ Pick's disease, ¹³⁶ corticobasal degeneration, ¹³⁷ and chronic traumatic encephalopathy ¹³⁸ has been determined. Cryo‐EM structure of in vitro tau filament assembly provides an optimistic model system to investigate the formation of different tau folds that define distinct tauopathies. The recent attainment for reporting fabrication of recombinant tau (297–391) into filaments identical to those of AD and chronic traumatic encephalopathy will play a crucial role in future investigations into the structural diversity of amyloids ²³ and understanding the mechanism behind tauopathies. ³⁸ Reweighted hierarchical chain growth (RHCG) algorithm method is a computationally common efficient alternative for MD where it combines pre‐sampled chain fragments in a statistically reproducible manner into ensembles of full‐length atomically detailed biomolecular structures. ¹³⁹ RHCG was used to determine global structure of tau K18 protein by emerging from the local structure. RHCG integrates experimental data on local structure into the assembly process in a systematic manner and combines Bayesian ensemble refinement with importance sampling to end up with well‐defined structures. ²¹

Structural information about IDPs including tau conformations in solutions has been made by small‐angle x‐ray scattering. ¹¹⁷ Several studies investigated amyloid aggregation based on direct monitoring of oligomers, the slow processes of IDPs, protein dynamics, and intramolecular distance distributions using single molecule FRET (smFRET). ¹⁴⁰ , ¹⁴¹ , ¹⁴² , ¹⁴³ , ¹⁴⁴ Also, more information about conformational distributions and secondary structures identification of intrinsically disordered and partially folded proteins is obtained by using NMR spectroscopy. ¹⁰⁴ , ¹⁰⁵ , ¹⁰⁶

3.3. Isothermal titration calorimetry

ITC is an irreplaceable tool and is considered a gold standard for the estimation of thermodynamic parameters associated with protein–drug interactions and provides idea about molecular interaction mechanisms without any requirement for labeling. ¹⁴⁵ , ¹⁴⁶ Measurement of the affinity of ligand for the target protein is crucial for the selection of hit to lead optimization and hence is essential in the field of drug development. ¹⁰¹ , ¹⁰² , ¹⁰³ Affinity parameters provide the driving force responsible for association of ligand, whereas the kinetic parameters of ligand–protein interactions provide residence time which measures the lifetime of the drug‐target complex, where both kinetic and thermodynamic profiling of compounds could be proposed as predictive tool for selectivity of compounds, and efficacy of drugs in vivo. ¹⁴⁷ , ¹⁴⁸ , ¹⁴⁹ Novel therapeutic approaches acquire knowledge about protein–protein interactions between Aβ and its binding partners including tau and ApoE protein which has been possible by ITC. ¹⁵⁰ , ¹⁵¹ , ¹⁵² The interaction of Aβ42 with Na,K‐ATPase and subsequent oligomerization leads to inhibition of the enzyme activity in brain of AD patients and contributes to AD pathogenesis. ⁵⁴ The measured thermodynamic parameters for the formation of Na,K‐ATPase: Aβ42 complex at different conformations acquired by ITC are alike, which is in line with the data of MD. Similarity of all conformations of Na,K‐ATPase interaction interfaces with Aβ allowed to cross‐screen potential inhibitors for this interaction and find pharmaceutical compounds including ZINC153412538, ZINC153412622, and ZINC153412744 that can block it. ⁵⁴

3.4. Surface plasmon resonance

SPR technique is based on immobilization of one binding partner with biosensor. Quantitative estimation of interactions between biomolecules using SPR and BLI is a highly reliable technique in comparison with other techniques including ELISA and ITC. This is because of the ability to work in real time as well as through a high throughput method. ⁵² , ¹⁵³ Protein complexes associated with neuropathies such as Aβ, tau and their interacting partners, or ligands as lead molecules have been studied using SPR. Recent work reported that ApoE is binding laterally along the amyloid fibrils. ¹⁵⁴ , ¹⁵⁵ Immobilization procedures of monomeric or fibrillar form of amyloidogenic proteins on a sensor chip are different including covalent immobilization, high affinity capture binding, and hydrophobic attachment but can be used by slight modifications depending on the purposes of investigation to facilitate attachment and enhance signal. ¹⁵⁶ , ¹⁵⁷ , ¹⁵⁸ , ¹⁵⁹ SPR is a sensor based technique and could suffer from nonspecific binding which could be minimized and eliminated by careful experimental design, such as reducing the expected binding sites concentration. ¹⁶⁰ Zheng et al. developed Au nanoparticle‐based dual aptamer‐based SPR biosensor to detect picomolar concentrations of Aβ40 oligomers and fibrils. ¹⁵⁶

3.5. Biolayer interferometry

The fundamental importance of cellular function/dysfunction is associated with intra‐ and inter‐protein associations including various other biochemical reactions such as enzymatic reactions. Studies of multivalent interactions associated with protein functions and exploration in the field of novel therapeutics can be possible either by immobilization or fluorescence labeling based interactive modality techniques. ¹⁴⁶ BLI is reliable for high affinity multimeric peptides as it is independent of concentration for immobilization and measurements in contrast to low affinity dimeric peptides working in concentration dependent manner and high signal to noise ration. ¹⁴⁶ , ¹⁶¹ In a high throughput screening study employing BLI and other techniques for active small molecules from natural extract against Aβ42, potential amyloid inhibitors were identified including baicalein, polyporenic acid C, and other components which accelerates the development of therapeutics for AD. ⁵¹ , ⁸² , ¹⁶²

3.6. NMR spectroscopy

As a significant outcome of recent advances and breakthroughs in NMR spectroscopy including instrumentation, isotope labeling, pulse sequences, ³⁰ 2D methods, ²⁹ and solid‐state NMR probes; NMR has become a significant tool for studying dynamic protein complexes, IDPs structures (TDP‐43) and specific conformations which are hard to be obtained with other techniques. ¹⁶³ , ¹⁶⁴ NMR is a versatile tool where it encompasses solid‐state NMR at the atomic structure level, solution NMR for dynamic molecular structures and the in vivo biochemistry of proteins (hemoglobin, TDP‐43), ³¹ in addition to the membrane and cellular level for structural biology. ²⁸ , ³² , ¹⁶⁴ Recent NMR studies provided deeper insights into the protein's functional mechanisms, tau filaments specific structures, Aβ‐40 oligomers cross seeded by Aβ‐42 oligomers motif srtuctres. ¹⁶⁵ , ¹⁶⁶ Recent studies are indicating the ability of NMR for binding affinity of ligand‐proteins through the NMR coupling constants and chemical shifts changes, ¹⁶⁷ aiding to depict oligomeric intermediates in an amyloid assembly. ¹⁶⁸ Recently, solid‐state ¹⁹F‐NMR underlined the recognition of the binding sites for a positron emission tomography (PET) tracer to Aβ40. ¹⁶⁹ Overall NMR is providing and revealing gap knowledge and current understanding of the neurodegenerative diseases causing structures and underlying mechanisms in addition to aiding to design aggregation inhibitors. ¹⁶⁸

3.7. Characterization of amyloid deposits in living cells

In vivo time resolved monitoring of cellular and molecular events became accessible after the emergence of fluorescent protein that is sensitive toward cellular conditions as pH, stability, photoreactivity, and biosensors. ¹⁷⁰ , ¹⁷¹ , ¹⁷² Real‐time and accurate information about cerebral Aβ deposition which is pathological hallmark of AD, and its noninvasive early diagnostics became possible only with fluorescence imaging, ¹⁷³ , ¹⁷⁴ , ¹⁷⁵ PET, ¹⁶⁹ and MRI. ¹⁷⁶ Tao et al. developed ultrasensitive probe AH2, a derivative of Thioflavin T for the fluorescence‐based early detection and spatial distribution of Aβ deposition in mice brain as well as access the efficacy of antiaging medicines. ¹⁷⁷ For conformational dynamics and biomolecular interaction at scale of 1–10 nm distance in steady‐state condition, smFRET has been extensively used based on transfer of energy between two chromophores. ³⁵ Advantages of smFRET over other structural techniques includes high sensitivity, quality, structural transition measurement in equilibrium, high specificity to labeled domain of macromolecules, ¹⁷⁸ cell organelles ¹⁷⁹ even whole native cells. ¹⁸⁰ , ¹⁸¹ For successful drug discovery landscape, it became demanding to have meticulous knowledge about disease relevant amyloids at early stages of proteinopathies, for better drug protein interactions which is achieved by applying in vivo FRET. Hippocampal neurons were reported to contain Aβ in the early stages of AD. MircoRNA‐125b(miR‐125b) can be a promising biomarker for the detection of early AD as it is associated with tau hyperphosphorylation using biosensor for miR‐125b. ¹⁸² , ¹⁸³

4. AMYLOID POLYMORPHISM

Large numbers of evidence are now pointing out toward the amyloid polymorphism accountable for the occurrence of different strains of amyloids with varied toxicity and pathological spreading. ¹²⁸ Structural polymorphism in amyloid forming protein is now recognized as common property. ¹⁸⁴ , ¹⁸⁵ , ¹⁸⁶ Prion protein with abnormal β‐sheet rich form (PrP^sc) is hallmark of prion disease deposited in brain by autocatalytic conversion of PrP^c triggered by lipid and small RNA. ¹⁸⁷ Molecular structure of self‐assembled amyloid fibrils and aggregates of monomeric proteins differing in structural composition are responsible for existence of multiple strains of prion diseases which causes variation in the disease toxicity. ¹⁸⁸ , ¹⁸⁹ , ¹⁹⁰ , ¹⁹¹ In the case of AD and other amyloid disease, pattern of deposition of amyloid fibrils is responsible for generation of different strains. ¹⁹² Paravastu et al. describe the structural model of periodically twisted fibrils formed by Aβ‐40 by solid‐state NMR and TEM associated with AD also known as twisted pair morphology. ¹⁹³ Tau and α‐synuclein are now being considered as prion like because of their inherent templating property for misfolding and aggregation of monomeric proteins and ability to form distinct stains. ¹⁹⁴ Different downstream effects have been observed due to tau aggregates having conformational differences. ¹⁹⁵ , ¹⁹⁶ From the transsynaptic pattern of tau seed spreading, it has been proposed that tau protein also follow prion‐like mechanism of disease propagation. ¹⁹⁷ , ¹⁹⁸ On the basis of atomic structure of amyloid like fibers, three different models have been proposed and illustrated in Figure 2, ¹⁸⁴ : (i) packing polymorphism, amyloid segment arranged in a pair of zipper connected by double headed arrow results in different structural and function properties; (ii) segmental polymorphism in which steric zipper spines are formed by two or more amyloid proteins; (iii) heterosteric zipper is formed by on‐identical beta sheets through interdigitation. Models derived from solid‐state NMR and cryo‐EM are best describing the heterotypic interactions between sheets. ¹⁸⁴ , ¹⁹⁹ , ²⁰⁰ , ²⁰¹

FIGURE 2.

Three different models for the Tau amyloid polymorph: (A) Packing polymorphism (PDB: 2ON9 and 4NP8), (B) Segmental polymorphism (PDB: 2Y3J), and (C) Steric zipper (PDB: 3FVA) protofilament based on composition and pattern of amyloid segments.

5. PREVENTIVE MEASURE FOR AD PROGRESSION

Despite the enormous efforts and extensive research for finding novel therapeutics to prevent, halt or reverse the progression of AD, ²⁰² still substantially cureless. ⁴ , ²⁵ , ²⁰³ , ²⁰⁴ , ²⁰⁵ , ²⁰⁶ There are still no effective neurodegenerative diseases therapeutics that are able to halt or counteract the disease progression and impairment of mental activities but have short term symptoms of reliving from memory loss and thinking complications. ⁴ , ¹⁶ , ²⁰⁷ Most of these suggested medications and strategies ended with disappointments in early stages of development or in clinical trials. ⁶ , ⁷ , ⁸ , ¹⁷ , ¹⁹ , ²⁰⁵ , ²⁰⁸ , ²⁰⁹ , ²¹⁰ , ²¹¹ , ²¹² , ²¹³ Many previously reported reviews herein discussed thoroughly immunotherapy against tau and amyloids and related monoclonal antibodies as a future therapy. ⁸ , ¹³ , ¹⁷ , ¹⁹ , ³⁹ , ²⁰⁴ , ²⁰⁵ , ²⁰⁹ , ²¹¹ , ²¹² , ²¹³ , ²¹⁴ , ²¹⁵ , ²¹⁶ Researchers faces many hurdles from two decades of failing attempts, unpleasant results for clinical trials of vaccine for the toxic amyloid and over many years of controversy and doubts regarding the efficacy, safety, tolerance of immunotherapy for neurodegenerative diseases. Finally some of these obstacles was relieved by the recent FDA approval for intravenous drug Aducanumab in June 2021 and the accelerated approval pathway for Lecanemab in January 2023. ⁵ Aducanumab (Aduhelm, BIIB037, humanized mAbs derived B cells selective to Aβ sponsored by Biogen, Neurimmune) lecanemab (BAN2401, mAb158, IgG1 mAb sponsored by BioArctic AB, Biogen, Eisai Co., Ltd.). ²¹⁷ , ²¹⁸ , ²¹⁹ These two humanized monoclonal antibodies selectively bind with oligomeric and fibrillar states with signs of reduction and clearance of Aβ plaques and soluble Aβ protofibrils and as a result neuronal function and calcium uptake were restored in AD patient without significant toxicity. Lecanemab had tenfold stronger affinity with protofibrils than fibrils whereas Aducanumab preferred binding to fibrils over protofibrils. These results could explain some of the findings and outcomes in the clinical trials with regard to efficacy and adverse effects and type of mAb used. ¹⁷ The breakthrough for Aducanumab for clearance of Aβ plaques was achieved by its ability to cross brain–blood barrier (BBB), selective and preferred bounding to parenchymal Aβ over vascular Aβ aggregates. Intensified recruitment of f Iba‐1‐positive microglia associated with reduced potency of the glycosylated form of aducanumab that suggested that FcγR‐mediated microglial participation and phagocytosis contributed a major event in Aβ clearance. ²²⁰ The reported clearance ability of both Lecanemab and Aducanumab for Aβ plaques is bringing back the legacy of amyloid hypothesis of AD that was doubted and lapsed behind years ago as consequence of repeated setbacks in Aβ immunotherapy clinical trials. ⁴⁹ , ²²¹ , ²²² The FDA approval initiated, sparked once again the monoclonal antibodies as potential promising topline forefront therapeutics and brought back the concept of Aβ and tau or combination of them as major immunotherapeutic targets for the prevention and/or treatment of AD. ²¹² , ²²³ Still the AD is irreversible and inevitable and the scenario of ending AD dilemma is not fulfilled, ²²⁴ where other paradigms concerned with therapeutics and research development pipelines are pursuing and focusing on the use of small molecules to tackle these neurodegenerative disorders. ³ , ⁵ , ³⁹ , ⁴⁷ , ⁵⁰ , ²¹⁵ , ²²⁵

5.1. Therapeutic interventions using small molecules as drugs

Recent and earlier significant scientific reports acknowledged the toxicity, heterogenicity, and seeding ability of tau oligomers for disease propagation in neurodegenerative diseases including AD. ²²⁶ , ²²⁷ , ²²⁸ Also, parallel studies reported the significance of Aβ oligomers (Aβ) ²²⁹ in neuropathogenesis and formation of plaques. Also considerations for the prion like α‐synuclein ²³⁰ in addition to other cascades theories. ²³¹ , ²³² , ²³³ Based on these streamlines that emanated from scientific research, spectroscopic and modeling data, innovative therapeutics for protein misfolding diseases including AD in the present time are targeting these disease pertinent inducers, through different routes and mechanisms including inhibition of oligomerization/aggregation, enhancing the recurrent of nonpathogenic species, and inhibition/alternation of soluble tau oligomers or nontoxic entities, ⁸ , ²²⁵ , ²²⁶ , ²³⁴ , ²³⁵ , ²³⁶ , ²³⁷ herein this review is highlighting some small molecules.

5.2. Reduction of Aβ production

To selectively prevent brain toxicity, and disordered brain functions due to Aβ oligomers and plaques accumulation, curative strategies were developed to decrease Aβ production by inhibition of the APP proteolytic enzymes including (the beta‐site APP‐cleaving enzyme 1) BACE1 and the β and γ‐secretase. ⁵⁰ , ²³⁸ This strategy is important to tackle the protein misfolding disease from the very beginning. The γ‐secretase inhibitor Semagacestat (NCT00594568) was tested in phase 3 clinical trials in 2021 and terminated due several to causes including depletion of endogenous proteins, deterioration of motor functional capability, higher occurrence of malignant melanoma and cutaneous melanoma, increased infections in clinical trials patients acquiring drug as compared to placebo ²³⁸ , ²³⁹ and successive loss of associated cellular functions. ²³⁸ Another two BACE1 inhibitors were atabecestat (JNJ‐54861911) and E2609 which showed high tendency to decrease Aβ in the rodents' CSF, but were discounted in phase 2/3 clinical trials in 2021for safety issues and adverse effects including a loss of brain volume, a transient drop in white blood cells, and elevation of liver enzymes in some people. ²¹⁵ , ²⁴⁰ Verubecestat (MK‐8931) and Umibecestat (CNP‐520) are other small‐molecule β‐secretase inhibitors which reached phase 3 clinical trials and discontinued in 2018 due to worsening cognitive functions. ²⁴¹ , ²⁴²

5.3. Inhibiting protein aggregation

Anti‐aggregation agents are developed to exert their actions by forming different types of bonds between an inhibitor or small drug molecule and the backbone or side chain residues of the pathogenic conformation of related target protein that may influence one or all phases of the aggregation processes. ²⁴³ Some of these molecules can break off the formation of oligomer aggregates, and initiate the disaggregation of toxic oligomers into the native monomers consequently forestalling the buildup of oligomeric species. ²⁵ Tau hyperphosphorylation and dismantling from MTs and consequently going through undesirable folding and accumulation to form PHFs and neurofibrillary tangles (NFTs) is substantially contributing to neurodegeneration disorders. ²⁴⁴ , ²⁴⁵ PHFs are the hallmark in tauopathies including AD ²⁰² , ²⁴⁶ occurring from the abnormal aggregation of tau protein through nucleated assembly mechanism ²⁴⁷ ; and have double‐helical ribbon morphology. ²⁴⁸

To date, there are continued efforts on research pipelines aiming at the progress of cytotoxic aggregates and mechanisms and hindering the intracellular accumulation of tau aggregates considering the heterogenicity of the tau structures and isoforms in AD patients ²⁴ in addition to Aβ or both. ⁵⁰ , ²⁴⁹ , ²⁵⁰ Toxic tau oligomers impeding and modulation is still considered an effective promising drug development array for AD treatments. ⁴⁷ , ²⁵¹ , ²⁵²

Conclusively in this direction many preclinical studies were reported to explore many synthetic and natural products including polyphenol compounds such as, curcumin and its soluble bioavailable derivatives and analogs. ¹⁹⁴ Some of the curcumin derivatives proved to reduce tau aggregation and could be used as potential tau PET tracers for early detection of tau oligomers. ⁴⁷ Decreasing the population of brain derived toxic tau oligomers and enhancing the formation of higher order tau oligomers from small toxic oligomers through different interactions pathways, ¹⁹⁴ ending up with modified structural conformations correlating with toxicity decline. ⁴⁷ , ¹⁹⁴ , ²²⁶ , ²⁵³ Raloxifene and R− (−)‐apomorphine indicated early their ability to maintain the p‐tau‐treated cells with inhibition pathway and showed consistent cryoprotection. In addition to their ability to penetrate the BBB and to maintain the cognitive functions, making them potential AD future treatments. ²⁵⁴ Natural product protein amyloid/tau inhibitors listing here resveratrol, epigallocatechin gallate (EGCG), morin, amentoflavone, bilobetin, sequoiaflavone, sotetsuflavone, podocarpuflavone, ginkgetin, isoginkgetin, sciadopitysin, ²⁵ retinol, tannic acid, and caffeine have been shown to reduce aggregation proven by in vitro and cell‐based approaches. ²³⁸ , ²⁴³ Scyllo‐inositol, hopeahainol A, and rifampicin, in addition to other natural products tested on animals showed to be helpful for the recovery from the memory impairments and synaptic dysfunctions with alleviating the Aβ levels and senile plaque accumulations. ²⁵⁵

In vitro and preclinical research reported compounds like methylene blue with different formulations, N744, rhodamines, aminothienopyridazines, cinnamaldehyde, epicatechin oxidized form, crocin, VB‐008, and N‐(3‐chloro‐1,4‐dihydro‐1,4‐dioxo‐2‐naphthalenyl)‐l‐tryptophan that showed potential inhibition and targeting of tau oligomers or Aβ aggregation or combined action. ²²⁵ , ²⁵⁶ , ²⁵⁷ , ²⁵⁸ Still, the majority of these target compounds and drugs are not well developed or discontinued. ²²⁴ These disappointments in the scene of drug development and discovery could be attributable in part to lack in understanding of the structural information associated with the IDPs and amyloids. This includes cellular or in vivo pathways of disease relevant amyloid inhibition by small molecules, the presence of dynamic heterogeneous structural conformation in AD brain patients, triggering the cytotoxicity pathways and cell death progamming ²⁴ ; in addition to the drug delivery issues. ⁵⁰ , ²²⁵ , ²⁵⁹

The modified form of the methylthioninium (MT) moiety LMTX (TRx0237) which is an ongoing promising therapeutic is being investigated more often as an inhibitor for tau causing disease aggregation with failing attempts in phase 2/3 clinical trials due to low efficicy, ⁴ , ⁸ , ²⁶⁰ and slow cognitive or functional decline in subjects with mild to moderate AD (reported 2022). A new phase 3 clinical trial to acquire the safety and efficacy of LMTX (8 mg/day and 16 mg/day) in the treatment of AD patients compared to the placebo ⁵⁰ , ²²⁵ , ²⁵² , ²⁶⁰ , ²⁶¹ is still under evaluation. A phase II clinical trials (NCT02033941) to investigate the effect of grape seed polyphenolic extract GSPE on AD tau aggregation are still going on. ²⁶² The target drug NPT200‐11(C₂₈H₃₉N₅O₃) sponsored by neuropore therapies for the curative treatments of PD which had completed phase 1 clinical trial was reported to beat back the oligomerization, misfolding, buildup of α‐synuclein. It also lowers down the toxicity and pathology in cortex, reduction of astrogliosis, and improves brain motor function in preclinical studies ²⁶³ but did not exceed this stage as a proved drug or of label medication. In 2020, an 18‐month phase 2a double‐blind, placebo‐controlled study was initiated by UCB Biopharma SRL, for evaluation of the efficacy, tolerability, safety, and pharmacokinetics of oral UCB0599 (the R‐enantiomer of NPT200‐11) in study participants with early PD and is expected to completed in 2024. ²⁰⁶

Tramiprosate or Homotaurine (ALZ‐801) as a small therapeutic molecule that inhibits amyloid aggregation in the brain by binding selectively to Asp23, Aβ42, Lys16, and Lys28 showed in clinical trials for orally administrated drug to reduce in the AD tau and fibrillary amyloid aggregations. This was achieved by providing monomer stability in the brain with enhanced cholinergic functions and stable cognition performance; still it failed to comply with phase 3 clinical trials and terminated due to efficiency issues in 2009. ²⁶⁴ Few years later phase 3/4 clinical trials on AD apolipoprotein E carriers ApoE3 and ApoE4 (NCT04770220) based on differences in cortical thickness and positive results were initiated by Alzheon Inc. in 2019 running at 85 sites across the United States, Canada, and Europe, and still trials under investigation and expected to be completed in 2024. ⁷³ , ²⁶⁴

5.4. Agents modulating tau posttranslational modifications

Several scientific works and publications reported the use of small molecule to tackle the tauopathies including AD by alternation and manipulation of the tau posttranslational modifications like hyperphosphorylation, truncation, glycosylation, and acetylation. Herein, the most important modification is hyperphosphorylation where the p‐tau is the predominant protein in most AD cases. It accounts for disruption of synaptic function, causing tau self‐assembly and intraneuronal accumulation of PHFs, aggregation in the form of NFTs and eventually leading to sever AD symptoms. Many phosphate activators like phosphoprotein phosphatase 2A (PP2A)‐related drugs were developed to reduce tau phosphorylation including the ionic compound sodium selenite. It was tested in vitro and in vivo to activate the PP2A, the sub catalytic enzymatic unit, reversing memory deficits and reducing tau phosphorylation in animal models of AD and showed in vivo selectivity toward p‐tau. ²²⁵ , ²⁶⁵ Randomized controlled trials of oral supplementation may potentially slow neurodegeneration in AD. ²⁶⁶ Other drugs were developed to work as kinase inhibitors targeting tau phosphorylation enzymes ²²⁵ such as saracatinib (AZD0530) which is a fyn inhibitor and tideglusib (NP031112) which is an inhibitor of glycogen synthase kinase 3β (GSK‐3β). ²⁶⁷ In animal studies, it was reported that tideglusib reduced a range of disease factors and symptoms, including tau phosphorylation, amyloid aggregation and deposition, neuron loss, and gliosis in mouse brain. Tideglusib showed some reduction in brain atrophy in a studied a subgroup; however, this trial was negative on the primary outcome. ²⁶⁸ All studies up to date for tideglusib were with limited success and did not exceed the phase II trials endpoints regarding mid and mild AD. ²⁶⁹

Blarcamesine (ANAVEX 2‐73) another (GSK3‐β) functioning as an agonist of sigma‐1 chaperone protein and developed for AD, PD, and related dementia is in the clinical trials for AD (phase 2/3), PD dementia (phase 2), PD endpoints (reported 2022) data outcomes indicated that it could be restoring neural cell homeostasis and enhancing neuroplasticity. Clinical trials indicated reduction in early and mild cognitive impairment ¹⁴ with association of AD and dementia in clinical trials patients. Blarcamesine is now in clinical trials to evaluate the effects of ANAVEX2‐73‐AD‐004 on safety and efficacy of daily treatment (NCT04314934) and expected to be completed in 2024. ²⁷⁰

5.5. Molecular docking‐molecular dynamics simulation for potential small molecular therapeutics

Recently, to overcome some of the hurdles in drugs discovery scenario in relation to many neurodegenerative diseases; computer‐based molecular dynamics (MD)‐molecular docking researchers to observe protein–drug interaction events exhibiting different methodologies. ²⁷¹ , ²⁷² , ²⁷³ The significance of these methods is to predict the atomic and molecular 3D functional structures of biomolecules at different disease‐associated stages, ²⁷⁴ , ²⁷⁵ fast effective identification of potential safe drugs, ²⁷⁶ relating structural disorders to cellular functions, ²⁷⁷ and envision binding modes, investigating interaction of tau and Aβ with cellular membranes and peptides insertions in model membrane, ²⁷⁸ , ²⁷⁹ reducing the use of animal models. ²⁷³ Many research groups are aiming to design innovative small molecules therapeutics, predict, simulate and screen synthetic and natural drugs ²⁸⁰ with optimum affinity for active site of target proteins and/or folding intermidiates. ²⁸⁰ , ²⁸¹ , ²⁸² , ²⁸³ Another way, some research groups actively employing network biology and polypharmacology (network pharmacology), for data integration and multitarget drug development. ²⁸² , ²⁸⁴ , ²⁸⁵ , ²⁸⁶ This will be providing a library for potential multi‐target multifunction candidate drugs to tackle neurodegenerative diseases, proposing new AD treatment strategies, and an acceleration pathway for drug development. ²⁸⁰ , ²⁸¹ , ²⁸² , ²⁸⁶ Recent studies are showing that it is possible through the in vitro, machine learning and experimental and computational studies to predict novel inhibitors of aggregation (lead compounds) and acetylcholinesterase inhibitors. ²⁸⁷ , ²⁸⁸ Aβ42 monomer is in random coil conformation including either helices or β‐strand content whereas Aβ42 oligomers are in the form of amorphous aggregates containing few β‐sheets. ²⁸⁷ This outcome could be important for search of matching small molecules that prevent aggregation and toxicity. MD simulations of the antagonist ADH‐31 against Aβ42 aggregation resolved the inhibitory mechanism of ADH‐31 and demonstrated future potential use as protofibril destabilizer. Experimental and MD simulation studies of β‐casein displays chaperone like activity and inhibits Aβ40/Aβ42 aggregation and extracellular plaque formation, therefore can be used as novel drug for AD. ²⁹⁰ Kinetic modulation of Aβ42 aggregation based on biophysical approaches including x‐ray and in silico experiments is another recent addition to disclose structure based rational design of Aβ42 fibril inhibitor. ²⁹¹ Murray et al. research group screened small molecule library using EGCG pharmacophore by molecular docking and found two novel compounds (CNS‐11 and CNS‐11g) and experimentally demonstrate the ability to disaggregate tau PHF and α‐synuclein fibrils. ²⁹² MD simulation was performed to reveal the putative binding sites disaggregation mechanism of CNS‐11 and CNS‐11g in bound form and unbound form, ²⁹² where these compounds could be potential specific targets for treatment of synucleinopathies. ²⁹² Similar study disclosed the inhibition mechanisms of EGCG and Genistein on the AD amyloid‐beta 42 peptide conformational changes employing MD and molecular docking. ²⁹³ For visualizing the morphology of amyloidogenic protein aggregates, TEM ⁴⁶ and AFM ⁴⁷ , ⁴⁸ are frequently used in vitro. Both these techniques provide quantitative and qualitative information at the nanometer scale for quaternary structure features including the length, width, and surface feature of aggregates. ²⁹⁴ , ²⁹⁵ , ²⁹⁶

6. CONCLUSION

With the intricate pathophysiology of the neurodegenerative diseases including AD and hindrance for drug development trajectories, application of computational methods with structural and biophysical techniques, can significantly contribute to developing small molecules targeting distinct protein aggregates associated with disease pathogenesis. Availability of well‐defined structures of biomolecules, kinetics, and their biochemistry accelerate the progress of drug discovery. Detection and revealing different pathogenic structural variants pose a new challenge to prepare and replicate them for further testing, which could be a critical step for drug development trajectories and effective treatments of AD. This review pinpoint about recent development in structural and biophysical techniques especially for studying amyloid formation and its dynamics, where such obtained data could be critical for revealing the mystery of the amyloids and its role in pathogenic processes.

AUTHOR CONTRIBUTIONS

Sharif Arar: Writing – original draft. Md. Anzarul Haque: Writing – original draft. Rakez Kayed: Conceptualization; validation; supervision; funding acquisition; writing – review and editing; writing – original draft.

PEER REVIEW

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1002/prot.26561.

Arar S, Haque MA, Kayed R. Protein aggregation and neurodegenerative disease: Structural outlook for the novel therapeutics. Proteins. 2025;93(8):1314‐1329. doi: 10.1002/prot.26561

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.