Aggregation Inhibitory Effect Research Articles

An 11-mer Synthetic Peptide Suppressing Aggregation of Aβ25-35 and Resolving Its Aggregated Form Improves Test Performance in an Aβ25-35-Induced Alzheimer's Mouse Model.

There is a high demand for the development of drugs against Alzheimer's disease (AD), which is related to the misfolding and aggregation of Amyloid-β (Aβ), due to the increasing number of patients with AD. In our present study, we aimed to assess the aggregation inhibitory effect of various synthetic YS-peptides on Aβ25-35 to identify an applicable peptide for clinical use for AD treatment and prevention. Suppression and aggregate resolution activities of YS-peptides against Aβ25-35 were evaluated using a Thioflavin T assay and scanning electron microscopy (SEM). Structure-activity relationship studies revealed that YS-RD11 (RETLVYLTHLD) and YS-RE16 (RETLVYLTHLDYDDTE) showed suppression and aggregate-resolution activities. The effect of YS-peptides on phagocytosis in microglial cells (BV-2 cells) demonstrated that YS-RD11 and YS-RE16 activated the phagocytic ability of microglia. In the Aβ25-35-induced AD mouse model, YS-RD11 prevented and improved the deficits in short-term memory. In conclusion, YS-RD11 is a suitable candidate therapeutic drug against AD and uses a strategy similar to that used for antibodies.

Why Is Arginine the Only Amino Acid That Inhibits Polyglutamine Monomers from Taking on Toxic Conformations?

Polyglutamine (polyQ) diseases are devastating neurodegenerative disorders characterized by abnormal expansion of glutamine repeats within specific proteins. The aggregation of polyQ proteins is a critical pathological hallmark of these diseases. Arginine was identified as a promising inhibitory compound because it prevents polyQ-protein monomers from forming intra- and intermolecular β-sheet structures and hinders polyQ proteins from aggregating to form oligomers. Such an aggregation inhibitory effect was not observed in other amino acids. However, the underlying molecular mechanism of the aggregation inhibition and the factors that differentiate arginine from other amino acids, in terms of the inhibition of the polyQ-protein aggregation, remain poorly understood. Here, we performed replica-permutation molecular dynamics simulations to elucidate the molecular mechanism by which arginine inhibits the formation of the intramolecular β-sheet structure of a polyQ monomer. We found that the intramolecular β-sheet structure with more than four β-bridges of the polyQ monomer with arginine is more unstable than without any ligand and with lysine. We also found that arginine has 1.6-2.1 times more contact with polyQ than lysine. In addition, we revealed that arginine forms more hydrogen bonds with the main chain of the polyQ monomer than lysine. More hydrogen bonds formed between arginine and polyQ inhibit polyQ from forming the long intramolecular β-sheet structure. It is known that intramolecular β-sheet structure enhances intermolecular β-sheet structure between proteins. These effects are thought to be the reason for the inhibition of polyQ aggregation. This study provides insights into the molecular events underlying arginine's inhibition of polyQ-protein aggregation.

Inhibition of protein misfolding and aggregation by steroidal quinoxalin-2(1H)-one and their molecular docking studies

A series of D-ring fused 16-substituted steroidal quinoxalin-2(1H)-one attached to an electron-releasing (ER) or electron-withdrawing (EW) groups via steroidal oxoacetate intermediate were synthesized to investigate their protein aggregation inhibition potential using human lysozyme (HLZ). The influence of the type of substituent at the C-6 positions of the quinoxalin-2(1H)-one ring on the protein aggregation inhibition potential was observed, showing that the EW moiety improved the protein aggregation inhibition potency. Of all the evaluated compounds, NO2-substituted quinoxalin-2(1H)-one derivative 13 was the most active compound and had a maximum protein aggregation inhibition effect. Significant stabilization effects strongly support the binding of the most biologically active steroidal quinoxalin-2(1H)-one with docking studies. The predicted physicochemical and ADME properties lie within a drug-like space which shows no violation of Lipinski's rule of five except compounds 12 and 13. Combined, our results suggest that D-ring fused 16-substituted steroidal quinoxalin-2(1H)-one has the potential to modulate the protein aggregation inhibition effect.

Total Synthesis of (-)-Amovillosumin A and Structure Correction of (-)-Amovillosumin C Using Chemical Synthesis.

Norlignans are a rare class of natural products isolated from a diverse range of plant species, many of which have interesting biological activities including antibacterial, antioxidant, phytotoxic, platelet aggregation inhibitory effects, and more. Isolated from Amomum villosum (Amomi Fructus), amovillosumins A (1) and C (3) are norlignans which were of interest to synthesize, due to their interesting bioactivities, specifically their ability to increase stimulation of glucagon-like peptide-1 (GLP-1) secretion. In this research, key intermediate 15 was used to stereoselectively synthesize (7R,8R)-amovillosumins A (1) and C (3). The developed method includes a Mitsunobu coupling, a modified rhodium-catalyzed Miyaura arylation, and an acid-catalyzed cyclization in key bond-forming steps. After synthesis, the structure of 1 was confirmed, but it was revealed that the benzodioxane-containing structure of amovillosumin C (3) that had been proposed in the literature was incorrect. Thus, with further investigation a structure correction of 3 was achieved by synthesis, the correct structure being 8-O-4'-oxynorlignan.

Fluorescence quenching-based immunological probe for ticagrelor monitoring

Introduction: Ticagrelor is extensively utilized for the treatment of acute coronary syndromes (ACS), but its platelet aggregation inhibitory effects can potentially result in tissue bleeding, posing a serious risk to patients’ lives.Methods: In this study, we developed highly sensitive full length anti-ticagrelor Quenchbodies (Q-bodies) for fast monitoring of ticagrelor both in solution and serum for the first time. Ticagrelor coupled with N- hydroxysuccinimide (Ticagrelor-NHS) ester was also designed and synthesized for interaction and biological activity detection.Results: Both ATTO-labeled MEDI2452 (2452A) Q-body and TAMRA-labeled IgG 152 (152T) Q-body demonstrated efficient detection of ticagrelor and its active metabolite (TAM). The 2452A Q-body exhibited a broader detection range, while the 152T Q-body displayed a lower limit of detection (LOD). Under physiological conditions (Ticagrelor:TAM, 3:1), the concentration of ticagrelor was further measured, yielding LOD values of 4.65 pg/mL and 2.75 pg/mL for the two Q-bodies, with half-maximal effect concentrations of 8.15 ng/mL and 3.0 ng/mL, respectively.Discussion: Compared with traditional liquid chromatography-mass spectrometry (LC-MS) methods, anti-ticagrelor Q-bodies have higher sensitivity and detection speed. It enabled the completion of analysis within 3 min, facilitating rapid preoperative detection of blood drug concentration in ACS to determine the feasibility of surgery and mitigate the risk of intraoperative and postoperative hemorrhage. The swift detection of ticagrelor holds promise for enhancing individualized drug administration, preventing adverse reactions, and providing preoperative guidance.

Improving Chuanxiong Rhizoma quality standards using an effect-constituent index based bioassay

Chuanxiong Rhizoma is a traditional Chinese medicine (TCM) that is used to promote blood circulation. We set out to improve Chuanxiong Rhizoma quality standards using a bioassay-based Effect-constituent Index (ECI). We performed high performance liquid chromatography (HPLC) analysis to determine the chemical constituents of 10 Chuanxiong Rhizoma samples from different locations. We then constructed a direct bioassay method to investigate each sample’s antiplatelet aggregation effects. To screen for active ingredients that promote antiplatelet aggregation, we carried out Pearson correlation analyses between biopotency and compounds identified in the HPLC data. We developed an ECI of platelet aggregation inhibition using a multi-indicator synthetic evaluation method based on the integration of biopotency and active constituents. To further assess the biopotency-based Chuanxiong Rhizoma quality evaluation result accuracy, we compared the ECI with the chemical indicator’ method. Eight common chemical fingerprints peaks indicated notable content variation among samples. Biological evaluation showed that all 10 samples could inhibit platelet aggregation, although they had significantly different biological potencies. Using spectrum-effect relationships, we determined that Ligustilide was the significant active constituent responsible for antiplatelet aggregation. Using correlation analysis, we found that ECI correlated with the Chuanxiong Rhizoma extract’s platelet aggregation inhibitory effect. Additionally, ECI proved to be a good indicator of Chuanxiong Rhizoma quality, whereas chemical indicators failed to distinguish and predict the biopotency-based quality grade. This work indicates that ECI is a useful tool for associating sample quality with chemical markers linked to TCM clinical effects. ECI also provides a paradigm for improving the quality control of other TCMs that invigorate blood circulation.

Theoretical Analysis of the Aggregation-Inhibition Effect of Arginine on Polyglutamine Protein by the Generalized-Ensemble Method

Theoretical Analysis of the Aggregation-Inhibition Effect of Arginine on Polyglutamine Protein by the Generalized-Ensemble Method

Synthesis, and docking studies of arylhydrazone compounds and evaluation of their platelet aggregation inhibitory effect and cytotoxicity

Synthesis, and docking studies of arylhydrazone compounds and evaluation of their platelet aggregation inhibitory effect and cytotoxicity

Structural basis of Alzheimer β-amyloid peptide recognition by engineered lipocalin proteins with aggregation-blocking activity.

We describe the structural analysis of two Anticalin® proteins that tightly bind Aβ40, a peptide involved in the pathophysiology of Alzheimer's disease. These anticalins, US7 and H1GA, were engineered on the basis of the human lipocalin 2, thus yielding compact single-domain binding proteins as an alternative to antibodies. Albeit selected under different conditions and mutually deviating in 13 amino acid positions within the binding pocket (of 17 mutated residues in total), both crystallised anticalins recognize the same epitope in the middle of the β-amyloid peptide. In the two complexes with the Aβ40 peptide, its central part comprising residues LysP16 to LysP28 shows well defined electron density whereas the flanking regions appear structurally disordered. The compact zigzag-bend conformation which is seen in both structures may indicate a role during conversion of the soluble monomeric form into pathogenic Aβ state(s) and, thus, explain the aggregation-inhibiting effect of the anticalins. In contrast to solanezumab, which targets the same Aβ region in a different conformation, the anticalin H1GA does not show cross-reactivity with sequence-related human plasma proteins. Consequently, anticalins offer promising reagents to prevent oligomerization of Aβ peptides to neurotoxic species in vivo and their small size may enable new routes for brain delivery.

Beyond Lipid-Lowering: Effects of Statins on Cardiovascular and Cerebrovascular Diseases and Cancer.

The 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, also known as statins, are administered as first-line therapy for hypercholesterolemia, both as primary and secondary prevention. Besides the lipid-lowering effect, statins have been suggested to inhibit the development of cardiovascular disease through anti-inflammatory, antioxidant, vascular endothelial function-improving, plaque-stabilizing, and platelet aggregation-inhibiting effects. The preventive effect of statins on atherothrombotic stroke has been well established, but statins can influence other cerebrovascular diseases. This suggests that statins have many neuroprotective effects in addition to lowering cholesterol. Furthermore, research suggests that statins cause pro-apoptotic, growth-inhibitory, and pro-differentiation effects in various malignancies. Preclinical and clinical evidence suggests that statins inhibit tumor growth and induce apoptosis in specific cancer cell types. The pleiotropic effects of statins on cardiovascular and cerebrovascular diseases have been well established; however, the effects of statins on cancer patients have not been fully elucidated and are still controversial. This review discusses the recent evidence on the effects of statins on cardiovascular and cerebrovascular diseases and cancer. Additionally, this study describes the pharmacological action of statins, focusing on the aspect of ‘beyond lipid-lowering’.

Synthesis of novel benzothiazole derivatives and investigation of their enzyme inhibitory effects against Alzheimer's disease.

The use of dual acetylcholinesterase (AChE)–monoamine oxidase B (MAO-B) inhibitors is a new approach in the treatment of Alzheimer disease (AD). In this work, 14 new benzothiazoles (4a–4n) were designed and synthesized. In biological activity studies, the AChE, butyrylcholinesterase (BChE), MAO-A and MAO-B inhibitory potentials of all compounds were evaluated using the in vitro fluorometric method. Additionally, amyloid beta (Aβ)-aggregation inhibitory effects of active compounds were evaluated by means of an in vitro kit-based method. The biological evaluation showed that compounds 4a, 4d, 4f, 4h, 4k and 4m displayed significant activity against AChE and MAO-B enzymes. Compound 4f displayed inhibitory activity against AChE and MAO-B enzyme with IC50 values of 23.4 ± 1.1 nM and 40.3 ± 1.7 nM, respectively. It has been revealed that compound 4f may have the potential to inhibit AChE and MAO-B enzymes, as well as the ability to prevent the formation of beta amyloid plaques accumulated in the brains of patients suffering from AD. In silico studies also support the obtained biological activity findings. Compound 4f provided strong interactions with the active site of both enzymes. In particular, the interaction of compound 4f with flavin adenine dinucleotide (FAD) in the MAO-B enzyme active site is a promising and exciting finding.

Functional and binding studies of gallic acid showing platelet aggregation inhibitory effect as a thrombin inhibitor

ObjectiveThis study was devoted to identifying natural thrombin inhibitors from traditional Chinese medicine (TCM) and evaluating its biological activity in vitro and binding characteristics. MethodsA combination strategy containing molecular docking, thrombin inhibition assay, surface plasmon resonance (SPR) and molecular dynamics simulation were applied to verify the study result. ResultsGallic acid was confirmed as a direct thrombin inhibitor with IC50 of 9.07 μmol/L and showed a significant inhibitory effect on thrombin induced platelet aggregation. SPR-based binding studies demonstrated that gallic acid interacted with thrombin with a KD value of 8.29 μmol/L. Molecular dynamics and binding free energy analysis revealed that thrombin-gallic acid system attained equilibrium rapidly with very low fluctuations, the calculated binding free energies was −14.61 kcal/mol. Ala230, Glu232, Ser235, Gly258 and Gly260 were the main amino acid residues responsible for thrombin inhibition by gallic acid, providing a mechanistic basis for further optimization. ConclusionThis study proved that gallic acid is a direct thrombin inhibitor with platelet aggregation inhibitory effect, which could provide a basis for the follow-up research and development for novel thrombin inhibitors.

Inhibition of alpha-synuclein aggregation by AM17, a synthetic resveratrol derivative

Parkinson's disease (PD) is linked to the aberrant self-assembly of the amyloid protein, α-synuclein (αS), where αS monomers aggregate to form oligomers and fibrils. Out of the three conformers, αS oligomers are the major toxic agents in PD, while αS fibrils may work as a reservoir for toxic oligomeric conformers. Thus, compounds that inhibit aggregation of αS monomers and disaggregate αS oligomers and fibrils may serve as therapeutic agents against PD. In this regard, resveratrol and its synthetic derivatives (e.g., AM17, which contains a copper ion-selective ionophoric motif) have previously been examined for their inhibitory effects on aggregation of amyloid proteins, such as the β-amyloid peptide implicated in Alzheimer's disease. In the current study, we employed an array of experimental tools, such as Thioflavin T fluorescence, transmission electron microscopy, immuno-dot blot assays, SDS- and native-PAGE, and circular dichroism, to determine the impact of AM17 and resveratrol on αS aggregation. To the best of our knowledge, we show for the first time that AM17 not only inhibits aggregation of αS monomers but also disaggregates αS oligomers and fibrils, independent of the copper ions. Similar αS aggregation inhibitory effects were observed with resveratrol only in the presence of the copper ion. The present study supports the high promise of applicability of AM17 as an effective amyloid aggregation inhibitor for various conformers and protein sequences.

Antithrombotic Potential of Red Allium cepa and Angelica gigas Nakai

Abstract: Introduction: Red Allium cepa and Angelica gigas Nakai is widely used in oriental medicine and also has various physiological activities. The main components of two substances will have potential benefits to blood flow improvement. Objectives: This study evaluated the cell viability and antioxidant effects of Red Allium cepa extract, Angelica gigas Nakai extract and their mixture and observed anticoagulation activity and antiplatelet aggregation activity. Methods: Red Allium cepa was bioconverted through the use of the Bacillus subtilis KJ-3 (BS3) strain after ethanol extraction (Red-BCQ). Angelica gigas Nakai extract (Agnex) contains a large amount of coumarins such as decursin and decursinol angelate (D/DA). Here, we observed antioxidant and antithrombotic effects after treatment with Red-BCQ, Agnex and mixture of these two compounds. Results: The quercetin content of Red-BCQ increased by about 7.4-fold after bioconversion. A 1 mg portion of Agnex contained 0.4146 mg of D and 0.3659 mg of DA. Red-BCQ and Agnex exerted strong antioxidant activities due to their high polyphenol and flavonoid content. In our study, Red-BCQ and Agnex exhibit beneficial effects to external coagulation pathways by significantly expanding aPTT. However, a combination of Red-BCQ and Agnex did not exert any synergistic effect on anti-coagulation activity. Additionally, our results concerning platelet aggregation inhibitory effects revealed that these effects increased by treatment with Agnex alone or in combination with Red-BCQ. Conclusion: Further research is required to clarify the underlying mechanism. However, Red-BCQ and Agnex are considered to possess antioxidant effects either alone or as a mixture and also exert antithrombotic effects. Key words: Red Allium cepa, Angelica gigas Nakai, Bacillus subtilis KJ-3, Bioconversion, Anti-coagulation.

Efficacy of Monitoring for Multiple Antiplatelet Therapy during Intracranial Stent Placement: A Preliminary Study.

During cerebral aneurysm embolization using intracranial stents, platelet aggregation increases owing to increased wall shear stress and a loss of vascular endothelial function at the stent implantation site. Preoperative multiple antiplatelet therapy was introduced to prevent severe thromboembolic complications due to increased platelet aggregation. However, specific guidelines for the administration and pharmacological evaluation of this therapy do not exist currently. We examined the benefits of perioperative platelet aggregation monitoring in a cohort of patients. We had 377 patients with unruptured intracranial aneurysms who underwent stent-assisted embolization at our hospital between December 2012 and November 2019. We ultimately included 181 patients in our final analysis. These patients were continuously administered aspirin (100 mg/day) and clopidogrel (75 mg/day) for more than 5 days before the procedure to the post-procedural period. Of these patients, 30 patients who underwent light transmission aggregometry (LTA) before procedure, post-procedure (3 days after procedure), and at first post-discharge clinic visit were included as the subjects. The following characteristics were studied: age; sex; presence/absence of hypertension, dyslipidemia, and/or diabetes mellitus; location of aneurysm; type/number of stent; technique for stent placement; duration of preoperative multiple antiplatelet therapy; perioperative platelet aggregation test results; and postoperative ischemic or hemorrhagic complications. Among these 30 patients, the median duration of antiplatelet therapy prior to the preoperative platelet aggregation measurements was 7 (interquartile range [IQR]: 6-8) days, and post-discharge measurement of LTA was performed at a median period of 27 (IQR: 22-35.5) days after procedure. The preoperative, postoperative, and first post-discharge clinic visit LTA values for adenosine diphosphate (ADP)-induced platelet aggregation were 50% (IQR: 44-54%), 42.5% (IQR: 36-48%), and 36% (IQR: 32-40%), respectively. These results represented gradual decrease in LTA values and a significant difference between the preoperative and post-discharge values. The LTA values for collagen aggregation showed a significant difference evident between the preoperative and post-discharge values; preoperative 38% (IQR: 27-60%), postoperative 42% (IQR: 30-58%), post-discharge 28% (IQR: 20-42%), respectively. We had one thromboembolic complication and one hemorrhagic complication. The results indicated that appropriate platelet aggregation monitoring during multiple antiplatelet therapy prevents thromboembolic complications such as stent thrombosis. However, we also found that many patients demonstrated increased postoperative platelet aggregation inhibitory effects due to the postoperative continuation of the same multiple antiplatelet therapy that was used preoperatively. This study demonstrates that postoperative, continuous, oral antiplatelet therapy induces increased platelet aggregation inhibition effects, which may lead to hemorrhagic complications. Therefore, continued platelet aggregation monitoring after surgery may be important to allow for any necessary alterations to the therapeutic dose and regimen.

Semi-Mechanistic Modeling of HY-021068 Based on Irreversible Inhibition of Thromboxane Synthetase.

Background: HY-021068 [4-(2-(1H-imidazol-1-yl) ethoxy)-3-methoxybenzoate], developed by Hefei Industrial Pharmaceutical Institute Co., Ltd. (Anhui, China), is a potential thromboxane synthetase inhibitor under development as an anti-platelet agent for the treatment of stroke. A semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was developed to characterize the PK of HY-021068 and its platelet aggregation inhibitory effect in beagle dogs. Method: Beagle dogs received single oral administration of 2.5 mg/kg HY-021068 or consecutively oral administration of 5 mg/kg HY-021068 once daily for 7 days. The plasma concentration of HY-021068 and the platelet aggregation rate (PAR) were determined by liquid chromatography tandem-mass spectrometry (LC‐MS/MS) assay and a photometric method, respectively. The PK/PD data was sequentially fitted by Phoenix NLME. The PK/PD parameters of HY-021068 in beagle dogs were estimated by 2.5 and 5 mg/kg dosing on the 1st day, and then used to simulate the PAR of HY-021068 on the 7th day after 5 mg/kg dosing daily. Result: A one-compartment model with saturable Michaelis-Menten elimination was best fitted to the PK of HY-021068. A mechanistic PD model based on irreversible inhibition of thromboxane synthetase was constructed to describe the relationship between plasma concentration of HY-021068 and PAR. Diagnostic plots showed no obvious bias. Visual predictive check confirmed the stability and reliability of the model. Most of PK/PD observed data on the 7th day after 5 mg/kg dosing fell in the 90% prediction interval. Conclusion: We established a semi-mechanistic PK/PD model for characterizing the PK of HY-021068 and its anti-platelet effect in beagle dogs. The model can be used to predict the concentration and PAR under different dosage regimen of HY-021068, and might be served as a reference for dose design in the future clinical studies.

Graphene oxide sheets and quantum dots inhibit α-synuclein amyloid formation by different mechanisms.

Aggregation and amyloid formation of the 140-residue presynaptic and intrinsically disordered protein α-synuclein (α-syn) is a pathological hallmark of Parkinson's disease (PD). Understanding how α-syn forms amyloid fibrils, and investigations of agents that can prevent their formation is therefore important. We demonstrate herein that two types of graphene oxide nanoparticles (sheets and quantum dots) inhibit α-syn amyloid formation by different mechanisms mediated via differential interactions with both monomers and fibrils. We have used thioflavin-T fluorescence assays and kinetic analysis, circular dichroism, dynamic light scattering, fluorescence spectroscopy and atomic force microscopy to asses the kinetic nature and efficiency of this inhibitory effect. We show that the two types of graphene oxide nanoparticles alter the morphology of α-syn fibrils, disrupting their interfilament assembly and the resulting aggregates therefore consist of single protofilaments. Our results further show that graphene oxide sheets reduce the aggregation rate of α-syn primarily by sequestering of monomers, thereby preventing primary nucleation and elongation. Graphene quantum dots, on the other hand, interact less avidly with both monomers and fibrils. Their aggregation inhibitory effect is primarily related to adsorption of aggregated species and reduction of secondary processes, and they can thus not fully prevent aggregation. This fine-tuned and differential effect of graphene nanoparticles on amyloid formation shows that rational design of these nanomaterials has great potential in engineering materials that interact with specific molecular events in the amyloid fibril formation process. The findings also provide new insight into the molecular interplay between amyloidogenic proteins and graphene-based nanomaterials in general, and opens up their potential use as agents to manipulate fibril formation.

Amyloid-β Aggregation Inhibitory and Neuroprotective Effects of Xanthohumol and its Derivatives for Alzheimer's Diseases.

Xanthohumol has been reported to have cytoprotection through activation of Nrf2-ARE signaling pathway and; it has capability of scavenging free radicals, suggesting its potential for the prevention of neurodegeneration. However, the bio-incompatibility and blood-brain barrier impermeability of xanthohumol hindered its in vivo efficacy potential for treating Alzheimer's disease (AD). We designed and prepared a series of xanthohumol derivatives to enhance the desirable physical, biological and pharmacological properties in particular the blood-brain barrier permeability for intervention of AD. We designed and synthesized a novel series of 9 xanthohumol derivatives. Their inhibitory effect on amyloid-β (1-42), Aβ1-42, oligomerization and fibrillation as well as neuroprotection against amyloid-β induced toxicities, were explored. Among the 9 xanthohumol derivatives, some of them exhibited a moderate to high inhibitory effect on Aβ1-42 oligomerization and fibrillation. They were biocompatible and neuroprotective to the SH-SY5Y cells by reducing the ROS generation and calcium uploading that were induced by the amyloid- β. Importantly, two of the derivatives were found to be blood-brain barrier permeable showing promising potential for AD treatment. Two derivatives have been identified to be biocompatible, non-toxic, neuroprotective against Aβ-induced toxicities and blood-brain barrier permeable highlighting their promising potential as AD drug candidates for future clinical use.

Adaptation and diversification of venomous snake proteins

According to the World Health Organization, approximately 1.8–2.7 million people worldwide suffer from venomous snake bites each year and at least 138,000 of these incidents are fatal. Whereas, snakebite envenoming poses a serious threat to public health, yet the snake venom toxins endow several pharmacological effects, including presynaptic neurotoxicity, myotoxicity, and cardiotoxicity, as well as anticoagulant, hemolytic, hemorrhagic, edema-inducing, and platelet aggregation-inhibiting effects. Duplication and mutation of the genes encoding these toxins play an important role in generating molecular diversity. Curiously, venomous snakes are not lethal to the viper itself because the viper’s resistance against its own venom. However, endogenous inhibitor proteins evolutionarily acquired by venomous snakes to protect themselves have not yet been fully characterised because it is unclear how to inhibit for target toxin due to the lack of information including mutation analysis and the three-dimensional structures of the inhibitors. This review provides an overview of endogenous inhibitors of venomous snake as regulation systems for the toxin proteins. Recently, we isolated some inhibitors targeting different toxins from the sera of the Japanese vipers. We investigated the evolution of these endogenous inhibitors, which have been significantly influenced by positive selection. Directional mutagenesis, where mutation hotspots are found in genes encoding molecular surface proteins and functional domains of these proteins, acts as a diversifying mechanism for the exquisite biological targeting necessary to protect the host from its own venom.

Bioactivity-guided identification of flavonoids with cholinesterase and β-amyloid peptide aggregation inhibitory effects from the seeds of Millettia pachycarpa

Millettia pachycarpa Benth, a widely used anthelminthic drug in folk, is rich in flavonoids with various bioactivities. This study aimed to identify active flavonoids with anti-Alzheimer’s disease (AD) effect from its seeds by a bioassay-guided isolation. A novel rotenoid with unusual oxidative ring-opening skeleton (10) and nine known flavonoids (1–9) were obtained, and their structures were elucidated by NMR and HR-ESIMS analysis. Among all isolates, 7 and 8 showed selective butyrylcholinesterase (BChE) inhibitory activities (IC50 = 2.34 and 11.49 μM, respectively), while 3 was classified as a dual-action inhibitor against acetylcholinesterase (AChE) and BChE (IC50 AChE = 17.14 μM, IC50 BChE = 5.68 μM). Further kinetic study revealed that 3, 7, and 8 were mixed-type BChE inhibitors, but 3 was a competitive AChE inhibitor. Their strong binding affinities to BChE were confirmed by fluorescence quenching analysis. Additionally, 3 and 8 exhibited potent inhibitory effects against β-amyloid peptide aggregation. These results revealed M. pachycarpa could be a valuable source for anti-AD leads development, and compounds 3, 7 and 8 were worthy of further study as multifunctional or specific agents for AD treatment.