Engineering a human IgG2 antibody stable at low pH

IgG2 subclass antibodies have unique properties that include low effector function and a rigid hinge region. Although some IgG2 subclasses have been clinically tested and approved for therapeutic use, they have a higher propensity than IgG1 for aggregation, which can curtail or abolish their biological activity and enhance their immunogenicity. In this regard, acid‐induced aggregation of monoclonal antibodies during purification and virus inactivation must be prevented. In the present study, we replaced the constant domain of IgG2 with that of IgG1, using anti‐2,4‐dinitrophenol (DNP) IgG2 as a model antibody, and investigated whether that would confer greater stability. While the anti‐DNP IgG2 antibody showed significant aggregation at low pH, this was reduced for the IgG2 antibody containing the IgG1 CH2 domain. Substituting three amino acids within the CH2 domain—namely, F300Y, V309L, and T339A (IgG2_YLA)—reduced aggregation at low pH and increased CH2 transition temperature, as determined by differential scanning calorimetric analysis. IgG2_YLA exhibited similar antigen‐binding capacity to IgG2, low affinity for FcγRIIIa, and low binding ability to C1q. The same YLA substitution also reduced the aggregation of panitumumab, another IgG2 antibody, at low pH. Our engineered human IgG2 antibody showed reduced aggregation during bioprocessing and provides a basis for designing improved IgG2 antibodies for therapeutic applications.     

Effects of subclass change on the structural stability of chimeric,humanized, and human antibodies under thermal stress

To address how changes in the subclass of antibody molecules affect their thermodynamic stability, we prepared three types of four monoclonal antibody molecules (chimeric, humanized, and human) and analyzed their structural stability under thermal stress by using size‐exclusion chromatography, differential scanning calorimetry (DSC), circular dichroism (CD), and differential scanning fluoroscopy (DSF) with SYPRO Orange as a dye probe. All four molecules showed the same trend in change of structural stability; the order of the total amount of aggregates was IgG1 < IgG2 < IgG4. We thus successfully cross‐validated the effects of subclass change on the structural stability of antibodies under thermal stress by using four methods. The T_h values obtained with DSF were well correlated with the onset temperatures obtained with DSC and CD, suggesting that structural perturbation of the CH2 region could be monitored by using DSF. Our results suggested that variable domains dominated changes in structural stability and that the physicochemical properties of the constant regions of IgG were not altered, regardless of the variable regions fused.     

Protein aggregation and mitigation strategy in low pH viral inactivation for monoclonal antibody purification

ABSTRACT

Significant amounts of soluble product aggregates were observed during low-pH viral inactivation (VI) scale-up for an IgG4 monoclonal antibody (mAb IgG4-N1), while small-scale experiments in the same condition showed negligible aggregation. Poor mixing and product exposure to low pH were identified as the root cause. To gain a mechanistic understanding of the problem, protein aggregation properties were studied by varying critical parameters including pH, hold time and protein concentration. Comprehensive biophysical characterization of product monomers and aggregates was performed using fluorescence-size-exclusion chromatography, differential scanning fluorimetry, fluorescence spectroscopy, and dynamic light scattering. Results showed IgG4-N1 partially unfolds at about pH 3.3 where the product molecules still exist largely as monomers owing to strong inter-molecular repulsions and favorable colloidal stability. In the subsequent neutralization step, however, the conformationally changed monomers are prone to aggregation due to weaker inter-molecular repulsions following the pH transition from 3.3 to 5.5. Surface charge calculations using homology modeling suggested that intra-molecular repulsions, especially between CH2 domains, may contribute to the IgG4-N1 unfolding at ≤ pH 3.3. Computational fluid dynamics (CFD) modeling was employed to simulate the conditions of pH titration to reduce the risk of aggregate formation. The low-pH zones during acid addition were characterized using CFD modeling and correlated to the condition causing severe product aggregation. The CFD tool integrated with the mAb solution properties was used to optimize the VI operating parameters for successful scale-up demonstration. Our research revealed the governing aggregation mechanism for IgG4-N1 under acidic conditions by linking its molecular properties and various process-related parameters to macroscopic aggregation phenomena. This study also provides useful insights into the cause and mitigation of low-pH-induced IgG4 aggregation in downstream VI operation.     

Understanding the relevance of local conformational stability and dynamics to the aggregation propensity of an IgG1 and IgG2 monoclonal antibodies

Aggregation of monoclonal antibodies is often a multi‐step process involving structural alterations in monomeric proteins and subsequent formation of soluble or insoluble oligomers. The role of local conformational stability and dynamics of native and/or partially altered structures in determining the aggregation propensity of monoclonal antibodies, however, is not well understood. Here, we investigate the role of conformational stability and dynamics of regions with distinct solvent exposure in determining the aggregation propensity of an IgG1 and IgG2 monoclonal antibody. The temperatures employed span the pre‐unfolding range (10–40°C) and the onset temperatures (T_onset) for exposure of apolar residues (～50°C), alterations in secondary structures (～60°C) and initiation of visible aggregate formation (～60°C). Solvent‐exposed regions were found to precede solvent‐shielded regions in an initiation of aggregation for both proteins. Such a process was observed upon alterations in overall tertiary structure while retaining the secondary structures in both the proteins. In addition, a greater dynamic nature of solvent‐shielded regions in potential intermediates of IgG1 and the improved conformational stability increased its resistance to aggregation when compared to IgG2. These results suggest that local conformational stability and fluctuations of partially altered structures can influence the aggregation propensity of immunoglobulins.     

A single mutation induces amyloid aggregation in the alpha-spectrin SH3 domain: analysis of the early stages of fibril formation

The Src-homology region 3 domain of chicken alpha-spectrin (Spc-SH3) is a small two-state folding protein, which has never been described to form amyloid fibrils under any condition investigated so far. We show here that the mutation of asparagine 47 to alanine at the distal loop, which destabilises similarly the native and folding transition states of the domain, induces the formation of amyloid fibrils under mild acid conditions. Amyloid aggregation of the mutant is enhanced by the increase in temperature, protein concentration and NaCl concentration. The early stages of amyloid formation have been monitored as a function of time and temperature using a variety of biophysical methods. Differential scanning calorimetry experiments under conditions of amyloid formation have allowed the identification of different thermal transitions corresponding to conformational and aggregation processes as well as to the high-temperature disaggregation and unfolding of the amyloid fibrils. Aggregation is preceded by a rapid conformational change in the monomeric domain involving about 40% of the global unfolding enthalpy, considerable change in secondary structure, large loss of tertiary structure and exposure of hydrophobic patches to the solvent. The conformational change is followed by formation of a majority of oligomeric species with apparent hydrodynamic radius between 2.5 nm and 10nm, depending on temperature, together with the appearance and progressive growth of protofibrillar aggregates. After these early aggregation stages, long and curved fibrils of up to several micrometers start to develop by elongation of the protofibrils. The calorimetric data indicate that the specific enthalpy of fibril disaggregation and unfolding is relatively low, suggesting a low density of interactions within the fibril structure as compared to the native protein and a main entropy contribution to the stability of the amyloid fibrils.     

Influence of N-glycosylation on effector functions and thermal stability of glycoengineered IgG1 monoclonal antibody with homogeneous glycoforms

Glycosylation of the conserved asparagine residue in each heavy chain of IgG in the CH2 domain is known as N-glycosylation. It is one of the most common post-translational modifications and important critical quality attributes of monoclonal antibody (mAb) therapeutics. Various studies have demonstrated the effects of the Fc N-glycosylation on safety, Fc effector functions, and pharmacokinetics, both dependent and independent of neonatal Fc receptor (FcRn) pathway. However, separation of various glycoforms to investigate the biological and functional relevance of glycosylation is a major challenge, and existing studies often discuss the overall impact of N-glycans, without considering the individual contributions of each glycoform when evaluating mAbs with highly heterogeneous distributions. In this study, chemoenzymatic glycoengineering incorporating an endo-β-N-acetylglucosaminidase (ENGase) EndoS2 and its mutant with transglycosylation activity was used to generate mAb glycoforms with highly homogeneous and well-defined N-glycans to better understand and precisely evaluate the effect of each N-glycan structure on Fc effector functions and protein stability. We demonstrated that the core fucosylation, non-reducing terminal galactosylation, sialylation, and mannosylation of IgG1 mAb N-glycans impact not only on FcγRIIIa binding, antibody-dependent cell-mediated cytotoxicity, and C1q binding, but also FcRn binding, thermal stability and propensity for protein aggregation.     

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the C_H2 and distant segments in the V_H, C_H1, and V_L domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244–254 segment of the C_H2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (T_onset) and unfolding (T_m1) temperatures of 7°C and 6.7°C, respectively, for the C_H2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244–254 segment, providing a site-directed mutant example that this segment of the C_H2 domain is an aggregation hot spot in IgG1 mAbs.     

Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress

Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lag‐time behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size‐exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.     

Correlations between changes in conformational dynamics and physical stability in a mutant IgG1 mAb engineered for extended serum half-life

This study compares the local conformational dynamics and physical stability of an IgG1 mAb (mAb-A) with its corresponding YTE (M255Y/S257T/T259E) mutant (mAb-E), which was engineered for extended half-life in vivo. Structural dynamics was measured using hydrogen/deuterium (H/D) exchange mass spectrometry while protein stability was measured with differential scanning calorimetry (DSC) and size exclusion chromatography (SEC). The YTE mutation induced differences in H/D exchange kinetics at both pH 6.0 and 7.4. Segments covering the YTE mutation sites and the FcRn binding epitopes showed either subtle or no observable differences in local flexibility. Surprisingly, several adjacent segments in the C_H2 and distant segments in the V_H, C_H1, and V_L domains had significantly increased flexibility in the YTE mutant. Most notable among the observed differences is increased flexibility of the 244–254 segment of the C_H2 domain, where increased flexibility has been shown previously to correlate with decreased conformational stability and increased aggregation propensity in other IgG1 mAbs (e.g., presence of destabilizing additives as well as upon de-glycosylation or methionine oxidation). DSC analysis showed decreases in both thermal onset (T_onset) and unfolding (T_m1) temperatures of 7°C and 6.7°C, respectively, for the C_H2 domain of the YTE mutant. In addition, mAb-E aggregated faster than mAb-A under accelerated stability conditions as measured by SEC analysis. Hence, the relatively lower physical stability of the YTE mutant correlates with increased local flexibility of the 244–254 segment, providing a site-directed mutant example that this segment of the C_H2 domain is an aggregation hot spot in IgG1 mAbs.     

Impact of freezing on pH of buffered solutions and consequences for monoclonal antibody aggregation

Freezing of biologic drug substance at large scale is an important unit operation that enables manufacturing flexibility and increased use‐period for the material. Stability of the biologic in frozen solutions is associated with a number of issues including potentially destabilizing pH changes. The pH changes arise from temperature‐associated change in the pK_as, solubility limitations, eutectic crystallization, and cryoconcentration. The pH changes for most of the common protein formulation buffers in the frozen state have not been systematically measured. Sodium phosphate buffer, a well‐studied system, shows the greatest change in pH when going from +25 to ?30°C. Among the other buffers, histidine hydrochloride, sodium acetate, histidine acetate, citrate, and succinate, less than 1 pH unit change (increase) was observed over the temperature range from +25 to ?30°C, whereas Tris‐hydrochloride had an ～1.2 pH unit increase. In general, a steady increase in pH was observed for all these buffers once cooled below 0°C. A formulated IgG2 monoclonal antibody in histidine buffer with added trehalose showed the same pH behavior as the buffer itself. This antibody in various formulations was subject to freeze/thaw cycling representing a wide process (phase transition) time range, reflective of practical situations. Measurement of soluble aggregates after repeated freeze–thaw cycles shows that the change in pH was not a factor for aggregate formation in this case, which instead is governed by the presence or absence of noncrystallizing cryoprotective excipients. In the absence of a cryoprotectant, longer phase transition times lead to higher aggregation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Genetic and physical interaction of Ssp1 CaMKK and Rad24 14-3-3 during low pH and osmotic stress in fission yeast

The Ssp1 calmodulin kinase kinase (CaMKK) is necessary for stress-induced re-organization of the actin cytoskeleton and initiation of growth at the new cell end following division in Schizosaccharomyces pombe. In addition, it regulates AMP-activated kinase and functions in low glucose tolerance. ssp1⁻ cells undergo mitotic delay at elevated temperatures and G₂ arrest in the presence of additional stressors. Following hyperosmotic stress, Ssp1-GFP forms transient foci which accumulate at the cell membrane and form a band around the cell circumference, but not co-localizing with actin patches. Hyperosmolarity-induced localization to the cell membrane occurs concomitantly with a reduction of its interaction with the 14-3-3 protein Rad24, but not Rad25 which remains bound to Ssp1. The loss of rad24 in ssp1⁻ cells reduces the severity of hyperosmotic stress response and relieves mitotic delay. Conversely, overexpression of rad24 exacerbates stress response and concomitant cell elongation. rad24⁻ does not impair stress-induced localization of Ssp1 to the cell membrane, however this response is almost completely absent in cells overexpressing rad24.     

Contributions of a disulfide bond to the structure, stability, and dimerization of human IgG1 antibody CH3 domain

Recombinant human monoclonal antibodies have become important protein-based therapeutics for the treatment of various diseases. The antibody structure is complex, consisting of beta-sheet rich domains stabilized by multiple disulfide bridges. The dimerization of the C(H)3 domain in the constant region of the heavy chain plays a pivotal role in the assembly of an antibody. This domain contains a single buried, highly conserved disulfide bond. This disulfide bond was not required for dimerization, since a recombinant human C(H)3 domain, even in the reduced state, existed as a dimer. Spectroscopic analyses showed that the secondary and tertiary structures of reduced and oxidized C(H)3 dimer were similar, but differences were observed. The reduced C(H)3 dimer was less stable than the oxidized form to denaturation by guanidinium chloride (GdmCl), pH, or heat. Equilibrium sedimentation revealed that the reduced dimer dissociated at lower GdmCl concentration than the oxidized form. This implies that the disulfide bond shifts the monomer-dimer equilibrium. Interestingly, the dimer-monomer dissociation transition occurred at lower GdmCl concentration than the unfolding transition. Thus, disulfide bond formation in the human C(H)3 domain is important for stability and dimerization. Here we show the importance of the role played by the disulfide bond and how it affects the stability and monomer-dimer equilibrium of the human C(H)3 domain. Hence, these results may have implications for the stability of the intact antibody.     

Effect of 2‐hydroxypropyl‐β‐cyclodextrin on thermal stability and aggregation of glycogen phosphorylase b from rabbit skeletal muscle

The study of the kinetics of thermal aggregation of glycogen phosphorylase b (Phb) from rabbit skeletal muscles by dynamic light scattering at 48°C showed that 2‐hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) accelerated the aggregation process and induced the formation of the larger protein aggregates. The reason of the accelerating effect of HP‐β‐CD is destabilization of the protein molecule under action of HP‐β‐CD. This conclusion was supported by the data on differential scanning calorimetry and the kinetic data on thermal inactivation of Phb. It is assumed that destabilization of the Phb molecule is due to preferential binding of HP‐β‐CD to intermediates of protein unfolding in comparison with the original native state. The conclusion regarding the ability of the native Phb for binding of HP‐β‐CD was substantiated by the data on the enzyme inhibition by HP‐β‐CD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 986–993, 2010.     

α-Synuclein aggregation variable temperature and variable pH kinetic data: A re-analysis using the Finke–Watzky 2-step model of nucleation and autocatalytic growth

The aggregation of proteins is believed to be intimately connected to many neurodegenerative disorders. We recently reported an “Ockham's razor”/minimalistic approach to analyze the kinetic data of protein aggregation using the Finke–Watzky (F–W) 2-step model of nucleation (A → B, rate constant k₁) and autocatalytic growth (A + B → 2B, rate constant k₂). With that kinetic model we have analyzed 41 representative protein aggregation data sets in two recent publications, including amyloid β, α-synuclein, polyglutamine, and prion proteins (Morris, A. M., et al. (2008) Biochemistry 47, 2413-2427; Watzky, M. A., et al. (2008) Biochemistry 47, 10790–10800). Herein we use the F–W model to reanalyze protein aggregation kinetic data obtained under the experimental conditions of variable temperature or pH 2.0 to 8.5. We provide the average nucleation (k₁) and growth (k₂) rate constants and correlations with variable temperature or varying pH for the protein α-synuclein. From the variable temperature data, activation parameters ΔG^‡, ΔH^‡, and ΔS^‡ are provided for nucleation and growth, and those values are compared to the available parameters reported in the previous literature determined using an empirical method. Our activation parameters suggest that nucleation and growth are energetically similar for α-synuclein aggregation (ΔG^‡_nucleation = 23(3) kcal/mol; ΔG^‡_growth = 22(1) kcal/mol at 37 °C). From the variable pH data, the F–W analyses show a maximal k₁ value at pH ~ 3, as well as minimal k₁ near the isoelectric point (pI) of α-synuclein. Since solubility and net charge are minimized at the pI, either or both of these factors may be important in determining the kinetics of the nucleation step. On the other hand, the k₂ values increase with decreasing pH (i.e., do not appear to have a minimum or maximum near the pI) which, when combined with the k₁ vs. pH (and pI) data, suggest that solubility and charge are less important factors for growth, and that charge is important in the k₁, nucleation step of α-synuclein. The chemically well-defined nucleation (k₁) rate constants obtained from the F–W analysis are, as expected, different than the 1/lag-time empirical constants previously obtained. However, k₂ × [A]₀ (where k₂ is the rate constant for autocatalytic growth and [A]₀ is the initial protein concentration) is related to the empirical constant, k_app obtained previously. Overall, the average nucleation and average growth rate constants for α-synuclein aggregation as a function of pH and variable temperature have been quantitated. Those values support the previously suggested formation of a partially folded intermediate that promotes aggregation under high temperature or acidic conditions.     

Creating stable stem regions for loop elongation in Fcabs — Insights from combining yeast surface display,in silico loop reconstruction and molecular dynamics simulations

Fcabs (Fc antigen binding) are crystallizable fragments of IgG where the C-terminal structural loops of the CH3 domain are engineered for antigen binding. For the design of libraries it is beneficial to know positions that will permit loop elongation to increase the potential interaction surface with antigen. However, the insertion of additional loop residues might impair the immunoglobulin fold. In the present work we have probed whether stabilizing mutations flanking the randomized and elongated loop region improve the quality of Fcab libraries. In detail, 13 libraries were constructed having the C-terminal part of the EF loop randomized and carrying additional residues (1, 2, 3, 5 or 10, respectively) in the absence and presence of two flanking mutations. The latter have been demonstrated to increase the thermal stability of the CH3 domain of the respective solubly expressed proteins. Assessment of the stability of the libraries expressed on the surface of yeast cells by flow cytometry demonstrated that loop elongation was considerably better tolerated in the stabilized libraries. By using in silico loop reconstruction and mimicking randomization together with MD simulations the underlying molecular dynamics were investigated. In the presence of stabilizing stem residues the backbone flexibility of the engineered EF loop as well as the fluctuation between its accessible conformations were decreased. In addition the CD loop (but not the AB loop) and most of the framework regions were rigidified. The obtained data are discussed with respect to the design of Fcabs and available data on the relation between flexibility and affinity of CDR loops in Ig-like molecules.     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.     

Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast

We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and T_m-values up to 85 °C was developed. Besides library construction by error prone PCR, strong heat stress at 79 °C for 10 min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.     

Effect of poly(phosphate) anions on glyceraldehyde-3-phosphate dehydrogenase structure and thermal aggregation: comparison with influence of poly(sulfoanions)

Background

It is well documented that poly(sulfate) and poly(sulfonate) anions suppress protein thermal aggregation much more efficiently than poly(carboxylic) anions, but as a rule, they denature protein molecules. In this work, a polymer of different nature, i.e. poly(phosphate) anion (PP) was used to elucidate the influence of phosphate groups on stability and thermal aggregation of the model enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Methods

Isothermal titration calorimetry and differential scanning calorimetry were used for studying the protein–polyanion interactions and the influence of bound polyanions on the protein structure. The enzymatic activity of GAPDH and size of the complexes were measured. The aggregation level was determined from the turbidity.

Results

Highly polymerized PP chains were able to suppress the aggregation completely, but at significantly higher concentrations as compared with poly(styrenesulfonate) (PSS) or dextran sulfate chains of the same degree of polymerization. The effect of PP on the enzyme structure and activity was much gentler as opposed to the binding of dextran sulfate or, especially, PSS that denatured GAPDH molecules with the highest efficacy caused by short PSS chains. These findings agreed well with the enhanced affinity of polysulfoanions to GAPDH.

Conclusions

The revealed trends might help to illuminate the mechanism of control of proteins functionalities by insertion of charged groups of different nature through posttranslational modifications.

General significance

Practical implementation of the results could be the use of PP chains as promising tools to suppress the proteins aggregation without noticeable loss in the enzymatic activity.     

Conformational analysis by NMR and molecular dynamics of adamantane-doxorubicin prodrugs and their assemblies with β-cyclodextrin: A focus on the design of platforms for controlled drug delivery

Nanoscale design and construction of affinity-based drug delivery systems (ADDS) is an active research area with enormous potential for the improvement of cancer treatment. For the therapeutic load of these ADDS, a promising strategy is the design of pH-sensitive prodrugs based on the construction of conjugates between adamantane and doxorubicin (Ad-Dox), which stands out as an excellent model system to obtain novel supramolecular materials. Construction of these prodrugs involves a modification of three zones of doxorubicin which in principle does not affect the action mechanism: the carbonyl group C13 (hydrazone linker), the primary alcohol neighboring the carbonyl (ester linker) and the 3′ amino group of daunosamine sugar (amide linker). These modifications are aimed to improve the efficacy and reduce the systemic toxicity of the drug chemotherapy by controlling its release in cancer cells. In this work, we performed 2D NMR experiments and molecular dynamics simulations to characterize the conformational changes of three constructed prodrugs. Our results demonstrated that ring A and the daunsamine sugar of the hydrazone and amide linkers conserve the half-chair state ⁹H₈, while the ester linker disrupts this conformation. Our study also showed that the hydrazone-linked compound (Ad-h-Dox) does not modify the conformation of the original drug and maintains cytotoxic activity. Moreover, the inclusion complex (IC) of Ad-h-Dox with β-cyclodextrin (βCD) generated a highly soluble platform in water, whereas the ester-linked compound (Ad-e-Dox) causes the loss of biological activity. This study proves that Ad-h-Dox prodrug can be an optimum prodrug and act as a building block for a more complex drug transport system.