Cyclic Peptide Ligands Research Articles

ChemInform Abstract: Directed Evolution of Bicyclic Peptides for Therapeutic Application.

Many naturally occurring cyclic peptides or derivatives thereof are used as therapeutics such as the human hormones vasopressin and oxytocin or the antibiotics vancomycin and daptomycin. The success of cyclic peptide therapeutics is based on their ability to bind with high affinity, their good target selectivity and their low toxicity. As nature provides cyclic peptides to only a small number of disease targets, strategies have been developed to generate cyclic peptide ligands with tailored specificity de novo. Our laboratory is specialized on the directed evolution of bicyclic peptide ligands by phage display. In this article, we review our recent work to in vitro evolve bicyclic peptide antagonists, the binding and pharmacokinetic properties of bicyclic peptides, as well as efforts to generate bicyclic peptides for therapeutic application.

Directed Evolution of Bicyclic Peptides for Therapeutic Application

Many naturally occurring cyclic peptides or derivatives thereof are used as therapeutics such as the human hormones vasopressin and oxytocin or the antibiotics vancomycin and daptomycin. The success of cyclic peptide therapeutics is based on their ability to bind with high affinity, their good target selectivity and their low toxicity. As nature provides cyclic peptides to only a small number of disease targets, strategies have been developed to generate cyclic peptide ligands with tailored specificity de novo. Our laboratory is specialized on the directed evolution of bicyclic peptide ligands by phage display. In this article, we review our recent work to in vitro evolve bicyclic peptide antagonists, the binding and pharmacokinetic properties of bicyclic peptides, as well as efforts to generate bicyclic peptides for therapeutic application.

Reversible Cyclic Peptide Libraries for the Discovery of Affinity Ligands

A novel strategy is presented for the identification of cyclic peptide ligands from combinatorial libraries of reversible cyclic depsipeptides. A method for the solid-phase synthesis of individual cyclic depsipeptides and combinatorial libraries of these compounds is proposed, which employs lactic acid (Lact) and the dipeptide ester (Nα-Ac)-Ser(Ala)- as linkers for dilactonization. Upon alkaline treatment of the beads selected by screening a model library, the cyclic depsipeptides are linearized and released from the solid support to the liquid phase, to be sequenced via single-step tandem mass spectrometry (MS/MS). The protocol presented for library synthesis provides for wide structural diversity. Two model sequences, VVWVVK and AAWAAR, were chosen to present different structural examples for depsipeptide libraries and demonstrate the process of sequence determination by mass spectrometry. Further, a case study using the IgG binding cyclic depsipeptide cyclo[(Nα-Ac)-S(A)-RWHYFK-Lact-E] is presented to demonstrate the process of library screening and sequence determination on the selected beads. Finally, a method is shown for synthesis of the irreversible cyclic peptide corresponding to the proposed depsipeptide structure, to make the ligand stable to the aqueous acid and alkaline conditions encountered in affinity chromatographic applications. The cyclic peptide ligand was synthesized on a poly(methacrylate) resin and used for chromatographic binding of the target IgG.

English

The pathogenic dengue virus (DV) is a growing global threat for which there is no specific treatment and prevention. Several vaccines have been developed against the disease, but they only prevent the disease and reduce the risk of death. Consequently, the antiviral drug becomes the most powerful treatment to solve this problem. The virus possesses a two component NS2B-NS3 protease that cleaves viral precursor proteins, and therefore represents a target for the development of antiviral drugs. In many researches, several models of peptides inhibitor were generated in complexes with the NS2B-NS3 DENV2 protease by performing molecular docking. The goal of this research was to study the interaction of ligands as inhibitors for protein (enzyme) in solvent explicit condition by performing molecular dynamics simulation at 300 and 312 K. The simulations were performed on four disulfide cyclic tripeptide ligands (RGR, KRK, RRA and RKR) and two proline-proline based cyclic peptide ligands (RKK and KKR) compared with linear peptide inhibitor Bz-Nle-K-R-R-H as standard ligand. The results provide conformational changes of enzyme-inhibitor complex that is shown by root-mean-square deviation (RMSD) values. These results show that dynamic behavior of the complex occurs in the presence of solvent. The interaction between ligand and amino acid residue of enzyme changed during simulation. By these parameters, RKR becomes the best ligand compared with others. The ligand has hydrogen bond interaction with the residue of active side, and is stable during simulation at both temperatures. In conclusion, RKR ligand is proposed as an antiviral drug by performing molecular dynamics simulation. Keywords : Dengue virus, DENV2, NS2B-NS3 protease, inhibitor, cyclic peptide, ligand, molecular dynamics simulation, interaction, conformation, drug. African Journal of Biotechnology Vol. 12(28), pp. 4419-4431

Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer-related protease urokinase-type plasminogen activator (uPA), we observed a glycine residue that has a positive ϕ dihedral angle when bound to the target. We hypothesized that replacing it with a D-amino acid, which favors positive ϕ angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D-serine in the bicyclic peptide indeed improved inhibitory activity (1.75-fold) and stability (fourfold). X-ray-structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ϕ angles are found in many protein-bound peptides. These results suggest that the glycine-to-D-amino acid mutagenesis strategy could be broadly applied.

Designing cyclopentapeptide inhibitor as potential antiviral drug for dengue virus ns5 methyltransferase.

NS5 methyltransferase (Mtase) has a crucial role in the replication of dengue virus. There are two active sites on NS5 Mtase i.e., SAM and RNA-cap binding sites. Inhibition of the NS5 Mtase activity is expected to prevent the propagation of dengue virus. This study was conducted to design cyclic peptide ligands as enzyme inhibitors of dengue virus NS5 Mtase through computational approach. Cyclopentapeptides were designed as ligand of SAM binding site as much as 1635 and 736 cyclopentpeptides were designed as ligand of RNA-cap binding site. Interaction between ligand and NS5 Mtase has been conducted on the Docking simulation. The result shows that cyclopentapeptide CTWYC was the best peptide candidate on SAM binding site, with estimated free binding energy -30.72 kca/mol. Cyclopentapeptide CYEFC was the best peptide on RNA-cap binding site with estimated free binding energy -22.89 kcal/mol. Both peptides did not have tendency toward toxicity properties. So it is expected that both CTWYC and CYEFC ligands could be used as a potential antiviral drug candidates, which can inhibit the SAM and RNA-cap binding sites of dengue virus NS5 Mtase.

Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER.

Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct 'target' structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.

Selection of cyclic-peptide inhibitors targeting Aurora kinase A: Problems and solutions

The critical role of Aurora kinase in cell cycle progression and its deregulation in cancer has garnered significant interest. As such, numerous Aurora targeted inhibitors have been developed to date, almost all of which target the ATP cleft at the active site. These current inhibitors display polypharmacology; that is, they target multiple kinases, and some are being actively pursued as therapeutics. Currently, there are no general approaches for targeting Aurora at sites remote from the active site, which in the long term may provide new insights regarding the inhibition of Aurora as well as other protein kinases, and provide pharmacological tools for dissecting Aurora kinase biology. Toward this long term goal, we have recently developed a bivalent selection strategy that allows for the identification of cyclic peptides that target the surface of PKA, while the active site is blocked by an ATP-competitive compound. Herein, we extend this approach to Aurora kinase (Aurora A), which required significant optimization of selection conditions to eliminate background peptides that target the streptavidin matrix upon which the kinases are immobilized. Using our optimized selection conditions, we have successfully selected several cyclic peptide ligands against Aurora A. Two of these inhibitors demonstrated IC50 values of 10μM and were further interrogated. The CTRPWWLC peptide was shown to display a noncompetitive mode of inhibition suggesting that alternate sites on Aurora beyond the ATP and peptide substrate binding site may be potentially targeted.

Novel poly(ethylene‐co‐acrylic acid) nanofibrous biomaterials for peptide synthesis and biomedical applications

Poly(ethylene-co-acrylic acid) (PE-co-AA) fibers in sizes of 200-500 nm were prepared by using a novel melt-extrusion-extraction fabrication process. The thermoplastic nanofibers could be controllably dispersed and reassembled by a novel solvent exchange filtration method. The dispersed PE-co-AA nanofibers possess active surface areas and could directly conduct chemical reactions on surfaces. Surface modifications and organic synthesis on the nanofibers were proven effective and controllable after the dispersion. Multistep synthesis of biomolecules, such as peptide ligand HWRGWV against Fc portion of human IgG, was successful. The surface-anchored ligand has shown bioactivity through selective binding to and staining by human IgG-alkaline phosphatase conjugate. Another peptide, LXY3, a selective cyclic peptide ligand against alpha3beta1 integrin of MDA-MB-231 breast cancer cells, was also prepared on the surfaces of the dispersed nanofibers. The results showed that MDA-MB-231 cells were able to specifically bind to and grow on surfaces of the nanofibers that were functionalized with LXY3.

Small-Molecule Ligands of GD2 Ganglioside, Designed from NMR Studies, Exhibit Induced-Fit Binding and Bioactivity

Ganglioside GD2 is a cell surface glycosphingolipid. Targeting of GD2, i.e., by anti-GD2 mAb 3F8, is used clinically for cancer diagnosis, prognosis, and therapy. Here, the conformations of free GD2, and of GD2 bound to mAb 3F8, were resolved by saturation transfer difference NMR and molecular modeling. Then, three small-molecule cyclic peptide ligands that bind to GD2 selectively were designed. Transferred nuclear Overhauser enhancement of the GD2-bound conformation of the peptide ligands showed an induced-fit binding mechanism. The mAb 3F8 and the peptidic GD2 ligands mediate similar biological functions in cell-based assays of calcium fluxes and src activation. Thus, small molecules can selectively and functionally interact with a sugar head group. This work furthers the concept of rationally designing ligands for carbohydrate targets, and may be expanded to other clinically relevant gangliosides.

The use of RGDGWK-lipopeptide to selectively deliver genes to mouse tumor vasculature and its complexation with p53 to inhibit tumor growth

In vivo selection of phage display libraries have been exploited successfully in the past to isolate various high affinity conformationally strained cyclic peptide ligands (CX 5–7C, peptides flanked by a cysteine residue on each side) for integrin receptors capable of selectively homing to tumor vasculatures. Previously, such phase display library studies have shown that integrin α5β1 binds with high affinity to cyclic peptides containing CRGDGWC motif. Herein we show that a lipopeptide with just the RGDGW motif (without the flanking cysteine groups) covalently attached to the lysine residue of a monolysinylated cationic amphiphile (RGDGWK-lipopeptide 1) delivers genes to cultured cells preferably via α5β1 integrins. Importantly, remarkable tumor growth inhibition was observed when the electrostatic complex of the RGDGWK-lipopeptide 1 and the anti-cancer p53 gene was intravenously administered in C57BL/6 J mice bearing the aggressive B16F10 tumor. Immunohistochemical staining of mice tumor cryosections with vasculature markers combined with monitoring expression of the green fluorescence protein in the same tumor cryosections revealed that the RGDGWK-lipopeptide 1 targets genes to tumor vasculatures. The colocalization of the TUNEL ( terminal deoxy uridine triphosphate nick- end labeling, a widely used marker of apoptosis) and VE-cadherin (markers of tumor endothelial cells) positive cells in tumor cryosections support the notion that the remarkable tumor growth inhibition property of the RGDGWK-lipopeptide 1:p53 complex is initiated through apoptosis of the tumor endothelial cells.

Citrullination of Linear and Cyclic Altered Peptide Ligands from Myelin Basic Protein (MBP87−99) Epitope Elicits a Th1 Polarized Response by T Cells Isolated from Multiple Sclerosis Patients: Implications in Triggering Disease

Derangement of cellular immunity is central in the pathophysiology of multiple sclerosis (MS) and is often manifested by abnormal cytokine production. We investigated cytokine secretion in peripheral blood mononuclear cells (PBMC) of 18 MS patients and 15 controls and correlated cytokine polarization with the nature of antigenic stimulus. We synthesized two novel citrullinated peptides, linear [Cit(91), Ala(96), Cit(97)]MBP(87-99) and cyclo(87-99)[Cit(91), Ala(96), Cit(97)]MBP(87-99) that resulted from citrullination of 91,97 Arg residues in antagonists, linear [Arg(91), Ala(96)]MBP(87-99) and cyclo(87-99)[Arg(91), Ala(96)]MBP(87-99) peptides. PBMC from MS patients and controls were cultured with citrullinated peptides, and both peptides caused a Th1 polarization in all MS patients studied. In contrast, culture with noncitrullinated MBP peptides resulted in heterogeneous cytokine secretion that differed between individual patients. Thus, citrullination of self-antigens may potentially trigger disease in susceptible individuals. This finding may open new avenues in drug design of new substances that inhibit citrullination and arrest epitope spreading and worsening of MS.

Polyplex Micelles with Cyclic RGD Peptide Ligands and Disulfide Cross-Links Directing to the Enhanced Transfection via Controlled Intracellular Trafficking

Thiolated c(RGDfK)-poly(ethylene glycol)-block-poly(lysine) (PEG-PLys), a novel block polymer that has a cyclic RGD peptide in the PEG terminus and thiol groups in the PLys side chain, was prepared and applied to the preparation of targetable disulfide cross-linked polyplex micelles through ion complexation with plasmid DNA (pDNA). The obtained polyplex micelles achieved remarkably enhanced transfection efficiency against cultured HeLa cells possessing alpha(v)beta(3) integrin receptors, which are selectively recognized by cyclic RGD peptides, demonstrating the synergistic effect of cyclic RGD peptide ligands on the micelle surface and disulfide cross-links in the core to exert the smooth release of pDNA in the intracellular environment via reductive cleavage. This enhancement was not due to an increase in the uptake amount of polyplex micelles but to a change in their intracellular trafficking route. Detailed confocal laser scanning microscopic observation revealed that polyplex micelles with cyclic RGD peptide ligands were distributed in the perinuclear region in the early stages preferentially through caveolae-mediated endocytosis, which may be a desirable pathway for avoiding the lysosomal degradation of delivered genes. Hence, this approach to introducing ligands and cross-links into the polyplex micelles is promising for the construction of nonviral gene vectors that enhance transfection by controlling intracellular distribution.

Design of Novel Cyclic Altered Peptide Ligands of Myelin Basic Protein MBP83−99That Modulate Immune Responses in SJL/J Mice

The use of antagonist peptides derived from the myelin sheath constitutes a promising therapeutic approach for multiple sclerosis (MS). Cyclization of peptide analogues is of great interest, since the limited stability of linear peptides restricts their potential as therapeutic agents. Herein, we designed and synthesized a number of cyclic peptides by mutating TCR contact sites of the MBP 83-99 epitope. A number of cyclic analogues were tested for their ability to inhibit (antagonize) Th1 (IFN-gamma) responses, and cyclo(83-99)[A (91)]MBP 83-99 mutant peptide was found to be the most efficient inhibitor. We demonstrated that cyclo(83-99)[A (91)]MBP 83-99 peptide emulsified in CFA enhanced Th2 (IL-4) and antibody responses in vivo. Moreover, immunization of mice with antagonist cyclo(83-99)[A (91)]MBP 83-99 peptide conjugated to reduced mannan enhanced IL-4 responses compared to cyclo(83-99)MBP 83-99 peptide. Thus, cyclized peptides, which offer greater stability and enhanced responses, are novel leads for the immunotherapy of many diseases, such as MS. In particular, cyclo(83-99)[A (91)]MBP 83-99 is a promising mutant peptide analogue for the potential treatment of MS.

Abstract 4740: Selective ablation of metastatic disease with integrin ανβ3 targeted nanoparticle drug delivery

Abstract Angiogenic blood vessels found within growing tumors express high levels of integrin ανβ3 while mature vessels fail to express this integrin. Based on this we developed a ανβ3-targeted nanoparticle (NP) in an attempt to selectively deliver therapeutics to the tumor neovasculature. A cyclic peptide ligand (cRGDfK) coupled to a phospholipid and incorporated into a nanoparticle demonstrated specific binding to ανβ3 on cultured endothelial cells. To characterize the in vivo targeting capacity of these NPs, we monitored NP accumulation using intravital microscopy of tumor neovasculature. A dorsal skin-fold window chamber implanted with GFP-labeled melanoma cells lacking integrin ανβ3 were imaged at various times after NP injection into the bloodstream. By 5h post injection, the cRGDfK NPs targeted the neovasculature at the angiogenic tips adjacent to the tumor margin, whereas untargeted (cRADfK) NPs failed to bind. After establishing in vivo targeting, these ανβ3-targeted NPs were loaded with doxorubicin via an ammonium phosphate gradient for therapeutic studies. The cRGD-Dox particles disrupted angiogenesis in the mouse matrigel model leaving only small fragments of disrupted vessels within the FGF loaded plugs. Next, we utilized an orthotopic pancreatic cancer model to study the ability of the NPs to target the vasculature of both primary pancreatic tumors as well as metastatic sites including the hepatic hilar lymph node and mesenteric lymph nodes. cRGD-Dox particles injected systemically at 1 mg/kg produced a profound anti-metastatic effect while only marginally influencing the pre-established primary tumor within the pancreas. This is much improved compared to the free doxorubicin treatments in which 1 mg/kg of free doxorubicin had no effect and 15 mg/kg of free doxorubicin was required for a similar therapeutic effect in this orthotopic model. Animals treated with 15 mg/kg of free doxorubicin lost over 20% of their body weight within 7 days, whereas no decrease in body weight was observed in the cRGD-Dox treated animals, confirming the reduction in overall toxicity. Histology confirmed that the cRGD-Dox particles induced apoptosis in regions co-localized with β3 expressing tumor vasculature. Additionally, we tested the cRGD-Dox particles in an orthotopic renal cell carcinoma model. Similar to the pancreatic carcinoma, the cRGD-Dox particles had a slight effect on primary tumor growth but greatly reduced metastasis to several sites including the pancreas, liver, spleen, and diaphragm. These findings reveal that ανβ3 targeted NP drug delivery may preferentially disrupt newly forming metastatic lesions by delivery of chemotherapeutic agents to sites of active angiogenesis in which the endothelium expresses high levels of ανβ3.

A cyclodecapeptide ligand to vitamin B12

Libraries of cyclic decapeptides were screened with vitamin B(12) derivatives to give cyclic peptide ligands incorporating histidine and cysteine as coordinating residues and negatively charged amino acids. Two hits, cyclo-(HisAspGluProGlyIleAlaThrProdGln) and cyclo-(ValAspGluProGlyGluAspCysProdGln) were resynthesized in good yields for solution experiments. The peptides bind aquocobalamin with coordination of His or Cys to the cobalt with high affinities (K(a) approximately 10(5) M(-1)). Additional interactions between the peptide side chains and the vitamin B(12) corrin moiety were determined by studying the (1)H NMR solution structure. The cyclopeptide-cobalamin complex with the histidine residue showed enhanced stability towards cyanide exchange, demonstrating the shielding effect of the ligand on the metal center.

Design, Synthesis, and Evaluation of Linear and Cyclic Peptide Ligands for PDZ10 of the Multi-PDZ Domain Protein MUPP1

PDZ10 is the 10th of 13 PDZ domains found within MUPP1, a cytoplasmic scaffolding protein first identified as an endogenous binding partner of serotonin receptor type 2c (5HT2c). This association, as with those of several other interacting proteins that have subsequently been identified, is mediated through the C-terminal tail of the PDZ domain partner. Using isothermal titration calorimetry (ITC), we measured the thermodynamic binding parameters [changes in Gibbs free energy (DeltaG), enthalpy (DeltaH) and entropy (TDeltaS)] of the isolated PDZ10 domain for variable-length N-acetylated peptides from the 5HT2c serotonin receptor C-terminal sequence, as well as for octapeptides of eight other putative partner proteins of PDZ10 (5HT2a, hc-kit, hTapp1, mTapp2, TARP, NG2, claudin-1, and HPV-18 E6). In length dependence studies of the 5HT2c sequence, the maximal affinity of the peptides leveled off rapidly and further elongation did not significantly improve the dissociation constant (Kd) of 11 microM observed with the pentapeptide. Among the native partners of PDZ10, octapeptides derived from the hc-kit and 5HT2c proteins were the strongest binders, with Kd values of 5.2 and 8.5 microM, respectively. The heat capacity change (DeltaCp) for the 5HT2c octapeptide was determined to be -94 cal/mol, and a calculated estimate indicates burial of polar and apolar surface areas in equal measure upon ligand binding. Peptides with phosphoserine at either the P-1 or P-2 position experienced decreased affinity, which is in accord with the hypothesis that reversible phosphorylation is a possible mechanism for regulating PDZ domain-mediated interactions. Additionally, two conformationally constrained side chain-bridged cyclic peptide ligands were also designed, prepared, evaluated by ITC, and shown to bind PDZ10 primarily through a favorable change in entropy.

New Method for the Synthesis of a Mononucleating Cyclic Peptide Ligand, Crystal Structures of its Ni, Zn, Cu, and Co Complexes, and Their Inhibitory Bioactivity Against Urease

A novel cyclic peptide complex, NiL 1 (H2L = 12,24-dihydroxy-1,6-dioxo-2,5,14,17-tetraaza[6*6]metacyclophane-13,17-diene has been synthesized for the first time under solvothermal conditions through a one-pot synthetic procedure using nickel ion as the template reagent. It was found that other metal ions were not suitable for the direct template reagent in this reaction. The nickel ion was eliminated from the complex and the metal-free cyclic peptide ligand H2L was obtained through a series of reactions. Then, ZnII, CuII, and CoII were coordinated with H2L under the same solvothermal conditions to produce three isomorphous complexes ZnL 2, CuL 3, and CoL 4. Their inhibitory bioactivities against urease were then studied. The copper(ii) complex 3 was the strongest inhibitor against jack bean urease, while H2L, 2, and 4 showed weak or no inhibitory activity against this enzyme.

Equilibrium and Non-Equilibrium Molecular Dynamics Simulations Provide Potential Mechanisms for the Loss of Ligand Binding to αIIbβ3 Mutants Affecting the Ligand-Induced Metal Binding Site (LIMBS).

Although the role of the β3 MIDAS metal ion in ligand binding to αIIbβ3 is well established, serving as the site of interaction of the ligand Asp residue, the role of the nearby LIMBS metal ion is less well defined. Previous studies suggested a role for the LIMBS in ligand binding. We confirmed this by showing that HEK293 cells expressing normal αIIbβ3 adhered to both immobilized fibrinogen and the RGD-containing venom echistatin in the presence of either Mg++/Ca++ or Mn++, whereas two different αIIbβ3 LIMBS mutants (β3 N215A and D217A) failed to adhere to either protein. In addition, we found that both mutations also increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the β3 PSI domain (normal 7±4% vs N215A 46±12% and D217A 41±20% of mAb anti-αIIb (HIP8) binding; mean±SD, n=6, p<0.05 for both), indicating that the mutations caused allosteric changes in the conformation of the receptor. To define the mechanism(s) by which the LIMBS mutants affect ligand binding, we carried out equilibrium and non-equilibrium (steered) molecular dynamics (MD) simulations of the cyclic peptide ligand eptifibatide in complex with either the fully hydrated normal αIIbβ3 integrin headpiece (PDB 1TY6) or the equivalent β3 D217A mutant, with and without the LIMBS metal ion. Simulations were carried out using the GROMACS package with the OPLS all-atom force-field. During the simulation, the hybrid domain of the D217A mutant demonstrated greater structural fluctuations than the normal αIIbβ3. Although Craig et al. have reported the appearance of a new contact between the RGD peptide ligand Asp carboxyl and the LIMBS metal ion in αVβ3 after 10 ps of a 1 ns simulation, we did not observe the appearance of such an interaction between the eptifibatide carboxyl and the normal αIIbβ3 LIMBS metal ion even after 20 ns. We did, however, observe such an interaction with the LIMBS metal ion in the D217A mutant. This interaction was facilitated by the movement of the LIMBS ~ 2 Å closer to the MIDAS, and was accompanied by rearrangements of the LIMBS coordinating residues D158 and N215. When the D217A mutant simulation was performed in the absence of the LIMBS metal ion, changes in the orientation of E220 were also observed. The D217A mutant demonstrated increased fluctuations in the C177–C184 specificity-determining loop (SDL), which has been implicated in ligand binding, and decreased fluctuations in K209. Steered MD were used to investigate the pulling forces required to unbind eptifibatide from its binding site. Notably, although the unbinding force decreased modestly when the LIMBS metal ion was removed, it required removal of both the LIMBS and MIDAS metal ions to effect a marked reduction in unbinding force. The binding free energies of the association of the αIIb and β3 subunits were also calculated, and the D217A mutant in the presence of the LIMBS metal ion demonstrated much tighter binding than normal integrin αIIbβ3 (ΔGb −162±6 vs −119±6 Kcal/mol; mean±SD; n=500). We conclude that the LIMBS plays a crucial role in ligand binding to αIIbβ3, perhaps by virtue of its effects on the coordination of the MIDAS, the accentuated mobility of specific domains (e.g., the SDL and the hybrid domains), and/or the number and strength of contacts between αIIb and β3.

Functional and Computational Analysis of the Role of the Adjacent to Metal Ion-Dependent Adhesion Site (ADMIDAS) of Integrin αIIbβ3.

The role of the β3 MIDAS in αIIbβ3 ligand binding is well established, but the role of the nearby ADMIDAS is less well defined. Thus, we studied HEK293 cells expressing normal αIIbβ3 (normal cells) or the ADMIDAS mutants β3 D126A and D127A (mutant cells). Both mutant cells adhered as well or better than normal cells to immobilized fibrinogen under static conditions in the presence of either Ca2+/Mg2+ or Mn2+. Under low shear flow conditions (0.15 dyne/cm2), adhesion of normal cells and D126A mutant cells to fibrinogen was similar in the presence of either Ca2+/Mg2+ or Mn2+. Adherent D126A mutant cells, however, demonstrated greater resistance to detachment at increasing shear rates in the presence of Ca2+/Mg2+ (e.g., at 20.4 dynes/cm2, only 40 ± 10% of normal cells remained vs 85 ± 8% of D126A mutant cells; mean ± SD; p<0.001). Substituting Mn2+ for Ca2+/Mg2+ increased the resistance to detachment of the normal cells (60 ± 20% remaining at 20.4 dynes/cm2; p=0.01), but the value was still less than the mutant cells in the presence of either Ca2+/Mg2+ (see above; p<0.01) or Mn2+ (84 ± 4%; p<0.01). The increased strength of adhesion we observed in the αIIbβ3 ADMIDAS mutant cells is similar to that found in α4β7 ADMIDAS mutant cells (Chen et al, JBC 2004) and is consistent with the findings in isolated β3 βA (I-like) domains (Pesho et al. JBC 2006). The binding of 7E3, whose epitope is near the ADMIDAS, to the D126 mutant cells was similar to its binding to the normal cells, but 7E3 binding to the D127A mutant cells was reduced by 89 ± 7% (n = 4; p<0.001). 7E3 decreased adhesion of normal cells to fibrinogen by 88 ± 4%, but it only decreased adhesion of D126A mutant cells by 3 ± 9%, and it did not inhibit adhesion of D127A cells at all. To provide a structural context for the role of the ADMIDAS in ligand binding to αIIbβ3, we compared results from nanosecond time-scale molecular dynamics (MD) simulations of the cyclic peptide ligand eptifibatide in complex with either the fully hydrated normal αIIbβ3 or the D126A mutant in the presence of Ca2+/Mg2+. Calculations were carried out using the OPLS all-atom force-field of the GROMACS simulation package. With respect to normal αIIbβ3, the mutant receptor demonstrated reduced fluctuations in the β3 207–210 and 335 regions and increased fluctuations in the β3 282–284 region. In addition, the ADMIDAS metal ion moved ~3 Å away from the MIDAS and became more solvent exposed. Rearrangements of the coordination of the ADMIDAS involving S123, D251, and D127 were also observed in the D126A mutant compared to normal αIIbβ3. Steered MD simulations were used to investigate the unbinding of eptifibatide from its binding site. The unbinding force for the D126A mutant was similar to that for the normal αIIbβ3. Quantitative estimations of the binding energies of eptifibatide to normal and D126A mutant αIIbβ3 from Molecular Mechanics/Poisson Boltzman Surface Area analysis of the MD trajectories also yielded similar results. Thus, the much greater resistance of D126A mutant cells to detachment from fibrinogen at increasing shear rates does not appear to be explained by differences in fibrinogen-αIIbβ3 interactions at the sites involved in the binding of eptifibatide. Potential alternative mechanisms involve differences in fibrinogen's access to the binding site, interactions with other sites, or changes in fibrinogen avidity due to receptor clustering.