Local Dynamics and Their Alteration by Excipients Modulate the Global Conformational Stability of an lgG1 Monoclonal Antibody

A molecular understanding of excipient effects on the interrelationship(s) between dynamics and conformational stability of proteins, such as monoclonal antibodies (mAbs), can be important for their pharmaceutical development. The current study examines stabilizing and destabilizing effects of excipients on the conformational stability and local dynamics of distinct solvent‐exposed regions within an IgG1 monoclonal antibody (mAb‐B). The principles of site‐selective photoselection upon red‐edge excitation, accompanied by acrylamide quenching of tryptophan fluorescence were employed in this study. The initiation of mAb‐B thermal unfolding occurs by structural alterations in the more solvent‐exposed regions of the antibody, which subsequently leads to a cascade of structural alterations in its relatively more solvent‐shielded regions. In addition, an increase in internal dynamics of solvent‐shielded regions made mAb‐B more susceptible to thermally induced structural perturbations resulting in its global destabilization. Sucrose and arginine exert their stabilizing and destabilizing effects by predominantly influencing the conformational stability of solvent‐exposed regions in mAb‐B. The complex molecular effects of sucrose and arginine on local dynamics of different regions in mAb‐B and their correlation with the protein's conformational stability are described within the pretransition range, at the onset temperature (T_onset) and at the thermal melting temperature (T_M).     

Protein A does not induce allosteric structural changes in an IgG1 antibody during binding

Affinity chromatography is widely used for antibody purification in biopharmaceutical production. Although there is evidence suggesting that affinity chromatography might induce structural changes in antibodies, allosteric changes in structure have not been well-explored. Here, we used hydrogen exchange-mass spectrometry (HX-MS) to reveal conformational changes in the NIST mAb upon binding with a protein A (ProA) matrix. HX-MS measurements of NIST mAb bound to in-solution and resin forms of ProA revealed regions of the C_H2 and C_H3 domains with increased protection from HX upon ProA binding, consistent with the known ProA binding region. In-solution ProA experiments revealed regions in the F_ab with increased HX uptake when the ProA:mAb molar ratio was increased to 2:1, suggesting an allosterically induced increase in backbone flexibility. Such effects were not observed with lower ProA concentration (1:1 molar ratio) or when ProA resin was used, suggesting some kind of change in binding mode. Since all pharmaceutical processes use ProA bound to resin, our results rule out reversible allosteric effects on the NIST mAb during interaction with resin ProA. However, irreversible effects cannot be ruled out since the NIST mAb was previously exposed to ProA during its original purification.     

Investigating Monoclonal Antibody Aggregation Using a Combination of H/DX-MS and Other Biophysical Measurements

To determine how structural changes in antibodies are connected with aggregation, the structural areas of an antibody prone to and/or impacted by aggregation must be identified. In this work, the higher-order structure and biophysical properties of two different monoclonal antibody (mAb) monomers were compared with their simplest aggregated form, that is, dimers that naturally occurred during normal production and storage conditions. A combination of hydrogen/deuterium exchange mass spectrometry and other biophysical measurements was used to make the comparison. The results show that the dimerization process for one of the mAb monomers (mAb1) displayed no differences in its deuterium uptake between monomer and dimer forms. However, the other mAb monomer (mAb2) showed subtle changes in hydrogen/deuterium exchange as compared with its dimer form. In this case, differences observed were located in specific functional regions of the C_H2 domain and the hinge region between C_H1 and C_H2 domains. The importance and the implications of these changes on the antibody structure and mechanism of aggregation are discussed.     

Correlating Excipient Effects on Conformational and Storage Stability of an IgG1 Monoclonal Antibody with Local Dynamics as Measured by Hydrogen/Deuterium-Exchange Mass Spectrometry

The effects of sucrose and arginine on the conformational and storage stability of an IgG1 monoclonal antibody (mAb) were monitored by differential scanning calorimetry (DSC) and size-exclusion chromatography (SEC), respectively. Excipient effects on protein physical stability were then compared with their effects on the local flexibility of the mAb in solution at pH 6, 25°C using hydrogen/deuterium-exchange mass spectrometry (H/D-MS). Compared with a 0.1 M NaCl control, sucrose (0.5 M) increased conformational stability (T_m values), slowed the rate of monomer loss, reduced the formation of insoluble aggregates, and resulted in a global trend of small decreases in local flexibility across most regions of the mAb. In contrast, the addition of arginine (0.5 M) decreased the mAb's conformational stability, increased the rate of loss of monomer with elevated levels of soluble and insoluble aggregates, and led to significant increases in the local flexibility in specific regions of the mAb, most notably within the constant domain 2 of the heavy chain (C_H2). These results provide new insights into the effect of sucrose and arginine on the local dynamics of IgG1 domains as well as preliminary correlations between local flexibility within specific segments of the C_H2 domain (notably heavy chain 241–251) and the mAb's overall physical stability. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:2136–2151, 2013     

Understanding the Increased Aggregation Propensity of a Light-Exposed IgG1 Monoclonal Antibody Using Hydrogen Exchange Mass Spectrometry,Biophysical Characterization,and Structural Analysis

This work compares the conformational stability, backbone flexibility, and aggregation propensity of monomer and dimer fractions of an IgG1 monoclonal antibody (mAb) generated on UVA light exposure for up to 72 h collected by preparative size-exclusion chromatography, compared with unstressed control. UVA light exposure induced covalent aggregation, and fragmentation as measured by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and extensive oxidation of specific methionine residues (Met 257, Met 433, and Met 109) in both size fractions identified by reverse phase chromatography coupled to mass spectrometry. Compared with unstressed mAb, both the monomer and dimer fractionated from 72 h UVA light–exposed mAb had decreased thermal melting temperatures (T_m1) by 1.4°C as measured by differential scanning calorimetry, minor changes in tertiary structure as measured by near-UV CD, increased monomer loss, and aggregation on accelerated storage at 35°C. Hydrogen/deuterium exchange mass spectrometry identified local segments with increased flexibility in C_H2 and C_H3 domains of both size fractions, and decreased flexibility in few segments of F_ab and C_H1 domains in the dimer fraction. Segment 247-256 in heavy chain, an established aggregation hotspot in IgG1 mAbs had large increase in flexibility in both size fractions compared with unstressed mAb.     

Insights into the Conformation and Self-Association of a Concentrated Monoclonal Antibody using Isothermal Chemical Denaturation and Nuclear Magnetic Resonance

The purpose of this investigation was to highlight the utility of nuclear magnetic resonance (NMR) as a multi-attribute method for the characterization of therapeutic antibodies. In this case study, we compared results from isothermal chemical denaturation (ICD) and NMR with standard methods to relate conformational states of a model monoclonal antibody (mAb1) with protein-protein interactions (PPI) that lead to self – association in concentrated solutions. The increase in aggregation rate and relative viscosity for mAb1 was found to be both concentration and pH dependent. The free energy of unfolding (∆G⁰) from ICD and thermal analysis in dilute solutions indicated that although the native state predominated between pH 4 – pH 7, it was disrupted at the CH₂ and unfolded noncooperatively under acidic conditions. One-dimensional (1D) ¹H NMR and two-dimensional (2D) ¹³C-¹H NMR performed, in concentrated solutions, confirmed that PPI between pH 4–7 occurred while mAb1 was in the native state. NMR corroborated that mAb1 maintained a dominant native state at formulation-relevant conditions at the tested pH range, had increased global molecular tumbling dynamics at lower pH and confirmed increased PPI at higher pH conditions. This report aligns and compares typical characterization of an IgG1 with assessment of structure by NMR and provided a more precise assessment and deeper insight into the conformation of an IgG1 in concentrated solutions.     

Evaluation of Hydrogen Exchange Mass Spectrometry as a Stability-Indicating Method for Formulation Excipient Screening for an IgG4 Monoclonal Antibody

Antibodies are molecules that exhibit diverse conformational changes on different timescales, and there is ongoing interest to better understand the relationship between antibody conformational dynamics and storage stability. Physical stability data for an IgG4 monoclonal antibody (mAb-D) were gathered through traditional forced degradation (temperature and stirring stresses) and accelerated stability studies, in the presence of different additives and solution conditions, as measured by differential scanning calorimetry, size exclusion chromatography, and microflow imaging. The results were correlated with hydrogen exchange mass spectrometry (HX-MS) data gathered for mAb-D in the same formulations. Certain parameters of the HX-MS data, including hydrogen exchange in specific peptide segments in the C_H2 domain, were found to correlate with stabilization and destabilization of additives on mAb-D during thermal stress. No such correlations between mAb physical stability and HX-MS readouts were observed under agitation stress. These results demonstrate that HX-MS can be set up as a streamlined methodology (using minimal material and focusing on key peptide segments at key time points) to screen excipients for their ability to physically stabilize mAbs. However, useful correlations between HX-MS and either accelerated or real-time stability studies will be dependent on a particular mAb's degradation pathway(s) and the type of stresses used.     

Stability of Monoclonal Antibodies at High-Concentration: Head-to-Head Comparison of the IgG1 and IgG4 Subclass

Few studies have so far directly compared the impact of antibody subclass on protein stability. This case study investigates two mAbs (one IgG₁ and one IgG₄) with identical variable region. Investigations of mAbs that recognize similar epitopes are necessary to identify possible differences between the IgG subclasses. Both physical and chemical stability were evaluated by applying a range of methods to measure formation of protein aggregates [sizeexclusion chromatography (SEC)–HPLC and UV340 nm], structural integrity (circular dichroism and FTIR), thermodynamic stability (differential scanning calorimetry), colloidal interactions (dynamic light scattering), and fragmentation and deamidation (SEC–HPLC and capillary isoelectric focusing). The impact of pH (4–9) and ionic strength (10 and 150 mm) was investigated using highlyconcentrated (150 mg/mL) mAb formulations. Lower conformational stability was identified for the IgG₄ resulting in increased levels of soluble aggregates. The IgG₁ was chemically less stable as compared with the IgG₄, presumably because of the higher flexibility in the IgG₁ hinge region. The thermodynamic stability of individual mAb domains was also addressed in detail. The stability of our mAb molecules is clearly affected by the IgG framework, and this study suggests that subclass switching may alter aggregation propensity and aggregation pathway and thus potentially improve the overall formulation stability while retaining antigen specificity. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:115–127, 2014     

Effects of Salts and Surface Charge on the Biophysical Stability of a Low pI Monoclonal Antibody

The impact of five representative Hofmeister salts (NaCl, KCl, MgCl₂, Na₂SO₄, and NaSCN) on the thermal stability and aggregation kinetics of a slightly acidic monoclonal antibody (mAb) were investigated under different pH conditions. The thermal stability of the mAb was assessed by measuring the lowest unfolding transition temperature, T_m, with differential scanning fluorimetry. MgCl₂ and NaSCN significantly decreased T_m at all three charged states of the mAb, but to the greatest extent when the mAb surface charge was net positive. Non-native aggregation kinetics was monitored by measuring Rayleigh light scattering. When the mAb surface charge was net positive or net neutral, the nucleation rate increased non-monotonically with MgCl₂ and NaSCN but decreased monotonically with NaCl, KCl, and Na₂SO₄. By contrast, when the mAb surface was negatively charged, there were only minor changes in the nucleation rate with all salts tested. Furthermore, there was less structural perturbation and slower aggregation rates when the mAb was net negatively charged than when it was net neutrally or positively charged. The observed salt effects on thermal unfolding are consistent with ion-specific mechanisms dominated by short-range amide backbone binding. On the other hand, the salt effects on nucleation rates appear to be influenced by both amide backbone binding and long-range electrostatic binding of ions to charged amino acid side chains.     

Formulation design and high-throughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions

A systematic approach is presented to characterize and stabilize the higher order structural integrity of an immunoglobulin G (IgG1) monoclonal antibody (mAb) formulated at both low concentrations and as a highly concentrated solution. The conformational and colloidal stabilities of a recombinant humanized IgG1κ mAb at both 1 and 100 mg/mL were investigated as a function of solution temperature (10°C-87.5°C) and pH (3-8). Protein secondary structure was characterized using circular dichroism, whereas intrinsic (tryptophan) and extrinsic (8-anilino-1-naphthalenesulfonic acid) fluorescence spectroscopy measurements were used to evaluate the tertiary structure of the protein. Light scattering analysis was employed to monitor mAb aggregation behavior as a function of temperature and solution pH. These biophysical data sets were analyzed and summarized using a previously described empirical phase diagrams (EPDs) approach. The different phases observed in the EPD were correlated with the individual physical states of the IgG1 in solution (aggregated, native, unfolded, etc.). The temperature-dependent conformational stability profile of the mAb, at both 1 and 100 mg/mL, generally followed the order pH 6 ≥ pH 7 ≥ pH 8 > pH 5 > pH 4 ≥ pH 3. Analysis of the EPD apparent phase boundaries identified solution conditions of pH 4.5 near 60°C for the development of an excipient screening assay. A supplemented generally regarded as safe excipient library was screened using an aggregation assay (optical density at 350 nm) at low mAb concentrations (4 mg/mL) and potential stabilizers were identified. The ability of these excipients to prevent conformational alterations in high concentration mAb solutions (100 mg/mL) was determined by monitoring tertiary structure changes using an intrinsic fluorescence method. The results suggest that substantial increases in the onset temperature of thermal transitions (>5°C) are obtained in the presence of (a) 20% dextrose, (b) 20% sorbitol, and (c) 5% dextrose + 10% sorbitol. Similar stabilization effects were obtained at an intermediate (50 mg/mL) as well as low mAb concentrations (1 mg/mL).     

Comparative Evaluation of Disodium Edetate and Diethylenetriaminepentaacetic Acid as Iron Chelators to Prevent Metal -Catalyzed Destabilization of a Therapeutic Monoclonal Antibody

Understanding the effect of metal chelators with respect to their ability to inhibit metal catalyzed degradation in biologic products is a critical component for solution formulation development. Two metal chelators, disodium edetate (Na₂EDTA) and diethylenetriaminepentaacetic acid (DTPA), were evaluated for their ability to stabilize IgG2 mAb in solution formulations spiked with various levels of iron. Real-time stability attributes such as oxidation, soluble aggregate formation, deamidation and fragmentation demonstrated that DTPA was equivalent to Na₂EDTA with respect to inhibiting iron-induced degradation over the range of iron concentrations studied. When sufficient chelator was present to stoichiometrically complex trace iron contamination, both Na2EDTA and DTPA exhibited the capacity to reduce protein degradation. However, sub-stoichiometric ratios of both chelators were unable to inhibit the degradation induced by free iron ions, which were found to bind weakly to the mAb. This bound iron did not measurably alter the secondary or the tertiary structure of the mAb, but appeared to decrease its intrinsic thermodynamic stability, probably by causing subtle perturbations in the tertiary structure. These destabilization effects were not observed when the chelators were present at stoichiometric ratios highlighting the feasibility of using DTPA as an alternate trace metal chelator to Na₂EDTA in biologic protein formulations.     

Monoclonal antibodies for prophylaxis and therapy of infectious diseases

Monoclonal antibodies (mAb) are attractive biologic drugs due to their exquisite specificity and well understood mechanisms of action, which results in a higher predictability and lower attrition rate compared with other drugs. Therefore, it may seem surprising that only a single mAb is presently marketed for an infectious disease indication. However, the antibiotic resistance crisis, emerging viral diseases and bioterroristic threats have recently spurred the development of anti-infective mAbs, of which more than a dozen are being tested in clinical trials. Conceptually, and validated in many preclinical models, mAbs will be most effective when used prophylactically against acute viral infections and bacterial toxins. The acute bacterial and chronic viral infections, which are medically and economically far more important, are much more difficult to control by antibodies, as the recent clinical failure of some polyclonal antibody products has shown. In these situations, the synergistic action of two or more mAbs together with a small molecule drug will most likely be required for therapeutic efficacy. This review aims to highlight the scientific and economic opportunities and obstacles that are encountered in the quest to add mAbs to the armament of anti-infective drugs.     

Monoclonal antibodies for prophylaxis and therapy of infectious diseases

Monoclonal antibodies (mAb) are attractive biologic drugs due to their exquisite specificity and well understood mechanisms of action, which results in a higher predictability and lower attrition rate compared with other drugs. Therefore, it may seem surprising that only a single mAb is presently marketed for an infectious disease indication. However, the antibiotic resistance crisis, emerging viral diseases and bioterroristic threats have recently spurred the development of anti-infective mAbs, of which more than a dozen are being tested in clinical trials. Conceptually, and validated in many preclinical models, mAbs will be most effective when used prophylactically against acute viral infections and bacterial toxins. The acute bacterial and chronic viral infections, which are medically and economically far more important, are much more difficult to control by antibodies, as the recent clinical failure of some polyclonal antibody products has shown. In these situations, the synergistic action of two or more mAbs together with a small molecule drug will most likely be required for therapeutic efficacy. This review aims to highlight the scientific and economic opportunities and obstacles that are encountered in the quest to add mAbs to the armament of anti-infective drugs.     

The effect of conformation on the solution stability of linear vs. cyclic RGD peptides

Abstract: The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg-Gly-Asp-Phe-OH; 1 ) and a cyclic RGD peptide (cyclo-(1, 6)-Ac-Cys-Arg-Gly-Asp-Phe-Pen-NH₂; 2 ); as a function of pH. Previously, it was found that cyclic peptide 2 was 30-fold more stable than linear peptide 1 . Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2 , which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1 , especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2 . This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.     

Solvent constraints on the property space of acetylcholine. 2. Ordered media

The objective of this study was to investigate the conformational and property spaces of acetylcholine in hydrated octanol and in a membrane model. Molecular dynamics simulations of long duration (15 ns) were carried out, yielding 3000 conformers. For each, we calculated N(+)-C8 distance, solvent-accessible surface area (SAS), polar surface area (PSA), dipole moment, and lipophilicity (virtual logP). Their variations as a function of the dihedral angles tau(2) and tau(3) remained unexpectedly broad and comparable to those seen previously in a vacuum, in water, and in chloroform.(12) Thus, each of the seven conformational clusters was able to access a marked proportion of the lipophilicity space accessible to acetylcholine (0.40 in the logP scale). Histograms of logP distributions revealed two overlapping populations, namely more lipophilic and more hydrophilic. Their deconvolution into two Gaussian curves demonstrated solvent-mediated constraints on the lipophilicity space of acetylcholine, clearly showing how a polar medium favors polar conformers, whereas the opposite is true for media of low polarity.     

Monoclonal antibody:the corner stone of modern biotherapeutics

Worldwide sales of biologic drugs exceeded 100 billion USD in 2011.About 32% is from therapeutic monoclonal antibody(mAb).With many blockbuster biopharmaceutical patents expiring over the next decade,there is a great opportunity for biosimilar to enter the worldwide especially emerging market.Both European Medicines Agency(EMA) and Food and Drug Administration(FDA) have introduced regulatory frameworks for the potential approval of biosimilar mAb therapeutics.Rather than providing a highly abbreviated path,as in the case for small molecule chemical drug,approval for biosimilar mAb will require clinical trial and the details will be very much on a case-by-case basis.Since mAb is the dominant category of biologic drugs,mAb will be the focus of this review.First,the United States(US) and European Union(EU) approved mAb and those in phase 3 trials will be reviewed,then strategies on how to win biosimilar competition will be reviewed.     

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro

Aim:

To evaluate the biochemical features and activities of a glyco-engineered form of the anti-human epidermal growth factor receptor monoclonal antibody (EGFR mAb) cetuximab in vitro.

Methods:

The genes encoding the Chinese hamster bisecting glycosylation enzyme (GnTIII) and anti-human EGFR mAb were cloned and coexpressed in CHO DG44 cells. The bisecting-glycosylated recombinant EGFR mAb (bisec-EGFR mAb) produced by these cells was characterized with regard to its glycan profile, antiproliferative activity, Fc receptor binding affinity and cell lysis capability. The content of galactose-α-1,3-galactose (α-Gal) in the bisec-EGFR mAb was measured using HPAEC-PAD.

Results:

The bisec-EGFR mAb had a higher content of bisecting N-acetylglucosamine residues. Compared to the wild type EGFR mAb, the bisec-EGFR mAb exhibited 3-fold higher cell lysis capability in the antibody-dependent cellular cytotoxicity assay, and 1.36-fold higher antiproliferative activity against the human epidermoid carcinoma line A431. Furthermore, the bisec-EGFR mAb had a higher binding affinity for human FcγRIa and FcγRIIIa-158F than the wild type EGFR mAb. Moreover, α-Gal, which was responsible for cetuximab-induced hypersensitivity reactions, was not detected in the bisec-EGFR mAb.

Conclusion:

The glyco-engineered EGFR mAb with more bisecting modifications and lower α-Gal content than the approved therapeutic antibody Erbitux shows improved functionality in vitro, and requires in vivo validations.     

Effect of Peroxide- Versus Alkoxyl-Induced Chemical Oxidation on the Structure,Stability, Aggregation,and Function of a Therapeutic Monoclonal Antibody

Current guidelines indicate that the effects of oxidation should be included as part of forced degradation studies on protein drugs. We probed the effect of 3 commonly used oxidants, hydrogen peroxide, tert-butyl hydroperoxide, and 2,2'-Azobis(2-amidinopropane) dihydrochloride (AAPH), on a therapeutic monoclonal IgG1 antibody (mAb8). Upon oxidation, mAb8 did not show noticeable changes in its secondary structure but showed minor changes in tertiary structure. Significant decrease in conformational stability was observed for all the 3 oxidized forms. Both hydrogen peroxide and tert-butyl hydroperoxide destabilized mainly the C_H2 domain, whereas AAPH destabilized the variable domain in addition to C_H2. Increased aggregation was found for AAPH-oxidized mAb8. In addition, a significant decrease in Fc receptor binding was observed for all 3 oxidized forms. Antibody dependent cell-mediated cytotoxicity, binding to target protein receptor, and cell proliferation activity were significantly reduced in the case of AAPH-oxidized mAb8. The presence of free methionine in the formulation buffer seems to alleviate the effect of oxidation. The results of this study show that the 3 oxidants differ in terms of their effects on the structure and function of mAb8 because of chemical modification of different sets of residues located in Fab versus Fc.     

Assessment of Physical Stability of an Antibody Drug Conjugate by Higher Order Structure Analysis: Impact of Thiol- Maleimide Chemistry

Purpose

To provide a systematic biophysical approach towards a better understanding of impact of conjugation chemistry on higher order structure and physical stability of an antibody drug conjugate (ADC).

Methods

ADC was prepared using thiol-maleimide chemistry. Physical stabilities of ADC and its parent IgG1 mAb were compared using calorimetric, spectroscopic and molecular modeling techniques.

Results

ADC and mAb respond differently to thermal stress. Both the melting temperatures and heat capacities are substantially lower for the ADC. Spectroscopic experiments show that ADC and mAb have similar secondary and tertiary structures, but these are more easily destabilized by thermal stress on the ADC indicating reduced conformational stability. Molecular modeling calculations suggest a substantial decrease in the conformational energy of the mAb upon conjugation. The local surface around the conjugation sites also becomes more hydrophobic in the ADC, explaining the lower colloidal stability and greater tendency of the ADC to aggregate.

Conclusions

Computational and biophysical analyses of an ADC and its parent mAb have provided insights into impact of conjugation on physical stability and pinpointed reasons behind lower structural stability and increased aggregation propensity of the ADC. This knowledge can be used to design appropriate formulations to stabilize the ADC.     

Kappa-opioid receptor model in a phospholipid bilayer: molecular dynamics simulation

A three-dimensional molecular model of the transmembrane domain of the kappa-opioid receptor in a phospholipid bilayer is presented. The endogenous ligand, dynorphin A (1), and synthetic ligands, benzomorphan-based compounds (2a, 2b) (Figure 1), are docked into the model. We report the results of a 500 ps molecular dynamics simulation of these protein-ligand complexes in a simplified bilayer of 97 molecules of the lipid dipalmitoylphosphatidylcholine and 26 water molecules per lipid. The simulations explore the stability and conformational dynamics of the model in a phospholipid bilayer; we also investigate the interactions of the protein with its ligands. Molecular simulation of the receptor-ligand complexes, endogenous and synthetic, has confirmed the existence of different binding domains for peptide and non-peptide ligands. Similarities are found in the dynamics and binding mode of all conformations of the synthetic ligands studied. The protonated hydrogen of the benzomorphan is always involved in an H-bond with Asp138, and other potentially stabilizing receptor-ligand interactions found involve the hydroxyl substituent on the benzomorphan, which may form an H-bond with Tyr139 or Gly190 according to the different molecules. The ester group of 2a may therefore form an H-bond with Ile316, while the carbonyl group of 2b forms an H-bond with Gln115 and Tyr312. The remaining part of the ligand is located in the extracellular portion of the pocket. It is surrounded by hydrophobic residues in the transmembrane region (TM), and it interacts with different sets of residues. The results obtained are in general agreement with site-directed mutagenesis data that have highlighted the importance of all TM regions for synthetic-ligand affinity with the kappa-opioid receptor.