The structural basis of histone modifying enzyme specificity and promiscuity: Implications for metabolic regulation and drug design.

Histone modifying enzymes regulate chromatin architecture through covalent modifications and ultimately control multiple aspects of cellular function. Disruption of histone modification leads to changes in gene expression profiles and may lead to disease. Both small molecule inhibitors and intermediary metabolites have been shown to modulate histone modifying enzyme activity although our ability to identify successful drug candidates or novel metabolic regulators of these enzymes has been limited. Using a combination of large scale in silico screens and in vivo phenotypic analysis, we identified several small molecules and intermediary metabolites with distinctive HME activity. Our approach using unsupervised learning identifies the chemical fingerprints of both small molecules and metabolites that facilitate recognition by the enzymes active sites which can be used as a blueprint to design novel inhibitors. Furthermore, this work supports the idea that histone modifying enzymes sense intermediary metabolites integrating genes, environment and cellular physiology.

Prediction Machines: Applied Machine Learning for Therapeutic Protein Design and Development.

The rapid growth in technological advances and quantity of scientific data over the past decade has led to several challenges including data storage and analysis. Accurate models of complex datasets were previously difficult to develop and interpret. However, improvements in machine learning algorithms have since enabled unparalleled classification and prediction capabilities. The application of machine learning can be seen throughout diverse industries due to their ease of use and interpretability. In this review, we describe popular machine learning algorithms and highlight their application in pharmaceutical protein development. Machine learning models have now been applied to better understand the nonlinear concentration dependent viscosity of protein solutions, predict protein oxidation and deamidation rates, classify sub-visible particles and compare the physical stability of proteins. We also applied several machine learning algorithms using previously published data and describe models with improved predictions and classification. The authors hope that this review can be used as a resource to others and encourage continued application of machine learning algorithms to problems in pharmaceutical protein development.

Vibrational spectroscopy and chemometrics to characterize and quantitate trehalose crystallization.

Investigating the phase behavior of sugars in ice and lyophilized solids is of significant interest in the pharmaceutical industry. In this study, Raman and near infrared (NIR) spectroscopy are used to characterize and quantitate trehalose crystallization using several chemometric models. The predictive behaviors of partial least squares (PLS), principal component analysis (PCA), and multiple linear regression (MLR) models are compared. In general, PLS and PCA outperform linear and MLR models. Changes in specific vibrational modes associated with several coupled motions are described and assigned as a function of crystal content. In addition to characterization and quantitation, our method may be used to localize gradients of amorphous and/or crystallized trehalose within a sample.

Characteristics of rhVEGF release from topical hydrogel formulations.

PURPOSE: To study recombinant human vascular endothelial growth factor (rhVEGF), the release characteristics from topical gel formulations, and its interaction with the gelling agents. METHODS: The release kinetics were followed by quantifying rhVEGF that diffused into the receptor chamber of Franz cells. Analytical ultracentrifuge (AUC) was used to characterize the sedimentation velocity of rhVEGF experienced in the gel. The interactions were characterized by isothermal calorimetry (ITC), and rhVEGF conformation was assessed by circular dichroism (CD). RESULTS: The fraction of protein released was linear with the square root of time. The release rate constants did not show significant change within a wide range of bulk viscosities created by different concentrations of hydroxypropyl methylcellulose (HPMC) or MC gels. Sedimentation velocity determined by AUC generated comparable sedimentation coefficients of protein in these gels. AUC and ITC revealed no significant interaction between rhVEGF and HPMC and some change on secondary structure of the protein by Far UV CD, which was not the case with carboxymethyl cellulose (CMC). CONCLUSIONS: Microviscosity, not bulk viscosity, was the key factor for the release of rhVEGF from cellulosic gels such as HPMC. Interaction between rhVEGF and CMC resulted in slower, and reduced amount of, release from the gel.

Increasing IgG concentration modulates the conformational heterogeneity and bonding network that influence solution properties.

Multiple molecular driving forces mediate protein stability, association, and recognition in concentrated solutions. Here we investigate the interactions that modulate the nonideal solution behavior of two immunoglobulins (IgG1s) in highly concentrated solutions using two-dimensional vibrational correlation spectroscopy (2D-COS) and principal components analysis (PCA). A specific sequence of changes is observed in the concentration-dependent vibrational spectra of the highly viscous IgG solution that deviates from ideality, whereas that sequence is reversed for all other conditions examined. The asynchronous spectra reveal variation in beta-sheet and turn regions occur before intensity variations in disordered and alpha-helical regions as the concentration is increased for the highly viscous regime. This is in contrast to the sequence observed for all other conditions studied and to the idea that beta-sheet regions are resistant to concentration-dependent affects. Finally, we show that increased hydrogen bonding and electrostatics primarily modulate the intermolecular association and nonideal behavior. Specifically, 2D-COS and PCA analysis of the amide II region suggests that Glu and Asp residues trigger the change resulting in increased viscosity and association of one IgG.

Immunoglobulin dynamics, conformational fluctuations, and nonlinear elasticity and their effects on stability.

The relationships between protein dynamics, function, and stability are incompletely understood. Two external perturbations (temperature and pH) were used to modulate the flexibility and stability of an IgG1kappa monoclonal antibody (mAb) in an attempt to better understand the possible correlations between flexibility and stability. Ultrasonic velocimetry, densitometry, differential scanning calorimetry (DSC), and pressure perturbation calorimetry (PPC) were used to experimentally determine the adiabatic and isothermal compressibility, expansibility, fractional volumes of unfolding, and various nonlinear thermoacoustical parameters as a function of pH and temperature. By combining these results, state parameter fluctuations were calculated from fundamental statistical mechanical relationships. The most dynamic and rigid mAb ensemble is measured at pH 4 and 6, respectively, based on state parameter fluctuations and compressibility. The effect of pH appears to couple mAb dynamics to solvent fluctuations, which control its dynamics and stability. A nonlinear response to mechanical perturbation, comparable to that seen with many polymers, is observed for this monoclonal antibody at pH 4-8. This behavior is characterized as strongly anisotropic and anharmonic, especially at pH 4. The midpoint of thermal unfolding as measured by DSC does not necessarily correlate with flexibility.

The interaction of heparin/polyanions with bovine, porcine, and human growth hormone.

The interaction of polyanions with proteins is of potential pharmaceutical and cellular significance. A partial thermodynamic description of the interaction of four representative polyanions with human, bovine, and porcine growth hormone is described. A heparin bead-binding assay confirms all growth hormones bind to heparin but to varying extents. Moderate-binding constants and high ratios of bound protein to the more extended polyanions, heparin, and dextran sulfate were measured by isothermal titration calorimetry and dynamic light scattering. The binding constants and ratio of protein bound to ligand were significantly smaller for the low molecular weight polyanions phytic acid and sucrose octasulfate (SOS). The effect of polyanion binding on the bovine, porcine, and human growth hormone's (hGH) structural and colloidal stability was also explored. Heparin and dextran sulfate inhibit porcine somatotropin (pST) and bovine somatotropin (bST) aggregation to the greatest extent, as compared to phytic acid and SOS, while decreasing secondary and tertiary structural stability as measured by the temperature dependence of their circular dichroism and intrinsic fluorescence. Somewhat surprisingly, the polyanions do not appear to affect the structure or stability of hGH. The potential biological significance of growth hormone polyanion interactions is discussed.

Parathyroid hormone is a heparin/polyanion binding protein: binding energetics and structure modification.

The interaction of four representative polyanions with parathyroid hormone (PTH) residues 1-84 has been investigated utilizing a variety of spectroscopic and calorimetric techniques. Each of the polyanions employed demonstrate enthalpically driven binding to PTH (1-84) with significant affinity. The polyanions heparin, dextran sulfate, phytic acid, and sucrose octasulfate induce alpha-helical structure in PTH to varying extents depending on the ratio of polyanion to protein employed. Intrinsic and extrinsic fluorescence spectroscopy suggests significant protein tertiary structure alteration upon polyanion binding. Although structural modification occurred upon polyanion binding, PTH colloidal stability was increased depending on the ratio of polyanion to protein used. Nevertheless, the bioactivity of PTH in the presence of various ratios of heparin was not altered. The potential biological significance of PTH/polyanion interactions is discussed.

Conformational flexibility, hydration and state parameter fluctuations of fibroblast growth factor-10: effects of ligand binding.

Differential effects of ligand binding on local and global fibroblast growth factor-10 (FGF-10) flexibility and stability have been investigated utilizing a variety of experimental and computational techniques. Normal mode analysis was used to predict the low frequency motions and regional flexibility of FGF-10. Similarly, regional variations in local folding/unfolding equilibria were characterized with the COREX/BEST algorithm. Experimental adiabatic and isothermal compressibilities of FGF-10 alone and in the presence of polyanions are compared. Furthermore, the effect of polyanions on the coefficient of thermal expansion is compared. Measurements of density, heat capacity, compressibility, and expansibility were combined to calculate experimentally determined volume and enthalpy fluctuations. Global effects of polyanions on FGF-10 flexibility, thermodynamic fluctuations, and hydration vary depending on the size and charge density of the polyanion. Local effects of polyanions were investigated utilizing time-resolved fluorescence spectroscopy and red edge excitation spectroscopy (REES). Increased rigidity of the protein matrix or an increased solvent response surrounding the Trp residues is observed in the presence of polyanions. Similarly, time-resolved spectroscopy reveals increased ground state heterogeneity and increased dipole relaxation on the time scale of fluorescence for FGF-10 in the presence of polyanions. These polyanions increase heterogeneity, global flexibility, and fluctuations while increasing the melting temperature (Tm) of FGF-10.

Thermodynamic and structural characterization of an antibody gel.

Although extensively studied, protein-protein interactions remain highly elusive and are of increasing interest in drug development. We show the assembly of a monoclonal antibody, using multivalent carboxylate ions, into highly-ordered structures. While the presence and function of similar structures in vivo are not known, the results may present a possible unexplored area of antibody structure-function relationships. Using a variety of tools (e.g., mechanical rheology, electron microscopy, isothermal calorimetry, Fourier transform infrared spectroscopy), we characterized the physical, biochemical, and thermodynamic properties of these structures and found that citrate may interact directly with the amino acid residue histidine, after which the individual protein units assemble into a filamentous network gel exhibiting high elasticity and interfilament interactions. Citrate interacts exothermically with the monoclonal antibody with an association constant that is highly dependent on solution pH and temperature. Secondary structure analysis also reveals involvement of hydrophobic and aromatic residues.

Polar solvents decrease the viscosity of high concentration IgG1 solutions through hydrophobic solvation and interaction: formulation and biocompatibility considerations.

Low-volume protein dosage forms for subcutaneous injection pose unique challenges to the pharmaceutical scientist. Indeed, high protein concentrations are often required to achieve acceptable bioavailability and efficacy for many indications. Furthermore, high solution viscosities are often observed with formulations containing protein concentrations well above 150 mg/mL. In this work, we explored the use of polar solvents for reducing solution viscosity of high concentration protein formulations intended for subcutaneous injection. An immunoglobulin, IgG1, was used in this study. The thermodynamic preferential interaction parameter (Γ23 ) measured by differential scanning calorimetry, as well as Fourier transform infrared, Raman, and second-derivative UV spectroscopy, were used to characterize the effects of polar solvents on protein structure and to reveal important mechanistic insight regarding the nature of the protein-solvent interaction. Finally, the hemolytic potential and postdose toxicity in rats were determined to further investigate the feasibility of using these cosolvents for subcutaneous pharmaceutical formulations. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 102:1182-1193, 2013.

Coarse-grained modeling of the self-association of therapeutic monoclonal antibodies.

Coarse-grained computational models of two therapeutic monoclonal antibodies are constructed to understand the effect of domain-level charge-charge electrostatics on the self-association phenomena at high protein concentrations. The coarse-grained representations of the individual antibodies are constructed using an elastic network normal-mode analysis. Two different models are constructed for each antibody for a compact Y-shaped and an extended Y-shaped configuration. The resulting simulations of these coarse-grained antibodies that interact through screened electrostatics are done at six different concentrations. It is observed that a particular monoclonal antibody (hereafter referred to as MAb1) forms three-dimensional heterogeneous structures with dense regions or clusters compared to a different monoclonal antibody (hereafter referred to as MAb2) that forms more homogeneous structures (no clusters). These structures, together with the potential mean force (PMF) and radial distribution functions (RDF) between pairs of coarse-grained regions on the MAbs, are qualitatively consistent with the experimental observation that MAb1 has a significantly higher viscosity compared to MAb2, especially at concentrations >50 mg/mL, even though the only difference between the MAbs lies with a few amino acids at the antigen-binding loops (CDRs). It is also observed that the structures in MAb1 are formed due to stronger Fab-Fab interactions in corroboration with experimental observations. Evidence is also shown that Fab-Fc interactions can be equally important in addition to Fab-Fab interactions. The coarse-grained representations are effective in picking up differences based on local charge distributions of domains and make predictions on the self-association characteristics of these protein solutions. This is the first computational study of its kind to show that there are differences in structures formed by two different monoclonal antibodies at high concentrations.

The relative rate of immunoglobulin gamma 1 fragmentation.

The physicochemical stability of protein therapeutics is of significant pharmaceutical interest. Immunoglobulin gamma (IgG) hinge region fragmentation has recently garnered attention as an important degradation route of therapeutic monoclonal antibodies. In this work, the rates and relative amount of fragment species are compared for five different IgGs (IgG1-5) with widely varying solution properties. Native size-exclusion chromatography (SEC), sodium dodecyl sulfate (SDS)-based SEC, and capillary electrophoresis-SDS were used to characterize IgG1 fragmentation after storage at 30°C, 40°C, and 50°C. Two-dimensional correlation analysis of the chromatograms as a function of time was used to illustrate the relative rates of cleavage. Interestingly, the relative rate of Fab cleavage was greater than that of other species. An average apparent energy of activation for IgG1 fragmentation was also measured for all five molecules. This work suggests that IgG1 fragmentation is primarily hinge sequence dependent and other IgG1 molecules should behave similarly within the limits of the solution conditions used.

Protein-excipient interactions: mechanisms and biophysical characterization applied to protein formulation development.

The purpose of this review is to demonstrate the critical importance of understanding protein-excipient interactions as a key step in the rational design of formulations to stabilize and deliver protein-based therapeutic drugs and vaccines. Biophysical methods used to examine various molecular interactions between solutes and protein molecules are discussed with an emphasis on applications to pharmaceutical excipients in terms of their effects on protein stability. Key mechanisms of protein-excipient interactions such as electrostatic and cation-pi interactions, preferential hydration, dispersive forces, and hydrogen bonding are presented in the context of different physical states of the formulation such as frozen liquids, solutions, gels, freeze-dried solids and interfacial phenomenon. An overview of the different classes of pharmaceutical excipients used to formulate and stabilize protein therapeutic drugs is also presented along with the rationale for use in different dosage forms including practical pharmaceutical considerations. The utility of high throughput analytical methodologies to examine protein-excipient interactions is presented in terms of expanding formulation design space and accelerating experimental timelines.

Using empirical phase diagrams to understand the role of intramolecular dynamics in immunoglobulin G stability.

Understanding the relationship between protein dynamics and stability is of paramount importance to the fields of biology and pharmaceutics. Clarifying this relationship is complicated by the large amount of experimental data that must be generated and analyzed if motions that exist over the wide range of timescales are to be included. To address this issue, we propose an approach that utilizes a multidimensional vector-based empirical phase diagram (EPD) to analyze a set of dynamic results acquired across a temperature-pH perturbation plane. This approach is applied to a humanized immunoglobulin G1 (IgG1), a protein of major biological and pharmaceutical importance whose dynamic nature is linked to its multiple biological roles. Static and dynamic measurements are used to characterize the IgG and to construct both static and dynamic EPDs. Between pH 5 and 8, a single, pH-dependent transition is observed that corresponds to thermal unfolding of the IgG. Under more acidic conditions, evidence exists for the formation of a more compact, aggregation resistant state of the immunoglobulin, known as A-form. The dynamics-based EPD presents a considerably more detailed pattern of apparent phase transitions over the temperature-pH plane. The utility and potential applications of this approach are discussed.

The complex inter-relationships between protein flexibility and stability.

The ability to successfully formulate and manufacture therapeutic protein dosage forms requires a thorough understanding of their physico-chemical properties. Proteins are inherently dynamic molecules of marginal stability. These properties present unique challenges to the pharmaceutical scientist attempting to develop protein based therapeutics. The physicochemical stability and biological functions of proteins are thought to be intimately related to their global flexibility, intramolecular fluctuations and various other dynamic processes. Our understanding of these relationships, however, is incomplete but undeniably necessary for the development of efficacious therapies. Therefore, a better understanding of the complex inter-relationships between protein flexibility and stability should enable the rational design and optimization of protein formulation conditions based on protein dynamics. This review attempts to define protein dynamics and flexibility while summarizing a select number of studies of potential pharmaceutical interest that evaluate these relationships.

Two-dimensional correlation spectroscopy reveals coupled immunoglobulin regions of differential flexibility that influence stability.

Despite the well-accepted importance of protein flexibility and dynamics in molecular recognition and conformational stability, our understanding of these relationships is incomplete. Immunoglobulin flexibility is essential for antigen binding and adaptation to diverse molecular shapes and sizes. The inherent flexibility of immunoglobulins also renders these molecules suitable for investigating the possible relationships between protein flexibility and stability. To better understand these inter-relationships, we employ generalized perturbation-based two-dimensional correlation FTIR spectroscopy to monitor the time evolution of H-D exchange of an IgG1 as a function of pH. The differential flexibility of various immunoglobulin regions is described in response to an external perturbation and shown to vary widely. The greatest number of regions with differential exchange rates and, thus differential flexibility, is seen at pH 6. Approximately seven, six, five, and four separate states that exchange with different rates were observed at pH 6, 8, 4, and 2, respectively. The overall distribution of exchange rates calculated from the decays of the integrated Amide I and Amide II areas provides further evidence of multiple regions with differential flexibility. The sequence of events at pH 4 determined from the asynchronous vibrational patterns is of significant interest and suggests protonation of Glu and Asp side chains occurs first and initiates changes in the conformation and flexibility of different sheet and turns structure. A complex inter-relationship between differential regional flexibility and conformational coupling (i.e., cooperativity) initiated by changes in pH influences the stability of this IgG.