Molecular Dynamics Analysis of Antibody Recognition and Escape by Human H1N1 Influenza Hemagglutinin

The antibody immunoglobulin (Ig) 2D1 is effective against the 1918 hemagglutinin (HA) and also known to cross-neutralize the 2009 pandemic H1N1 influenza HA through a similar epitope. However, the detailed mechanism of neutralization remains unclear. We conducted molecular dynamics (MD) simulations to study the interactions between Ig-2D1 and the HAs from the 1918 pandemic flu (A/South Carolina/1/1918, 18HA), the 2009 pandemic flu (A/California/04/2009, 09HA), a 2009 pandemic flu mutant (A/California/04/2009, 09HA_mut), and the 2006 seasonal flu (A/Solomon Islands/3/2006, 06HA). MM-PBSA analyses suggest the approximate free energy of binding (ΔG) between Ig-2D1 and 18HA is −74.4 kcal/mol. In comparison with 18HA, 09HA and 06HA bind Ig-2D1 ∼6 kcal/mol (ΔΔG) weaker, and the 09HA_mut bind Ig-2D1 only half as strong. We also analyzed the contributions of individual epitope residues using the free-energy decomposition method. Two important salt bridges are found between the HAs and Ig-2D1. In 09HA, a serine-to-asparagine mutation coincided with a salt bridge destabilization, hydrogen bond losses, and a water pocket formation between 09HA and Ig-2D1. In 09HA_mut, a lysine-to-glutamic-acid mutation leads to the loss of both salt bridges and destabilizes interactions with Ig-2D1. Even though 06HA has a similar ΔG to 09HA, it is not recognized by Ig-2D1 in vivo. Because 06HA contains two potential glycosylation sites that could mask the epitope, our results suggest that Ig-2D1 may be active against 06HA only in the absence of glycosylation. Overall, our simulation results are in good agreement with observations from biological experiments and offer novel mechanistic insights, to our knowledge, into the immune escape of the influenza virus.     

A complex of influenza hemagglutinin with a neutralizing antibody that binds outside the virus receptor binding site.

The structure of a complex of influenza hemagglutinin (HA) with a neutralizing antibody shows that the antibody binds to HA at a distance from the virus receptor binding site. Comparison of the properties of this antibody and its Fab with those of an antibody that recognizes an epitope overlapping the receptor binding site leads to two main conclusions. First, inhibition of receptor binding is an important component of neutralization. Second, the efficiency of neutralization by the antibodies ranks in the same order as their avidities for HA, and their large size makes these antibodies highly efficient at neutralization, regardless of the location of their epitope in relation to the virus receptor binding site. These observations provide rationales for the range of antibody specificities that are detected in immune sera and for the distribution of sequence changes on the membrane-distal surface of influenza HAs that occur during 'antigenic drift.'     

Heptapeptide ligands against receptor-binding sites of influenza hemagglutinin toward anti-influenza therapy

The initial attachment of influenza virus to cells is the binding of hemagglutinin (HA) to the sialyloligosaccharide receptor; therefore, the small molecules that inhibit the sugar–protein interaction are promising as HA inhibitors to prevent the infection. We herein demonstrate that sialic acid-mimic heptapeptides are identified through a selection from a primary library against influenza virus HA. In order to obtain lead peptides, an affinity selection from a phage-displayed random heptapeptide library was performed with the HAs of the H1 and H3 strains, and two kinds of the HA-binding peptides were identified. The binding of the peptides to HAs was inhibited in the presence of sialic acid, and plaque assays indicated that the corresponding N-stearoyl peptide strongly inhibited infections by the A/Aichi/2/68 (H3N2) strain of the virus. Alanine scanning of the peptides indicated that arginine and proline were responsible for binding. The affinities of several mutant peptides with single-amino-acid substitutions against H3 HA were determined, and corresponding docking studies were performed. A Spearman analysis revealed a correlation between the affinity of the peptides and the docking study. These results provide a practicable method to design of peptide-based HA inhibitors that are promising as anti-influenza drugs.     

Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site

Influenza virus is a global health concern due to its unpredictable pandemic potential. This potential threat was realized in 2009 when an H1N1 virus emerged that resembled the 1918 virus in antigenicity but fortunately was not nearly as deadly. 5J8 is a human antibody that potently neutralizes a broad spectrum of H1N1 viruses, including the 1918 and 2009 pandemic viruses. Here, we present the crystal structure of 5J8 Fab in complex with a bacterially expressed and refolded globular head domain from the hemagglutinin (HA) of the A/California/07/2009 (H1N1) pandemic virus. 5J8 recognizes a conserved epitope in and around the receptor binding site (RBS), and its HCDR3 closely mimics interactions of the sialic acid receptor. Electron microscopy (EM) reconstructions of 5J8 Fab in complex with an HA trimer from a 1986 H1 strain and with an engineered stabilized HA trimer from the 2009 H1 pandemic virus showed a similar mode of binding. As for other characterized RBS-targeted antibodies, 5J8 uses avidity to extend its breadth and affinity against divergent H1 strains. 5J8 selectively interacts with HA insertion residue 133a, which is conserved in pandemic H1 strains and has precluded binding of other RBS-targeted antibodies. Thus, the RBS of divergent HAs is targeted by 5J8 and adds to the growing arsenal of common recognition motifs for design of therapeutics and vaccines. Moreover, consistent with previous studies, the bacterially expressed H1 HA properly refolds, retaining its antigenic structure, and presents a low-cost and rapid alternative for engineering and manufacturing candidate flu vaccines.     

Site-directed fragment-based generation of virtual sialic acid databases against influenza A hemagglutinin

In this study fragment-based drug design is combined with molecular docking simulation technique, to design databases of virtual sialic acid (SA) analogues with new substitutions at C2, C5 and C6 positions of SA scaffold. Using spaces occupied by C2, C5 and C6 natural moieties of SA when bound to hemagglutinin (HA) crystallographic structure, new fragments that are commercially available were docked independently in all the pockets. The oriented fragments were then connected to the SA scaffold with or without incorporation of linker molecules. The completed analogues were docked to the whole SA binding site to estimate their binding conformations and affinities, generating three databases of HA-bound SA analogues. Selected new analogues showed higher estimated affinities than the natural SA when tested against H3N2, H5N1 and H1N1 subtypes of influenza A. An improvement in the binding energies indicates that fragment-based drug design when combined with molecular docking simulation is capable to produce virtual analogues that can become lead compound candidates for anti-flu drug discovery program.     

Influenza PR8 HA-specific Fab fragments produced by phage display methods

Anti-influenza hemagglutinin (HA) Fabs were isolated from a phage display library using purified HA of influenza virus A/Puerto Rico/8/34 (PR8; H1N1) as an antigen. Four Fab clones displaying a 25-50-fold higher binding signal to PR8 HA than the control were selected for further analysis and comparison with anti-PR8 monoclonal antibody (mAb). All four Fabs and mAb recognized the PR8 HA under non-reducing conditions but rarely bound to reduced PR8 HA. Inhibition of influenza virus infection on MDCK cells was observed with Fab1 and mAb in a dose-dependent manner while Fab3 and 4 exhibited only a partial inhibitory effect. Moreover, Fab1 clone and mAb exhibited cross-reactivity with the A/Peking/262/95 (A/Peking; H1N1) strain. The inhibitory effects of mAb on both influenza strains were more potent than Fab1, which is likely attributed to its higher affinity for the antigen. SPR analyses, in fact, revealed that Fab1 and mAb have K_D of 1.5 × 10⁻⁸ and 3.2 × 10⁻⁹ M, respectively. These results strongly suggest that phage library-derived Fabs can be readily prepared and that such HA-specific Fabs with inhibitory action on influenza infection may be used to treat influenza patients.     

Sequence analysis and receptor specificity of the hemagglutinin of a recent influenza H2N2 virus isolated from chicken in North America

Influenza viruses bind host cells following an interaction between the viral hemagglutinin (HA) protein and host cell sialylated glycoproteins and glycolipids. Differences in binding affinities of the HAs for different types of sialic acid linkages (α2-3 vs. α2-6) contribute to determining the host range of an influenza virus. The ability of an avian influenza virus HA to bind the human form of the receptor may be one requirement for an avian virus to propagate in the human population. In this paper, we describe the characterization of the HA from an H2N2 virus isolated from a Pennsylvania chicken farm in 2004. Sequence analysis revealed that this HA is a member of the Eurasian clade, and receptor binding studies show that it maintains its specificity for the avian influenza virus α2-3 linked sialic acid receptor.     

A novel peptide inhibits the influenza virus replication by preventing the viral attachment to the host cells

Avian influenza viruses (AIV), the causative agent of avian flu or bird flu, cause widespread morbidity and mortality in poultry. The symptoms of the disease range from mild flu like symptoms to death. These viruses possess two important surface glycoproteins, namely hemagglutinin (HA) and neuraminidase (NA) against which neutralizing antibodies are produced. Due to the highly mutative nature of the genes which encode these proteins, the viruses often confer resistance to the current anti-viral drugs making the prevention and treatment of infection challenging. In our laboratory, we have recently identified a novel anti-viral peptide (P1) against the AIV H9N2 from a phage displayed peptide library. This peptide inhibits the replication of the virus in ovo and in vitro by its binding to the HA glycoprotein. In the current study, we demonstrate that the peptide inhibits the virus replication by preventing the attachment to the host cell but it does not have any effect on the viral fusion. The reduction in the viral nucleoprotein (NP) expression inside the host cell has also been observed during the peptide (P1) treatment. This novel peptide may have the potential to be developed as a therapeutic agent for the treatment and control of avian influenza virus H9N2 infections.     

Isolation of Recombinant Phage Antibodies Targeting the Hemagglutinin Cleavage Site of Highly Pathogenic Avian Influenza Virus

Highly pathogenic avian influenza (HPAI) H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS) in the hemagglutinin protein (HA). Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.     

Oral Priming with Replicating Adenovirus Serotype 4 Followed by Subunit H5N1 Vaccine Boost Promotes Antibody Affinity Maturation and Expands H5N1 Cross-Clade Neutralization

A Phase I trial conducted in 2009–2010 demonstrated that oral vaccination with a replication competent Ad4-H5 (A/Vietnam) vector with dosages ranging from 10⁷-10¹¹ viral particles was well tolerated. HA-specific T-cell responses were efficiently induced, but very limited hemagglutination-inhibiting (HI) humoral responses were measured. However, a single boost of Ad4-H5-Vtn vaccinated individuals with a unadjuvanted licensed H5N1 (A/Vietnam) subunit vaccine resulted in superior HI titers compared with unprimed subjects. In the current study, the impact of Ad4-H5 priming on the quality of the polyclonal humoral immune response was evaluated using a real-time kinetics assay by surface plasmon resonance (SPR). Total binding of serum polyclonal antibodies from the Ad4-H5-Vtn primed groups against both homologous H5N1-A/Vietnam/1194/2004 (clade 1) and heterologous A/Indonesia-5/2005 (clade 2.1) HA1 head domain was significantly higher compared with sera from individuals that received subunit H5N1 vaccination alone. SPR measurements also demonstrated that the antigen-antibody complex dissociation rates (a surrogate for antibody affinity) of serum antibodies against the HA1 of H5N1-A/Vietnam were significantly higher in the Ad4-H5 primed groups compared with those from the unprimed group. Furthermore, strong correlations were observed between the antibody affinities for HA1 (but not HA2) and the virus neutralization titers against the homologous strain and a panel of heterologous clade 2 H5N1 strains. These findings support the concept of oral prime-boost vaccine approaches against pandemic influenza to elicit long-term memory B cells with high affinity capable of rapid response to variant pandemic viruses likely to emerge and adapt to human transmissions.     

Molecular modeling, docking and dynamics simulations of GNA-related lectins for potential prevention of influenza virus (H1N1)

The Galanthus nivalis agglutinin (GNA)-related lectin family exhibit significant anti-HIV and anti-HSV properties that are closely related to their carbohydrate-binding activities. However, there is still no conclusive evidence that GNA-related lectins possess anti-influenza properties. The hemagglutinin (HA) of influenza virus is a surface protein that is involved in binding host cell sialic acid during the early stages of infection. Herein, we studied the 3D-QSARs (three-dimensional quantitative structure–activity relationships) of lectin– and HA–sialic acid by molecular modeling. The affinities and stabilities of lectin– and HA–sialic acid complexes were also assessed by molecular docking and molecular dynamics simulations. Finally, anti-influenza GNA-related lectins that possess stable conformations and higher binding affinities for sialic acid than HAs of human influenza virus were screened, and a possible mechanism was proposed. Accordingly, our results indicate that some GNA-related lectins, such as Yucca filamentosa lectin and Polygonatum cyrtonema lectin, could act as drugs that prevent influenza virus infection via competitive binding. In conclusion, the GNA-related lectin family may be helpful in the design of novel candidate agents for preventing influenza A infection through the use of competitive combination against sialic acid specific viral infection.     

Accurate PDZ/Peptide Binding Specificity with Additive and Polarizable Free Energy Simulations

PDZ domains contain 80–100 amino acids and bind short C-terminal sequences of target proteins. Their specificity is essential for cellular signaling pathways. We studied the binding of the Tiam1 PDZ domain to peptides derived from the C-termini of its Syndecan-1 and Caspr4 targets. We used free energy perturbation (FEP) to characterize the binding energetics of one wild-type and 17 mutant complexes by simulating 21 alchemical transformations between pairs of complexes. Thirteen complexes had known experimental affinities. FEP is a powerful tool to understand protein/ligand binding. It depends, however, on the accuracy of molecular dynamics force fields and conformational sampling. Both aspects require continued testing, especially for ionic mutations. For six mutations that did not modify the net charge, we obtained excellent agreement with experiment using the additive, AMBER ff99SB force field, with a root mean square deviation (RMSD) of 0.37 kcal/mol. For six ionic mutations that modified the net charge, agreement was also good, with one large error (3 kcal/mol) and an RMSD of 0.9 kcal/mol for the other five. The large error arose from the overstabilization of a protein/peptide salt bridge by the additive force field. Four of the ionic mutations were also simulated with the polarizable Drude force field, which represents the first test of this force field for protein/ligand binding free energy changes. The large error was eliminated and the RMS error for the four mutations was reduced from 1.8 to 1.2 kcal/mol. The overall accuracy of FEP indicates it can be used to understand PDZ/peptide binding. Importantly, our results show that for ionic mutations in buried regions, electronic polarization plays a significant role.     

A Unique and Conserved Neutralization Epitope in H5N1 Influenza Viruses Identified by an Antibody against the A/Goose/Guangdong/1/96 Hemagglutinin

Despite substantial efforts to control and contain H5N1 influenza viruses, bird flu viruses continue to spread and evolve. Neutralizing antibodies against conserved epitopes on the viral hemagglutinin (HA) could confer immunity to the diverse H5N1 virus strains and provide information for effective vaccine design. Here, we report the characterization of a broadly neutralizing murine monoclonal antibody, H5M9, to most H5N1 clades and subclades that was elicited by immunization with viral HA of A/Goose/Guangdong/1/96 (H5N1), the immediate precursor of the current dominant strains of H5N1 viruses. The crystal structures of the Fab′ fragment of H5M9 in complexes with H5 HAs of A/Vietnam/1203/2004 and A/Goose/Guangdong/1/96 reveal a conserved epitope in the HA1 vestigial esterase subdomain that is some distance from the receptor binding site and partially overlaps antigenic site C of H3 HA. Further epitope characterization by selection of escape mutants and epitope mapping by flow cytometry analysis of site-directed mutagenesis of HA with a yeast cell surface display identified four residues that are critical for H5M9 binding. D53, Y274, E83a, and N276 are all conserved in H5N1 HAs and are not in H5 epitopes identified by other mouse or human antibodies. Antibody H5M9 is effective in protection of H5N1 virus both prophylactically and therapeutically and appears to neutralize by blocking both virus receptor binding and postattachment steps. Thus, the H5M9 epitope identified here should provide valuable insights into H5N1 vaccine design and improvement, as well as antibody-based therapies for treatment of H5N1 infection.     

Theoretical Investigation on the Binding Specificity of Sialyldisaccharides with Hemagglutinins of Influenza A Virus by Molecular Dynamics Simulations

Recognition of cell-surface sialyldisaccharides by influenza A hemagglutinin (HA) triggers the infection process of influenza. The changes in glycosidic torsional linkage and the receptor conformations may alter the binding specificity of HAs to the sialylglycans. In this study, 10-ns molecular dynamics simulations were carried out to examine the structural and dynamic behavior of the HAs bound with sialyldisaccharides Neu5Acα(2–3)Gal (N23G) and Neu5Acα(2–6)Gal (N26G). The analysis of the glycosidic torsional angles and the pair interaction energy between the receptor and the interacting residues of the binding site reveal that N23G has two binding modes for H1 and H5 and a single binding mode for H3 and H9. For N26G, H1 and H3 has two binding modes, and H5 and H9 has a single binding mode. The direct and water-mediated hydrogen bonding interactions between the receptors and HAs play dominant roles in the structural stabilization of the complexes. It is concluded from pair interaction energy and Molecular Mechanic-Poisson-Boltzmann Surface Area calculations that N26G is a better receptor for H1 when compared with N23G. N23G is a better receptor for H5 when compared with N26G. However, H3 and H9 can recognize N23G and N26G in equal binding specificity due to the marginal energy difference (≈2.5 kcal/mol). The order of binding specificity of N23G is H3 > H5 > H9 > H1 and N26G is H1 > H3 > H5 > H9, respectively. The proposed conformational models will be helpful in designing inhibitors for influenza virus.     

Design and Structure of an Engineered Disulfide-Stabilized Influenza Virus Hemagglutinin Trimer

We engineered a disulfide-stabilized influenza virus hemagglutinin (HA) trimer, termed HA₃-SS, by introducing cysteine residues into the HA stem to covalently bridge the three protomers. HA₃-SS has increased thermostability compared to wild-type HA, and binding of head- and stem-targeted antibodies (Abs) is preserved; only minor structural changes are found in the vicinity of the additional disulfide. This platform has been applied to H1 and H3 HAs and provides prospects for design of intact, stabilized influenza virus HA immunogens.     

Docking of sialic acid analogues against influenza A hemagglutinin: a correlational study between experimentally measured and computationally estimated affinities

In this study fragment-based drug design is combined with molecular docking simulation technique, to design databases of virtual sialic acid (SA) analogues with new substitutions at C2, C5 and C6 positions of SA scaffold. Using spaces occupied by C2, C5 and C6 natural moieties of SA when bound to hemagglutinin (HA) crystallographic structure, new fragments that are commercially available were docked independently in all the pockets. The oriented fragments were then connected to the SA scaffold with or without incorporation of linker molecules. The completed analogues were docked to the whole SA binding site to estimate their binding conformations and affinities, generating three databases of HA-bound SA analogues. Selected new analogues showed higher estimated affinities than the natural SA when tested against H3N2, H5N1 and H1N1 subtypes of influenza A. An improvement in the binding energies indicates that fragment-based drug design when combined with molecular docking simulation is capable to produce virtual analogues that can become lead compound candidates for anti-flu drug discovery program.     

Effects of municipal waste compost and sewage sludge on proton binding behavior of humic acids from Portuguese sandy and clay loam soils

The effects of amendment with municipal solid waste compost (MSWC) and sewage sludge (SS) on acid-base properties of soil humic acids (HAs) were investigated. For this purpose, HAs were isolated from MSWC and SS and two different Portuguese soils, one sandy and the other clay loam, either unamended or amended with MSWC or SS at a rate of 60 t ha(-1), and analysed by potentiometric titrations at various ionic strengths (0.01, 0.05, 0.1 and 0.3M) over the pH range from 3.5 to 10.5. All titration data were fitted with the NICA-Donnan model and the variations of model parameters between the various HA samples were discussed. The HAs from MSWC and SS had lower acidic functional group contents and higher proton binding affinities than the control soil HAs. Amending soils with MSWC and SS determined a decrease of acidic functional group contents and an increase on proton binding affinities of soil HAs. These effects were more evident in SS-amended soil HAs than in MSWC-amended soil HAs, and in clay loam soil HA than in sandy soil HA.     

Cooperation between the Hemagglutinin of Avian Viruses and the Matrix Protein of Human Influenza A Viruses

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.     

<Emphasis Type="Italic">Ab initio</Emphasis> fragment molecular orbital studies of influenza virus hemagglutinin–sialosaccharide complexes toward chemical clarification about the virus host range determination

If we predict the host range of new or mutant influenza virus in advance, we are able to measure against pandemic human influenza immediately after the new virus emerges somewhere. Influenza viral hemagglutinin(HA)–sialoside receptor interaction is a target event for in silico chemical prediction studies about the virus host range determination. We theoretically studied avian and human influenza A virus HA H3 subtype complexed with avian or human type receptor Neu5Acα(2-3 or 2-6)Gal analogues by ab initio fragment molecular orbital (FMO) method at the second order Møller–Plesset (MP2)/6–31G level, which can evaluate correctly not only electrostatic interactions but also lipophilic interactions based on van der Waals dispersion force. Avian H3 bound to avian α2-3 11.4 kcal/mol stronger than to human α2-6 in the model complexes with taking account of intermolecular lipophilic interaction. A substitution at the position 226 between Gln(avian) and Leu(human) on influenza H3 HA1 has altered its virus host range between avian and human. In the ab initio FMO studies, binding energy of avian Gln226Leu H3–human α2-6 was quite similar to that in the human H3–human α2-6 complex with amino acid sequence differences at nine positions in the models. This similarity indicates that avian Gln226Leu H3 virus can infect human with the same level as human H3 virus. Opposite mutation Leu226Gln in the human H3 gave the moderate binding energies to avian α2-3 with similarity to avian H3–α2-3 complex that supported our previous virus-sialoside binding assay. Ab initio FMO studies have revealed the relationship between influenza H3 virus host range and H3–α(2-3 or 2-6) receptors binding. Our theoretical approach may predict the infectious level of new viruses and point out some unknown dangerous mutation positions on HA in advance.     

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (−7.2 to −7.0 kcal/mol) and the cowpea bean dipeptide Lys-Asp (−7.0 kcal/mol) had higher binding affinities than ezetimibe (−6.6 kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (−7.2 kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption.