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Polymorphism is common in both in vitro and in vivo amyloid fibrils formed by the same peptide/protein. However, the differences in their self-assembled structures at the amino acid level remain poorly understood. In this study, we utilized isotope-edited vibrational circular dichroism (VCD) on a well-known amyloidogenic peptide fragment (N 22 FGAIL 27 ) of human islet amyloid polypeptide (IAPf) to investigate the structural polymorphism. Two individual isotope-labeled IAPf peptides were used, with a 13 C label on the carbonyl group of phenylalanine (IAPf-F) and glycine (IAPf-G). We compared the amyloid-like nanofibril of IAPf induced by solvent casting (fibril B) with our previous report on the same IAPf peptide fibril but with a different fibril morphology (fibril A) formed in an aqueous buffer solution. Fibril B consisted of entangled, laterally fused amyloid-like nanofibrils with a relatively shorter diameter (15-50 nm) and longer length (several microns), while fibril A displayed nanofibrils with a higher diameter (30-60 nm) and shorter length (500 nm-2 μm). The isotope-edited VCD analysis indicated that fibrils B consisted of anti-parallel β-sheet arrangements with glycine residues in the registry and phenylalanine residues out of the registry, which was significantly different from fibrils A, where a mixture of parallel β-sheet and turn structure with the registry at phenylalanine and glycine residues was observed. The VCD analysis, therefore, suggests that polymorphism in amyloid-like fibrils can be attributed to the difference in the packing/arrangement of the individual β-strands in the β-sheet and the difference in the amino acid registry. Our findings provide insights into the structural aspects of fibril polymorphism related to various amyloid diseases and may aid in designing amyloid fibril inhibitors for therapeutic purposes.

In this work, anion-π interactions between sulfate groups (SO 4 2- ) and protein aromatic amino acids (AAs) (histidine protonated (HisP), histidine neutral (HisN), tyrosine (Tyr), tryptophan (Trp), and phenylalanine (Phe)) in an aqueous environment have been analyzed using quantum chemical (QC) calculations and molecular dynamics (MD) simulations. Sulfates can occur naturally in solution and can be contained in biomolecules playing relevant roles in their biological function. In particular, the presence of sulfate groups in glycosaminoglycans such as heparin and heparan sulfate has been shown to be relevant for protein and cellular communication and, consequently, for tissue regeneration. Therefore, anion-π interactions between sulfate groups and aromatic residues represent a relevant aspect to investigate. QC results show that such an anion-π mode of interaction between SO 4 2- and aromatic AAs is only possible in the presence of water molecules, in the absence of any other cooperative non-covalent interactions. Protonated histidine stands out in terms of its enhancement in the magnitude of interaction strength on solvation. Other AAs such as non-protonated histidine, tyrosine, and phenylalanine can stabilize anion-π interactions on solvation, albeit with weak interaction energy. Tryptophan does not exhibit any anion-π mode of interaction with SO 4 2- . The order of magnitude of the interaction of aromatic AAs with SO 4 2- on microsolvation is HisP > HisN > Tyr > Trp > Phe. Atoms in molecules (AIM) analysis illustrates the significance of water molecules in stabilizing the divalent SO 4 2- anion over the π surface of the aromatic AAs. MD simulation analysis shows that the order of magnitude of the interaction of SO 4 2- with aromatic AAs in macroscopic solvation is HisP > HisN, Tyr, Trp > Phe, which is very much in line with the QC results. Spatial distribution function analysis illustrates that protonated histidine alone is capable of establishing the anion-π interaction with SO 4 2- in the solution phase. This study sheds light on the understanding of anion-π interactions between SO 4 2- and aromatic AAs such as His and Tyr observed in protein crystal structures and the significance of water molecules in stabilizing such interactions, which is not feasible otherwise., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

We report the discovery of a novel class of compounds that function as dual inhibitors of the enzymes neutral sphingomyelinase-2 (nSMase2) and acetylcholinesterase (AChE). Inhibition of these enzymes provides a unique strategy to suppress the propagation of tau pathology in the treatment of Alzheimer's disease (AD). We describe the key SAR elements that affect relative nSMase2 and/or AChE inhibitor effects and potency, in addition to the identification of two analogs that suppress the release of tau-bearing exosomes in vitro and in vivo . Identification of these novel dual nSMase2/AChE inhibitors represents a new therapeutic approach to AD and has the potential to lead to the development of truly disease-modifying therapeutics.

Over the past decade, the available crystal structures have almost doubled in Protein Data Bank (PDB) providing the research community with a series of similar crystal structures to choose from for future docking studies. With the steady growth in the number of high-resolution three-dimensional protein structures, ligand docking-based virtual screening of chemical libraries to a receptor plays a critical role in the drug discovery process by identifying new drug candidates. Thus, identifying potential candidates among all the available structures in a database for docking studies is of utmost importance. Our work examined whether one could use the resolution of a number of known structures, without considering other parameters, to choose a good experimental structure for various docking studies to find more useful drug leads. We expected that a good experimental structure for docking studies to be the one that gave favorable docking with the largest number of ligands among the experimental structures to be selected. We chose three protein test systems for our study, all belonging to the family of MAPK: (1) JNK1, (2) JNK2, and (3) JNK3. On analysis of the results, the best resolution structures showed significant variations from the expected values in their result, whereas the poor resolution structures proved to be better candidates for docking studies., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Noncovalent interactions between the guanidinium cation (Gdm + ) and aromatic amino acids (AAs) in the water molecules have been studied using quantum chemical calculation and molecular dynamics (MD) simulations. Our studies show that there are two different modes of interactions between Gdm + and AAs with and without water molecules. It is observed that nonhydrated Gdm + interacts with AAs through N-H···π interactions, whereas hydrated clusters of Gdm + are stabilized by stacking interactions with the help of the water-mediated hydrogen bond. Thus, different hydration patterns have significant effects on the predominant cation···π interactions in AAs-Gdm + complexes. Findings from MD simulation elicit that the interaction pattern of Gdm + with AAs varies as Phe < Tyr < Trp. Both the QM and MD calculations show a similar trend in the interaction of AAs with Gdm + . Moreover, the interaction of AAs with Gdm + depends on the spatial orientation of AAs in the protein and the concomitant local structure, that is, the AAs present in the unstructured region of protein such as coils and bends exhibit higher binding for Gdm + when compared to the AAs present in the structured region of the protein such as the α-helix and the β-sheet. Our study clearly reveals that H-bonded water molecules and the hydration pattern of Gdm + as well as the positional presence of these AAs in the protein structure context play determining roles in the denaturation of protein by the Gdm + cation.

Humanin (HN), a 24-amino acid bioactive peptide, has been shown to increase cell survival of neurons after exposure to Aβ and NMDA-induced toxicity and thus could be beneficial in the treatment of Alzheimer's disease (AD). The neuroprotection by HN is reported to be primarily through its agonist binding properties to the gp130 receptor. However, the peptidic nature of HN presents challenges in its development as a therapeutic for AD. We report here for the first time the elucidation of the binding site of Humanin (HN) peptide to the gp130 receptor extracellular domain through modeling and the synthesis of small molecule mimetics that interact with the HN binding site on the gp130 receptor and provide protection against NMDA-induced neurotoxicity in primary hippocampal neurons. A brain permeable small molecule mimetic was identified through exploratory medicinal chemistry using microfluidic flow chemistry to facilitate the synthesis of new analogues for screening and SAR optimization.

Alzheimer's disease (AD) is the most common neurodegenerative disorder. Along with an increasing number of elderly worldwide, it poses a great challenge for the society and health care. Although sporadic AD is the common form of AD, 2-3% of the AD cases are expected to be due to mutations in the β region of the amyloid precursor protein, which is referred to as autosomal dominant AD (ADAD). These mutations may cause changes in the secondary structure of the amyloid β fibrils and may alter the fibrillization rate leading to changes in the disease development and could also affect the binding to tracers used in diagnosis. In particular, from some recent clinical studies using PET tracers for detection of fibrillar amyloids, it is evident that in ADAD patients with Arctic mutation no amyloid plaque binding can be detected with the 11 C-Pittsburgh Compound B ( 11 C-PIB). However, for in vitro conditions, significant binding of 3 H-PIB has been reported for the amyloid fibrils carrying the Arctic mutation. The aim of the present study is to investigate if there is any mutation specific binding of commonly used amyloid tracers, namely, florbetaben, florbetapir, FPIB, AZD4694, and AZD2184, by means of molecular modeling techniques. Other than Arctic, ADAD mutations, such as the Dutch, Italian, Iowa, and Flemish mutations, are considered in this study. We report that all tracers except florbetapir show reduced binding affinity toward amyloid β fibrils with the Arctic mutation when compared to the native type. Moreover, florbetapir is the only tracer that binds to all mutants with increased affinity when compared to the native fibril. The results obtained from these studies could increase the understanding of the structural changes caused by mutation and concomitant changes in the interaction pattern of the PET tracers with the mutated variants, which in turn can be useful in selecting the appropriate tracers for the purpose of diagnosis as well as for designing new tracers with desirable properties.

Positron emission tomography (PET) tracers play an important role in the diagnosis of Alzheimer's disease, a condition that leads to progressive dementia and memory loss. A high binding affinity and specificity of the PET tracers to amyloid oligomers and fibrils are crucial for their successful application as diagnostic agents. In this sense, it is essential to design PET tracers with enhanced binding affinities, which can lead to more precise and earlier detection of Alzheimer's disease conditions. The application of in silico methodology for the design and development of efficient PET tracers may serve as an important route to improved Alzheimer's disease diagnosis. In this work, the performance of widely used computational methods is explored for predicting experimental binding affinities of styrylbenzoxazole (SB) derivatives against a common amyloid protofibril. By performing docking, molecular dynamics, and quantum chemistry calculations in sequence their combined predictive performance is explored. The present work emphasizes the merits as well as limitations of these simulation strategies in the realm of designing PET tracers for Alzheimer's disease diagnosis.

The use of certain ionic liquids (ILs) as pretreatment solvents for lignocellulosic biomass has gained great interest in recent years due to the IL's capacity for efficient cellulose dissolution in aqueous solution as compared to other common pretreatment techniques. A fundamental understanding on how these ILs in aqueous environments act on cellulose, particularly at lower IL concentrations with water as a cosolvent, is essential for optimizing pretreatment efficiency, lowering pretreatment cost, and improving IL recyclability. The IL 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) is one of the most efficient cellulose solvents known, greatly altering cellulose structure for improved enzymatic saccharification. To understand the role of water as a cosolvent with [C2C1Im][OAc], we investigated the dissolution mechanism of microcrystalline cellulose, type Iβ, in different [C2C1Im][OAc]:water ratios at room (300 K) and pretreatment (433 K) temperatures using all atom molecular dynamics (MD) simulations. These simulations show that 80:20 ratios of [C2C1Im][OAc]:water should be considered as "the tipping point" above which [C2C1Im][OAc]:water mixtures are equally effective on decrystallization of cellulose by disrupting the interchain hydrogen bonding interactions. Simulations also reveal that the resulting decrystallized cellulose from 100% [C2C1Im][OAc] begins to repack in the presence of water but into a less crystalline, or more amorphous, form.

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in which P2' residue Leu17 (bovine pancreatic trypsin inhibitor numbering) is mutated to Arg selectively inhibits the active site of plasmin with ∼5-fold improved affinity. Thrombin cleavage (24 h extended incubation at a 1:50 enzyme-to-substrate ratio) of the KD1 mutant (Leu17Arg) yielded a smaller molecule containing the intact Kunitz domain with no detectable change in the active-site inhibitory function. The N-terminal sequencing and MALDI-TOF/ESI data revealed that the starting molecule has a C-terminal valine (KD1L17R-VT), whereas the smaller molecule has a C-terminal lysine (KD1L17R-KT). Because KD1L17R-KT has C-terminal lysine, we examined whether it could serve as a decoy receptor for plasminogen/plasmin. Such a molecule might inhibit plasminogen activation as well as the active site of generated plasmin. In surface plasmon resonance experiments, tissue plasminogen activator (tPA) and Glu-plasminogen bound to KD1L17R-KT (Kd ∼ 0.2 to 0.3 μM) but not to KD1L17R-VT. Furthermore, KD1L17R-KT inhibited tPA-induced plasma clot fibrinolysis more efficiently than KD1L17R-VT. Additionally, compared to ε-aminocaproic acid KD1L17R-KT was more effective in reducing blood loss in a mouse liver-laceration injury model, where the fibrinolytic system is activated. In further experiments, the micro(μ)-plasmin-KD1L17R-KT complex inhibited urokinase-induced plasminogen activation on phorbol-12-myristate-13-acetate-stimulated U937 monocyte-like cells, whereas the μ-plasmin-KD1L17R-VT complex failed to inhibit this process. In conclusion, KD1L17R-KT inhibits the active site of plasmin as well as acts as a decoy receptor for the kringle domain(s) of plasminogen/plasmin; hence, it limits both plasmin generation and activity. With its dual function, KD1L17R-KT could serve as a preferred agent for controlling plasminogen activation in pathological processes.

Graphene-based nanomaterials (GBNMs) [graphene oxide (GO), reduced graphene oxide (rGO), and graphene] have been recognized as potential candidates for various biomedical applications ranging from biosensing platform to cellular delivery of proteins and peptides. However, GBNMs induced conformational changes in proteins are the major concerns in realizing their full potential in aforementioned applications. Despite several studies, the effect of GBNMs on the conformation of proteins is still not well understood. Therefore, an attempt was made to investigate the effect of GBNMs on the adsorption and conformation of positively charged cytoplasmic protein using molecular dynamics (MD) simulations. Our study showed that the adsorption of protein on GO was highly selective and mediated through electrostatic interactions (hydrogen bond/salt bridge interactions), whereas the van der Waals and π-π stacking interactions were the major driving forces for the adsorption of protein on rGO and graphene. The secondary structure analysis showed the conformational stability of the protein on GO may be attributed to the extensive hydration of GO surface and the absence of tyrosine residues in π-π stacking with π regions of GO. The GO surface acts as a hydrogen bond acceptor similar to the protein's natural receptor present in a physiological environment. This computational study has also explored the artificial protein receptor like potential of GO.