Multiepitope Subunit Peptide-Based Nanovaccine against Porcine Circovirus Type 2 (PCV2) Elicited High Antibody Titers in Vaccinated Mice.

Porcine circovirus 2 (PCV2) infection is one of the most serious threats to the swine industry. While the disease can be prevented, to some extent, by commercial PCV2a vaccines, the evolving nature of PCV2 necessitates the development of a novel vaccine that can compete with the mutations of the virus. Thus, we have developed novel multiepitope vaccines based on the PCV2b variant. Three PCV2b capsid protein epitopes, together with a universal T helper epitope, were synthesized and formulated with five delivery systems/adjuvants: complete Freund's adjuvant, poly(methyl acrylate) (PMA), poly(hydrophobic amino acid), liposomes and rod-shaped polymeric nanoparticles built from polystyrene-poly(N-isopropylacrylamide)-poly(N-dimethylacrylamide). Mice were subcutaneously immunized with the vaccine candidates three times at three-week intervals. All vaccinated mice produced high antibody titters after three immunizations as analyzed by the enzyme-linked immunosorbent assay (ELISA), while mice vaccinated with PMA-adjuvanted vaccine elicited high antibody titers even after a single immunization. Thus, the multiepitope PCV2 vaccine candidates designed and examined here show strong potential for further development.

Comparative Evaluation of the Antioxidant and Anti-Alzheimer's Disease Potential of Coumestrol and Puerarol Isolated from Pueraria lobata Using Molecular Modeling Studies.

The current study assesses the antioxidant effects of two similar isoflavonoids isolated from Pueraria lobata, coumestrol and puerarol, along with the cholinergic and amyloid-cascade pathways to mitigate Alzheimer's disease (AD). Antioxidant activity was evaluated via 1,1-diphenyl-2-picryhydrazyl (DPPH) and peroxynitrite (ONOO-) scavenging ability further screened via ONOO--mediated nitrotyrosine. Similarly, acetyl- and butyrylcholinesterase (AChE/BChE) and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitory activities were assessed together with docking and kinetic studies. Considering DPPH and ONOO- scavenging activity, coumestrol (EC50 values of 53.98 and 1.17 µM) was found to be more potent than puerarol (EC50 values of 82.55 and 6.99 µM) followed by dose dependent inhibition of ONOO--mediated nitrotyrosine. Coumestrol showed pronounced AChE and BChE activity with IC50 values of 42.33 and 24.64 µM, respectively, acting as a dual cholinesterase (ChE) inhibitor. Despite having weak ChE inhibitory activity, puerarol showed potent BACE1 inhibition (28.17 µM). Kinetic studies of coumestrol showed AChE and BChE inhibition in a competitive and mixed fashion, whereas puerarol showed mixed inhibition for BACE1. In addition, docking simulations demonstrated high affinity and tight binding capacity towards the active site of the enzymes. In summary, we undertook a comparative study of two similar isoflavonoids differing only by a single aliphatic side chain and demonstrated that antioxidant agents coumestrol and puerarol are promising, potentially complementary therapeutics for AD.

Structure⁻Activity Relationship of the Tyrosinase Inhibitors Kuwanon G, Mulberrofuran G, and Albanol B from Morus Species: A Kinetics and Molecular Docking Study.

Kuwanon G (KG) and benzofuran flavonoids such as mulberrofuran G (MG) and albanol B (AB) isolated from Morus sp. are reported to exhibit anti-Alzheimer's disease, anti-inflammatory, fungicidal, anti-cancer, anti-bacterial, and anti-tyrosinase properties. We investigated the inhibition of mono- and diphenolase activity of mushroom tyrosinase by KG, MG, and AB. KG and MG displayed acceptable inhibition activity compared to kojic acid. AB did not show any activity up to 350 µM. MG displayed six-fold higher inhibition of l-tyrosine oxidation (IC50 = 6.35 ± 0.45 µM) compared to kojic acid (IC50 = 36.0 µM). Kinetic studies revealed that KG and MG inhibited monophenolase activity of tyrosinase in a competitive manner. Docking simulations of KG and MG demonstrated favorable binding energies with amino acid residues of the active sites of tyrosinase. Our investigation of the structure-activity relationship of the fused benzofuran flavonoids (MG vs. AB) implicated the methyl cyclohexene ring moiety in tyrosinase inhibition. The enzyme substrate and relative structural analyses demonstrated that KG and MG from Morus sp. could be useful natural tyrosinase inhibitors in foods or cosmetics.

Cholesterol as an inbuilt immunoadjuvant for a lipopeptide vaccine against group A Streptococcus infection.

Peptide-based vaccines can trigger highly specific immune responses, although peptides alone are usually unable to confer strong humoral or cellular immunity. Consequently, peptide antigens are administered with immunostimulatory adjuvants, but only a few are safe and effective for human use. To overcome this obstacle, herein a peptide antigen was lipidated to effectively anchor it to liposomes and emulsion. A peptide antigen B cell epitope from Group A Streptococcus M protein was conjugated to a universal T helper epitope, the pan DR-biding epitope (PADRE), alongside a lipidic moiety cholesterol. Compared to a free peptide antigen, the lipidated version (LP1) adopted a helical conformation and self-assembled into small nanoparticles. Surprisingly, LP1 alone induced the same or higher antibody titers than liposomes or emulsion-based formulations. In addition, antibodies produced by mice immunized with LP1 were more opsonic than those induced by administering the antigen with incomplete Freund's adjuvant. No side effects were observed in the immunized mice and no excessive inflammatory immune responses were detected. Overall, this study demonstrated how simple conjugation of cholesterol to a peptide antigen can produce a safe and efficacious vaccine against Group A Streptococcus - the leading cause of superficial infections and the bacteria responsible for deadly post-infection autoimmune disorders.

Self-assembled monovalent lipidated mannose ligand as a standalone nanoadjuvant.

Subunit vaccines require an immunostimulant (adjuvant) and/or delivery system to induce immunity. However, currently, available adjuvants are either too dangerous in terms of side effects for human use (experimental adjuvants) or have limited efficacy and applicability. In this study, we examined the capacity of mannose-lipopeptide ligands to enhance the immunogenicity of a vaccine consisting of polyleucine(L15)-antigen conjugates anchored to liposomes. The clinically tested Group A Streptococcus (GAS) B-cell epitope, J8, combined with universal T helper PADRE (P) was used as the antigen. Six distinct mannose ligands were incorporated into neutral liposomes carrying L15PJ8. While induced antibody titers were relatively low, the ligand carrying mannose, glycine/lysine spacer, and two palmitic acids as liposomal membrane anchoring moieties (ligand 3), induced significantly higher IgG titers than non-mannosylated liposomes. The IgG titers were significantly enhanced when positively charged liposomes were employed. Importantly, the produced antibodies were able to kill GAS bacteria. Unexpectedly, the physical mixture of only ligand 3 and PJ8 produced self-assembled nanorods that induced antibody titers as high as those elicited by the lead liposomal formulation and antigen adjuvanted with the potent, but toxic, complete Freund's adjuvant (CFA). Antibodies produced upon immunization with PJ8 + 3 were even more opsonic than those induced by CFA + PJ8. Importantly, in contrast to CFA, ligand 3 did not induce observable adverse reactions or excessive inflammatory responses. Thus, we demonstrated that a mannose ligand, alone, can serve as an effective vaccine nanoadjuvant.

Polymeric Nanoparticles as Oral and Intranasal Peptide Vaccine Delivery Systems: The Role of Shape and Conjugation.

Mucosal vaccines are highly attractive due to high patient compliance and their suitability for mass immunizations. However, all currently licensed mucosal vaccines are composed of attenuated/inactive whole microbes, which are associated with a variety of safety concerns. In contrast, modern subunit vaccines use minimal pathogenic components (antigens) that are safe but typically poorly immunogenic when delivered via mucosal administration. In this study, we demonstrated the utility of various functional polymer-based nanostructures as vaccine carriers. A Group A Streptococcus (GAS)-derived peptide antigen (PJ8) was selected in light of the recent global spread of invasive GAS infection. The vaccine candidates were prepared by either conjugation or physical mixing of PJ8 with rod-, sphere-, worm-, and tadpole-shaped polymeric nanoparticles. The roles of nanoparticle shape and antigen conjugation in vaccine immunogenicity were demonstrated through the comparison of three distinct immunization pathways (subcutaneous, intranasal, and oral). No additional adjuvant or carrier was required to induce bactericidal immune responses even upon oral vaccine administration.

Synthetic Anti-Cocaine Nanoaccine Successfully Prevents Cocaine-Induced Hyperlocomotion.

Cocaine is one of the most widely used and increasingly popular illicit psychoactive drugs. Unlike other commonly used substances of abuse, cocaine has no pharmacological therapies to treat addiction or aid in rehabilitation. Immunopharmacology has long been touted as a possible avenue to develop effective anticocaine therapies; however, lack of efficacy and designs which are not consistent with simple large-scale production have hindered vaccine translation. We have designed and synthesized a peptide-based anti-cocaine immunogen which we have shown is capable of inducing physiologically relevant immune responses in mice as part of a self-adjuvanting delivery system or in combination with the human-approved commercial adjuvant MF59. We have demonstrated that immunization with the reported vaccine elicits high titers of anti-cocaine IgG and prevents cocaine-induced hyperlocomotion in an in vivo murine model. This peptide-hapten immunogen along with self-adjuvanting liposomal-based delivery system provides a platform for the development of effective anti-drug vaccines.

Liposomal Formulations of a Polyleucine-Antigen Conjugate as Therapeutic Vaccines against Cervical Cancer.

Human papilloma virus (HPV) is responsible for all cases of cervical cancer. While prophylactic vaccines are available, the development of peptide-based vaccines as a therapeutic strategy is still under investigation. In comparison with the traditional and currently used treatment strategies of chemotherapy and surgery, vaccination against HPV is a promising therapeutic option with fewer side effects. A peptide derived from the HPV-16 E7 protein, called 8Qm, in combination with adjuvants showed promise as a therapeutic vaccine. Here, the ability of polymerized natural amino acids to act as a self-adjuvating delivery system as a therapeutic vaccine was investigated for the first time. Thus, 8Qm was conjugated to polyleucine by standard solid-phase peptide synthesis and self-assembled into nanoparticles or incorporated in liposomes. The liposome bearing the 8Qm conjugate significantly increased mice survival and decreased tumor growth after a single immunization. Further, these liposomes eradicated seven-day-old well-established tumors in mice. Dendritic cell (DC)-targeting moieties were introduced to further enhance vaccine efficacy, and the newly designed liposomal vaccine was tested in mice bearing 11-day-old tumors. Interestingly, these DCs-targeting moieties did not significantly improve vaccine efficacy, whereas the simple liposomal formulation of 8Qm-polyleucine conjugate was still effective in tumor eradication. In summary, a peptide-based anticancer vaccine was developed that stimulated strong cellular immune responses without the help of a classical adjuvant.

Activity Relationship of Poly(ethylenimine)-Based Liposomes as Group A Streptococcus Vaccine Delivery Systems.

Untreated group A Streptococcus (GAS) can lead to a range of life-threatening diseases, including rheumatic heart disease. To date, no therapeutic or prophylactic vaccines are commercially available to treat or prevent GAS infection. Development of a peptide-based subunit vaccine offers a promising solution, negating the safety issues of live-attenuated or inactive vaccines. Subunit vaccines administer small peptide fragments (antigens), which are typically poorly immunogenic. Therefore, these peptide antigens require formulation with an immune stimulant and/or vaccine delivery platform to improve their immunogenicity. We investigated polyelectrolyte complexes (PECs) and polymer-coated liposomes as self-adjuvanting delivery vehicles for a GAS B cell peptide epitope conjugated to a universal T-helper epitope and a synthetic toll-like receptor 2-targeting moiety lipid core peptide-1 (LCP-1). A structure-activity relationship of cationic PEC vaccines containing different external PEI-coatings (poly(ethylenimine); 10 kDa PEI, 25 kDa PEI, and a synthetic mannose-functionalized 25 kDa PEI) formed vaccines PEC-1, PEC-2, and PEC-3, respectively. All three PEC vaccines induced J8-specific systemic immunoglobulin G (IgG) antibodies when administered intranasally to female BALB/c mice without the use of additional adjuvants. Interestingly, PEC-3 induced the highest antibody titers among all tested vaccines, with the ability to effectively opsonize two clinically isolated GAS strains. A comparative study of PEC-2 and PEC-3 with liposome-based delivery systems was performed subcutaneously. LCP-1 was incorporated into a liposome formulation (DPPC, DPPG and cholesterol), and the liposomes were externally coated with PEI (25 kDa; Lip-2) or mannosylated PEI (25 kDa; Lip-3). All liposome vaccines induced stronger humoral immune responses compared to their PEC counterparts. Notably, sera of mice immunized with Lip-2 and Lip-3 produced significantly higher opsonic activity against clinically isolated GAS strains compared to the positive control, P25-J8 emulsified with the commercial adjuvant, complete Freund's adjuvant (CFA). This study highlights the capability of a PEI-liposome system to act as a self-adjuvanting vehicle for the delivery of GAS peptide antigens and protection against GAS infection.

Current Prospects in Peptide-Based Subunit Nanovaccines.

Vaccination renders protection against pathogens via stimulation of the body's natural immune responses. Classical vaccines that utilize whole organisms or proteins have several disadvantages, such as induction of undesired immune responses, poor stability, and manufacturing difficulties. The use of minimal immunogenic pathogen components as vaccine antigens, i.e., peptides, can greatly reduce these shortcomings. However, subunit antigens require a specific delivery system and immune adjuvant to increase their efficacy. Recently, nanotechnology has been extensively utilized to address this issue. Nanotechnology-based formulation of peptide vaccines can boost immunogenicity and efficiently induce cellular and humoral immune responses. This chapter outlines the recent developments and advances of nano-sized delivery platforms for peptide antigens, including nanoparticles composed of polymers, peptides, lipids, and inorganic materials.

Investigation of liposomal self-adjuvanting peptide epitopes derived from conserved blood-stage Plasmodium antigens.

Malaria is a vector born parasitic disease causing millions of deaths every year. Despite the high mortality rate, an effective vaccine against this mosquito-borne infectious disease is yet to be developed. Up to date, RTS,S/AS01 is the only vaccine available for malaria prevention; however, its efficacy is low. Among a variety of malaria antigens, merozoite surface protein-1(MSP-1) and ring-infected erythrocyte surface antigen (RESA) have been proposed as promising candidates for malaria vaccine development. We developed peptide-based Plasmodium falciparum vaccine candidates that incorporated three previously reported conserved epitopes from MSP-1 and RESA into highly effective liposomal polyleucine delivery system. Indeed, MSP-1 and RESA-derived epitopes conjugated to polyleucine and formulated into liposomes induced higher epitope specific antibody titres. However, immunized mice failed to demonstrate protection in a rodent malaria challenge study with Plasmodium yoelii. In addition, we found that the three reported P. falciparum epitopes did not to share conformational properties and high sequence similarity with P. yoelii MSP-1 and RESA proteins, despite the epitopes were reported to protect mice against P. yoelii challenge.

Structure-activity relationship of lipid, cyclic peptide and antigen rearrangement of physically mixed vaccines.

Currently there is no approved vaccine to prevent and/or treat group A Streptococcus (GAS) infection. With increasing reports of GAS antibiotic resistance, vaccine adjuvants and targeted delivery systems which induce a strong immune response are a widely acknowledged unmet need. Through extensive structure-activity studies, we investigated a cyclic decapeptide physically mixed with a GAS B cell peptide epitope (J8), a universal T helper epitope (PADRE), and different synthetic lipidic moieties as a conceivable self-adjuvanting GAS vaccine. We explored the structure (orientation)-relationship of the chemically-conjugated B cell epitope and T helper epitope peptide as part of this physically-mixed vaccine. Following in vivo assessment in mice, these cyclic lipopeptide vaccines showed successful induction of J8-specific systemic IgG antibodies when administered subcutaneously without additional adjuvant. Interestingly, an exposed C-terminus of the GAS B cell epitope and a 16-carbon alpha-amino fatty acid lipid was required for strong immunoreactivity, capable of effectively opsonising multiple strains of clinically-isolated GAS bacteria. Physicochemical assessment proved the alpha helix structure of the GAS B cell epitope was retained, impacting particle self-assembly and vaccine immunoreactivity. This study showed the capability for a self-adjuvanting cyclic delivery system to act as a vehicle for the delivery of GAS peptide antigens to treat GAS infection.

Development of Multilayer Nanoparticles for the Delivery of Peptide-Based Subunit Vaccine against Group A Streptococcus.

Peptide-based subunit vaccines include only minimal antigenic determinants, and, therefore, are less likely to induce allergic immune responses and adverse effects compared to traditional vaccines. However, peptides are weakly immunogenic and susceptible to enzymatic degradation when administered on their own. Hence, we designed polyelectrolyte complex (PEC)-based delivery systems to protect peptide antigens from degradation and improve immunogenicity. Lipopeptide (LCP-1) bearing J8 B-cell epitope derived from Group A Streptococcus (GAS) M-protein was selected as the model peptide antigen. In the pilot study, LCP-1 incorporated in alginate/cross-linked polyarginine-J8-based PEC induced high J8-specific IgG antibody titres. The PEC system was then further modified to improve its immune stimulating capability. Of the formulations tested, PEC-4, bearing LCP-1, alginate and cross-linked polylysine, induced the highest antibody titres in BALB/c mice following subcutaneous immunisation. The antibodies produced were more opsonic than those induced by mice immunised with other PECs, and as opsonic as those induced by antigen adjuvanted with powerful complete Freund's adjuvant.

Poly(hydrophobic Amino Acids) and Liposomes for Delivery of Vaccine against Group A Streptococcus.

Adjuvants and delivery systems are essential components of vaccines to increase immunogenicity against target antigens, particularly for peptide epitopes (poor immunogens). Emulsions, nanoparticles, and liposomes are commonly used as a delivery system for peptide-based vaccines. A Poly(hydrophobic amino acids) delivery system was previously conjugated to Group A Streptococcus (GAS)-derived peptide epitopes, allowing the conjugates to self-assemble into nanoparticles with self adjuvanting ability. Their hydrophobic amino acid tail also serves as an anchoring moiety for the peptide epitope, enabling it to be integrated into the liposome bilayer, to further boost the immunological responses. Polyleucine-based conjugates were anchored to cationic liposomes using the film hydration method and administered to mice subcutaneously. The polyleucine-peptide conjugate, its liposomal formulation, and simple liposomal encapsulation of GAS peptide epitope induced mucosal (saliva IgG) and systemic (serum IgG, IgG1 and IgG2c) immunity in mice. Polyleucine acted as a potent liposome anchoring portion, which stimulated the production of highly opsonic antibodies. The absence of polyleucine in the liposomal formulation (encapsulated GAS peptide) induced high levels of antibody titers, but with poor opsonic ability against GAS bacteria. However, the liposomal formulation of the conjugated vaccine was no more effective than conjugates alone self-assembled into nanoparticles.

Polyacrylate-GnRH Peptide Conjugate as an Oral Contraceptive Vaccine Candidate.

Contraceptive vaccines are designed to elicit immune responses against major components of animal reproductive systems. These vaccines, which are most commonly administered via injection, typically target gonadotropin-releasing hormone (GnRH). However, the need to restrain animals for treatment limits the field applications of injectable vaccines. Oral administration would broaden vaccine applicability. We explored contraceptive vaccine candidates composed of GnRH peptide hormone, universal T helper PADRE (P), and a poly(methylacrylate) (PMA)-based delivery system. When self-assembled into nanoparticles, PMA-P-GnRH induced the production of high IgG titers after subcutaneous and oral administration in mice. PADRE was then replaced with pig T helper derived from the swine flu virus, and the vaccine was tested in pigs. High levels of systemic antibodies were produced in pigs after both injection and oral administration of the vaccine. In conclusion, we developed a simple peptide-polymer conjugate that shows promise as an effective, adjuvant-free, oral GnRH-based contraceptive vaccine.

Physical mixture of a cyclic lipopeptide vaccine induced high titres of opsonic IgG antibodies against group A streptococcus.

Untreated or reoccurring group A Streptococcus (GAS) infection can lead to a number of post-infection complications, including rheumatic heart disease. There is no licenced vaccine for the treatment or prevention of GAS infection. We identified that a cyclic decapeptide plays a significant positive influence on the adjuvant activity of several lipid-antigen mixtures. Here, three synthetic vaccine components were synthesised: (1) J8-PADRE represents the GAS B cell antigen (J8) conjugated to the universal T helper epitope (PADRE); (2) a synthetic toll like receptor 2 (TLR2) ligand based on a C16 alkyl chain lipid moiety; and (3) a cyclic carrier deca-peptide. Previously, through structure-immune activity investigations, it was observed that a physical mixture of these three components had significantly higher IgG immune responses when compared to a fully conjugated vaccine construct. Expanding the scope of this structure-activity investigation, we show that the presence of the cyclic peptide is required for the induction of a strong, balanced Th1/Th2 immune response when compared with lipid and antigen only, and cyclic lipopeptide plus B/T cell antigen physical mixtures.

Carbohydrate Immune Adjuvants in Subunit Vaccines.

Modern subunit vaccines are composed of antigens and a delivery system and/or adjuvant (immune stimulator) that triggers the desired immune responses. Adjuvants mimic pathogen-associated molecular patterns (PAMPs) that are typically associated with infections. Carbohydrates displayed on the surface of pathogens are often recognized as PAMPs by receptors on antigen-presenting cells (APCs). Consequently, carbohydrates and their analogues have been used as adjuvants and delivery systems to promote antigen transport to APCs. Carbohydrates are biocompatible, usually nontoxic, biodegradable, and some are mucoadhesive. As such, carbohydrates and their derivatives have been intensively explored for the development of new adjuvants. This review assesses the immunological functions of carbohydrate ligands and their ability to enhance systemic and mucosal immune responses against co-administered antigens. The role of carbohydrate-based adjuvants/delivery systems in the development of subunit vaccines is discussed in detail.

Recent advances in pharmacological research on Ecklonia species: a review.

The genus Ecklonia (Lessoniaceae, Phaeophyceae), commonly called kelp (brown algae), is abundant on the coasts of Japan and Korea. During the past few decades, Ecklonia species have received tremendous attention for their wide range of therapeutic properties and multiple health benefits, such as great nutritional value and being rich in vitamins, minerals, dietary fiber, proteins, and polysaccharides. Several novel functional ingredients with diversified biological activities have been isolated and possess antimicrobial, antiviral, hepatoprotective, cardioprotective, anti-inflammatory, neuroprotective, anticarcinogenic, immunomodulatory, hypolipidemic, anti-diabetic, and antioxidant therapeutic properties. The present review discusses the phytochemical, pharmacological, therapeutic, nutritional, and health benefits of different species of genus Ecklonia, as well as their use in the prevention of disease and maintenance of good health.

Comparative molecular docking studies of lupeol and lupenone isolated from Pueraria lobata that inhibits BACE1: Probable remedies for Alzheimer's disease.

OBJECTIVE: To discover lead lupane triterpenoid's potential isolated from Pueraria lobata roots against ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), which serve as a rate limiting step in amyloid beta (Aß) production altering the course of Alzheimer's disease. In addition, enzyme kinetics study and molecular docking were conducted to establish the inhibition type and structure activity relationship. METHODS: A systematic study of 70% ethanolic P. lobata root extract was employed to identify its BACE1 inhibitory potential. Further, BACE1 inhibitory potential of two lupane terpenoids, yielded from ethanolic extract, was assessed. In order to determine their inhibition mode, Lineweaver-Burk plots and Michaelis-Menten model for BACE1 was performed. AutoDock 4.2 program in addition determined the molecular interaction of BACE1 with isolated terpenoids. RESULTS: Considering the inhibitory potential of 70% ethanolic extract of P. lobata against BACE1 (IC50 = 80.35 µg/mL), lupeol and lupenone were subsequently isolated and exhibited notable or moderate BACE1 inhibitory activity with IC50 values of 5.12 and 62.98 µmol/L, respectively, as compared to the positive control quercetin (IC50 = 21.28 µmol/L). The enzyme kinetics study enabled us to identify both compounds as competitive inhibitors, where lupeol displayed a very potent inhibition against BACE1 with low inhibition constant (Ki) value of 1.43 µmol/L, signifying greater binding affinity. In order to understand the binding mechanism and structure-activity relationship of two triterpene-based BACE1 inhibitors, we employed computer aided docking studies which evidently revealed that hydroxyl group of lupeol formed two hydrogen bonds with the ASP32 (catalytic aspartic residue) and SER35 residues of BACE1 with the binding energy of (-8.2 kcal/mol), while the ketone group of lupenone did not form any hydrogen bonds with BACE1 giving evidence for less binding affinity. These results in turn have predicted the dependence of the inhibitory activity in the presence of hydroxyl group which has provided a new basis for BACE1 blockade. CONCLUSIONS: Our results have successfully explored the molecular mechanism of lupane triterpenoids via BACE1 inhibition, suggesting that lupeol in particular could be utilized as a useful therapeutic and preventive agent to mitigate Alzheimer's disease.