Contribution of Phe112, Ser114, and Tyr115 to Drug-Binding Selectivity in the A Variant of α1-Acid Glycoprotein.

The plasma protein α1-acid glycoprotein (AGP) primarily affects the pharmacokinetics of basic drugs. There are two AGP variants in humans, A and F1*S, exhibiting distinct drug-binding selectivity. Elucidation of the drug-binding selectivity of human AGP variants is essential for drug development and personalized drug therapy. Herein, we aimed to establish the contribution of amino acids 112 and 114 of human AGP to drug-binding selectively. Both amino acids are located in the drug-binding region and differ between the variants. Phe112/Ser114 of the A variant and its equivalent residues in the F1*S variant (Leu112/Phe114) were swapped with each other. Binding experiments were then conducted using the antiarrhythmic drug disopyramide, which selectively binds to the A variant. A significant decrease in the bound fraction was observed in each singly mutated A protein (Phe112Leu or Ser114Phe). Moreover, the bound fraction of the double A mutant (Phe112Leu/Ser114Phe) was decreased to that of wild-type F1*S. Intriguingly, the double F1*S mutant (Leu112Phe/Phe114Ser), in which residues were swapped with those of the A variant, showed only partial restoration in binding. The triple F1*S mutant (Leu112Phe/Phe114Ser/Asp115Tyr), where position 115 is thought to contribute to the difference in pocket size between variants, showed a further recovery in binding to 70% of that of wild-type A. These results were supported by thermodynamic analysis and acridine orange binding, which selectively binds the A variant. Together, these data indicate that, in addition to direct interaction with Phe112 and Ser114, the binding pocket size contributed by Tyr115 is important for the drug-binding selectivity of the A variant.

Evaluation of the cyclic single-chain Fv antibody derived from nivolumab by biophysical analyses and in vitro cell-based bioassay.

Single-chain Fv (scFv) is a recombinant small antibody in which a polypeptide linker connects the variable regions of the light chain (VL) and the heavy chain (VH). The practical use of scFv, however, has been prevented by its tendency to aggregate due to interchain VL-VH interactions. We recently developed a cyclic scFv whose N-terminus and C-terminus were connected by protein ligation techniques. Biophysical comparisons between cyclic and linear scFv have been conducted, but cell biological evaluations remain unexplored. Here we studied the properties of cyclic and linear scFv derived from nivolumab. Biophysical studies revealed that the thermal stability was not changed but that the antigen-binding activity was approximately 3-fold higher as a result of circularization. A cell-based PD-1/PD-L1 interaction inhibitory assay revealed that the biological activity of scFv was markedly higher in the circularized form. In addition, biophysical analysis of scFv proteins incubated in the presence of serum revealed that circularization suppressed the decrease in antigen-binding activity. It could be assumed that circularization of scFv improved stability in the presence of serum, which in turn would suggest the applicability of cyclic scFv as a biopharmaceutical format.

Structure-based design, biophysical characterization, and biochemical application of the heterodimeric affinity purification tag based on the Schistosoma japonicum glutathione-S-transferase (SjGST) homodimer.

Schistosoma japonicum glutathione-S-transferase (SjGST), the so-called GST-tag, is one of the most widely used protein tags for the purification of recombinant proteins by affinity chromatography. Attachment of SjGST enables the purification of a protein of interest (POI) using commercially available glutathione-immobilizing resins. Here we produced an SjGST mutant pair that forms heterodimers by adjusting the salt bridge pairs in the homodimer interface of SjGST. An MD study confirmed that the SjGST mutant pair did not disrupt the heterodimer formation. The modified SjGST protein pair coexpressed in Escherichia coli was purified by glutathione-immobilized resin. The stability of the heterodimeric form of the SjGST mutant pair was further confirmed by size exclusion chromatography. Surface plasmon resonance measurements unveiled the selective formation of heterodimers within the pair, accompanied by a significant suppression of homodimerization. The heterodimeric SjGST exhibited enzymatic activity in assays employing a commercially available fluorescent substrate. By fusing one member of the heterodimeric SjGST pair with a fluorescent protein and the other with the POI, we were able to conveniently and sensitively detect protein-protein interactions using fluorescence spectroscopy in the pull-down assays. Thus, utilization of the heterodimeric SjGST would be a useful tag for protein science.

Molecular Dynamics-Based Design and Biophysical Evaluation of Thermostable Single-Chain Fv Antibody Mutants Derived from Pharmaceutical Antibodies.

Antibody drugs are denatured under physical stress, e.g., friction, heat, and freezing, which triggers formation of aggregates and resultant allergic reactions. Design of a stable antibody is thus critical for the development of antibody drugs. Here, we obtained a thermostable single-chain Fv (scFv) antibody clone by rigidifying the flexible region. We first conducted a short molecular dynamics (MD) simulation (3 runs of 50 ns) to search for weak spots in the scFv antibody, i.e., flexible regions located outside the CDR (complementarity determining region) and the interface between the heavy-chain and light-chain variable regions. We then designed a thermostable mutant and evaluated it by means of a short MD simulation (3 runs of 50 ns) based on reductions in the root-mean-square fluctuation (RMSF) values and formation of new hydrophilic interactions around the weak spot. Finally, we designed the VL-R66G mutant by applying our strategy to scFv derived from trastuzumab. Trastuzumab scFv variants were prepared by using an Escherichia coli expression system, and the melting temperature-measured as a thermostability index-was 5 °C higher than that of the wild-type trastuzumab scFv, while the antigen-binding affinity was unchanged. Our strategy required few computational resources, and would be applicable to antibody drug discovery.

Hydrogen bonds connecting the N-terminal region and the DE loop stabilize the monomeric structure of transthyretin.

Transthyretin (TTR) is a homo-tetrameric serum protein associated with sporadic and hereditary systemic amyloidosis. TTR amyloid formation proceeds by the dissociation of the TTR tetramer and the subsequent partial unfolding of the TTR monomer into an aggregation-prone conformation. Although TTR kinetic stabilizers suppress tetramer dissociation, a strategy for stabilizing monomers has not yet been developed. Here, we show that an N-terminal C10S mutation increases the thermodynamic stability of the TTR monomer by forming new hydrogen bond networks through the side chain hydroxyl group of Ser10. Nuclear magnetic resonance spectrometry and molecular dynamics simulation revealed that the Ser10 hydroxyl group forms hydrogen bonds with the main chain amide group of either Gly57 or Thr59 on the DE loop. These hydrogen bonds prevent the dissociation of edge strands in the DAGH and CBEF ß-sheets during the unfolding of the TTR monomer by stabilizing the interaction between ß-strands A and D and the quasi-helical structure in the DE loop. We propose that introducing hydrogen bonds to connect the N-terminal region to the DE loop reduces the amyloidogenic potential of TTR by stabilizing the monomer.

A Cost-Effective Immobilization Method for MBP Fusion Proteins on Microtiter Plates Using a Gelatinized Starch-Agarose Mixture and Its Application for Convenient Protein-Protein Interaction Analysis.

The detection and quantification of protein-protein interactions (PPIs) is a crucial technique that often involves the use of recombinant proteins with fusion protein tags, such as maltose-binding protein (MBP) and glutathione-S-transferase (GST). In this study, we improved the cohesive and sticky properties of gelatinized starch by supplementing it with agarose, resulting in a harder gel that could coat the bottom of a microtiter plate. The resulting gelatinized starch/agarose mixture allowed for the efficient immobilization of MBP-tagged proteins on the coated plates, enabling the use of indirect ELISA-like PPI assays. By using the enzymatic activity of GST as an indicator, we succeeded in determining the dissociation constants between MBP-tagged and GST-tagged proteins on 96-well microtiter plates and a microplate reader without any expensive specialized equipment.

Chlorine Atoms of an Aripiprazole Molecule Control the Geometry and Motion of Aripiprazole and Deschloro-aripiprazole in Subdomain IIIA of Human Serum Albumin.

Aripiprazole (ARP), an antipsychotic drug, binds more strongly to human serum albumin (HSA) than the other ARP derivatives. In addition, the signs for the extrinsic Cotton effects for HSA complexed with ARP or deschloro-ARP are reversed. In this study, we report on a structural-chemical approach using circular dichroism (CD) spectroscopic analysis, X-ray crystallographic analysis, and molecular dynamics simulations. The objective was to examine the relationship between the induced CD spectra and the structural features of the HSA complexes with ARP or deschloro-ARP. The intensity of the induced CD spectra of the HSA complexes with ARP or deschloro-ARP was reduced with increasing temperature. We determined the crystal structure of the HSA complexed with deschloro-ARP in this study and compared it to HSA complexed with ARP that we reported previously. The comparison of these structures revealed that both ARP and deschloro-ARP were bound at the site II pocket in HSA and that the orientation of the molecules was nearly identical. Molecular dynamics simulations indicated that the molecular motions of ARP and deschloro-ARP within the site II pocket were different from one another and the proportion of stacking interaction formations of Tyr411 with the dihydroquinoline rings of ARP and deschloro-ARP was also different. These findings indicate that the induced CD spectra are related to the molecular motions and dynamic interactions of ARP and deschloro-ARP in HSA and may help to understand the molecular recognition and motion that occurs within the binding site for the other HSA ligands more clearly.

Molecular recognition of a single-chain Fv antibody specific for GA-pyridine, an advanced glycation end-product (AGE), elucidated using biophysical techniques and synthetic antigen analogues.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by non-enzymatic reaction between reducing-sugar and Arg/Lys in proteins and are involved in various diabetic complications. GA-pyridine is derived from glycolaldehyde and is one of the most cytotoxic AGEs. Here, we established a single-chain Fv (scFv) antibody against GA-pyridine, 73MuL9-scFv, and examined the details of its specificity and antigen recognition by using various techniques involving biophysics, chemical biology and structural biology. We also synthesized several compounds that differ slightly in regard to the position and number of GA-pyridine substituent groups, and revealed that GA-pyridine was specifically bound to 73MuL9-scFv. Thermodynamic analysis revealed that the association of GA-pyridine to 73MuL9-scFv was an exothermic and enthalpy driven reaction, and thus that the antigen recognition involved multiple specific interactions. Crystallographic analysis of the Fv fragment of 73MuL9-scFv revealed that several CH-π and hydrogen bond interactions took place between the Fv-fragment and GA-pyridine, which was consistent with the results of thermodynamic analysis. Further studies using 73MuL9-scFv as a tool to clarify the relevance of GA-pyridine to diabetic complications are warranted.

Expression, Purification and Characterization of CAR/NCOA-1 Tethered Protein in E. coli Using Maltose-Binding Protein Fusion Tag and Gelatinized Corn Starch.

The constitutive active/androstane receptor (CAR) is a nuclear receptor that functions as a xenobiotic sensor, which regulates the expression of enzymes involved in drug metabolism and of efflux transporters. Evaluation of the binding properties between CAR and a drug was assumed to facilitate the prediction of drug-drug interaction, thereby contributing to drug discovery. The purpose of this study is to construct a system for the rapid evaluation of interactions between CAR and drugs. We prepared recombinant CAR protein using the Escherichia coli expression system. Since isolated CAR protein is known to be unstable, we designed a fusion protein with the CAR binding sequence of the nuclear receptor coactivator 1 (NCOA1), which was expressed as a fusion protein with maltose binding protein (MBP), and purified it by several chromatography steps. The thus-obtained CAR/NCOA1 tethered protein (CAR-NCOA1) was used to evaluate the interactions of CAR with agonists and inverse agonists by a thermal denaturation experiment using differential scanning fluorometry (DSF) in the presence and absence of drugs. An increase in the melting temperature was observed with the addition of the drugs, confirming the direct interaction between them and CAR. DSF is easy to set up and compatible with multiwell plate devices (such as 96-well plates). The use of DSF and the CAR-NCOA1 fusion protein together allows for the rapid evaluation of the interaction between a drug and CAR, and is thereby considered to be useful in drug discovery.

Author Correction: 1.2 MV/cm pulsed electric fields promote transthyretin aggregate degradation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

1.2 MV/cm pulsed electric fields promote transthyretin aggregate degradation.

Numerous theoretical studies have been conducted on the effects of high-voltage electric fields on proteins, but few have produced experimental evidence. To acquire experimental data for the amyloid disassemble theory, we exposed transthyretin aggregates to 1,000 ns 1.26 MV/cm pulsed electric fields (PEFs) to promote transthyretin degradation. The process produced no changes in pH, and the resulting temperature increases were < 1 °C. We conclude that the physical effects of PEFs, rather than thermal or chemical effects, facilitate aggregate degradation.

Spectroscopic Characterization of Halorhodopsin Reconstituted into Nanodisks Using Native Lipids.

We successfully reconstituted single Natronomonas pharaonis halorhodopsin (NpHR) trimers into a nanodisk (ND) using the native archaeal lipid (NL) and an artificial lipid having a zwitterionic headgroup, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Incorporation of single trimeric NpHR into NDs was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, size-exclusion chromatography, and visible circular dichroism spectroscopy. The Cl- binding affinity of NpHR in NDs using NL (NL-ND NpHR) or POPC (POPC-ND NpHR) was examined by absorption spectroscopy, showing that the Cl--releasing affinities (Kd,N↔O) of these ND-reconstituted NpHRs are more than 10 times higher than that obtained from native NpHR membrane fragments (MFs) harvested from a NpHR-overexpressing archaeal strain (MF NpHR). The photoreaction kinetics of these ND-reconstituted NpHRs revealed that the Cl- uptake was faster than that of MF NpHR. These differences in the Cl--releasing and uptake properties of ND-reconstituted NpHRs and MF NpHR may arise from suppression of protein conformational changes associated with Cl- release from the trimeric NpHR caused by ND reconstitution, conformational perturbation in the trimeric state, and loss of the trimer-trimer interactions. On the other hand, POPC-ND NpHR demonstrated accelerated Cl- uptake compared to NL-ND NpHR, suggesting that the negative charge on the archaeal membrane surface regulates the photocycle of NpHR. Although NL-ND NpHR and MF NpHR are embedded in the same lipid, the lower Cl--binding affinity at the initial state (Kd,initial) and faster recovering from the NpHR' state to the original state of the photoreaction cycle were observed for NL-ND NpHR, probably because of insufficient interactions with a chromophore in the native membrane, bacterioruberin in reconstituted NDs. Our results indicate that specific interactions of NpHR with surrounding lipids and bacterioruberin, structural flexibility of the membrane, and interactions between trimeric NpHRs may be necessary for efficient Cl- pumping.

Development of a long acting FGF21 analogue-albumin fusion protein and its anti-diabetic effects.

Fibroblast growth factor 21 (FGF21) is a hormone-like protein that improves blood glucose and lipid metabolism. However, its short half-life and instability are bottlenecks to its clinical applications. In this study, to extend its pharmacological action, we created a stabilized mutant FGF21 (mFGF21:ΔHPIP, P171G, A180E, L118C-A134C, S167A) and then genetically fused it with human albumin (HSA-mFGF21) via a polypeptide linker. Physicochemical analyses suggested that HSA-mFGF21 was formed from both intact HSA and mFGF21. Pharmacokinetic findings indicated the half-life of HSA-mFGF21 was 20 times longer than that of FGF21. In addition, HSA-mFGF21 was persistently distributed in adipose tissue as a target tissue. The in vivo hypoglycemic activity of HSA-mFGF21 using streptozotocin (STZ)-induced type I diabetes model mice, in which insulin secretion was suppressed, showed that a single intravenous administration of HSA-mFGF21 rapidly alleviated hyperglycemia. At that time, HSA-mFGF21 increased GLUT1 mRNA expression in adipose tissue without having any effect on insulin secretion. A twice weekly administration of HSA-mFGF21 continuously suppressed blood glucose levels and ameliorated the abnormalities of adipose tissue induced by STZ treatment. Interestingly, HSA-mFGF21 showed no hypoglycemic effects in healthy mice. Together, HSA-mFGF21 could be a novel biotherapeutic for the treatment of metabolic disorders including diabetes mellitus.

Convenient method of producing cyclic single-chain Fv antibodies by split-intein-mediated protein ligation and chaperone co-expression.

Single-chain Fv (scFv) is a recombinant antibody in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. Compared with monoclonal antibodies, scFvs have the advantages of low-cost production using Escherichia coli and easy genetic manipulation. ScFvs are, therefore, regarded as useful modules for producing next-generation medical antibodies. The practical use of scFvs has been limited due to their aggregation propensity mediated by interchain VH-VL interactions. To overcome this problem, we recently reported a cyclic scFv whose N-terminus and C-terminus were connected by sortase A-mediated ligation. Preparation of cyclic scFv is, however, a time-consuming process. To accelerate the application study of cyclic scFv, we developed a method to produce cyclic scFv by the combined use of a protein ligation technique based on protein trans-splicing reaction (PTS) by split intein and a chaperone co-expression system. This method allows for the preparation of active cyclic scFv from the cytoplasm of E. coli. The present method was applied to the production of cyclic 73MuL9-scFv, a GA-pyridine antibody, as a kind of advanced glycation end-product. These findings are expected to evoke further application study of cyclic scFv.

Preparation of single-chain Fv antibodies in the cytoplasm of Escherichia coli by simplified and systematic chaperone optimization.

A single-chain variable fragment (scFv) antibody is a recombinant protein in which a peptide linker connects the variable regions of the heavy chain and light chain. Due to its smaller molecular size, an scFv can be expressed using Escherichia coli. The presence of two disulphide bonds in the molecule often prevents expression of correctly folded scFv in the E. coli cytoplasm, making a refolding process necessary to regenerate scFv activity. The refolding process is time-consuming and requires large amounts of expensive reagents, such as guanidine hydrochloride, l-arginine and glutathione. Here, to conveniently obtain scFv proteins, we devised a simple and systematic method to optimize the co-expression of chaperone proteins and to combine them with specially engineered E. coli strains that permit the formation of stable disulphide bonds within the cytoplasm. Several scFv proteins were successfully obtained in a soluble form from E. coli cytoplasm. Thermal denaturation experiments and/or surface plasmon resonance measurements revealed that the thus-obtained scFvs possessed a stable tertiary structure and antigen-binding activity. The combined use of engineered E. coli with the simplified and systematic chaperone optimization can be useful for the production of scFv proteins.

Interaction of Aripiprazole With Human α1-Acid Glycoprotein.

We recently reported that aripiprazole binds strongly to human albumin. In continuing our investigations, we investigated the mechanism responsible for the binding and the related interactions of aripiprazole with α1-acid glycoprotein (AGP). The extrinsic Cotton effects for the binding of aripiprazole and its derivatives to AGP were generated, but the magnitudes of the induced circular dichroism intensities did not correlate with those for the binding affinities. It therefore appears that the binding mode of aripiprazole with AGP is somewhat complicated, compared with that of albumin. Isothermal titration calorimetry data obtained for the binding of aripiprazole with AGP were different from that for albumin systems in that the 3 driving reactions, entropy-driven, enthalpy-driven, and the entropy-enthalpy mixed type, were all found for the AGP system, but not albumin. Moreover, the weak binding mode of aripiprazole with the 2 proteins were supported by a molecular docking model analysis. The concentration of albumin in plasma is about 50 times higher than those of AGP, but AGP levels in plasma are increased by about 10 times under inflammatory disease. Therefore, the involvement of these 2 plasma proteins should be considered in more depth for understanding the pharmacokinetics of aripiprazole.

Cyclization of Single-Chain Fv Antibodies Markedly Suppressed Their Characteristic Aggregation Mediated by Inter-Chain VH-VL Interactions.

Single-chain Fv (scFv) antibodies are recombinant proteins in which the variable regions of the heavy chain (VH) and light chain (VL) are connected by a short flexible polypeptide linker. ScFvs have the advantages of easy genetic manipulation and low-cost production using Escherichia coli compared with monoclonal antibodies, and are thus expected to be utilized as next-generation medical antibodies. However, the practical use of scFvs has been limited due to low homogeneity caused by their aggregation propensity mediated by inter-chain VH-VL interactions. Because the interactions between the VH and VL domains of antibodies are generally weak, individual scFvs are assumed to be in equilibrium between a closed state and an open state, in which the VH and VL domains are assembled and disassembled, respectively. This dynamic feature of scFvs triggers the formation of dimer, trimer, and larger aggregates caused by the inter-chain VH-VL interactions. To overcome this problem, the N-terminus and C-terminus were herein connected by sortase A-mediated ligation to produce a cyclic scFv. Open-closed dynamics and aggregation were markedly suppressed in the cyclic scFv, as judged from dynamic light scattering and high-speed atomic force microscopy analyses. Surface plasmon resonance and differential scanning fluorometry analysis revealed that neither the affinity for antigen nor the thermal stability was disrupted by the scFv cyclization. Generality was confirmed by applying the present method to several scFv proteins. Based on these results, cyclic scFvs are expected to be widely utilized in industrial and therapeutic applications.

SIRT7 has a critical role in bone formation by regulating lysine acylation of SP7/Osterix.

SP7/Osterix (OSX) is a master regulatory transcription factor that activates a variety of genes during differentiation of osteoblasts. However, the influence of post-translational modifications on the regulation of its transactivation activity is largely unknown. Here, we report that sirtuins, which are NAD(+)-dependent deacylases, regulate lysine deacylation-mediated transactivation of OSX. Germline Sirt7 knockout mice develop severe osteopenia characterized by decreased bone formation and an increase of osteoclasts. Similarly, osteoblast-specific Sirt7 knockout mice showed attenuated bone formation. Interaction of SIRT7 with OSX leads to the activation of transactivation by OSX without altering its protein expression. Deacylation of lysine (K) 368 in the C-terminal region of OSX by SIRT7 promote its N-terminal transactivation activity. In addition, SIRT7-mediated deacylation of K368 also facilitates depropionylation of OSX by SIRT1, thereby increasing OSX transactivation activity. In conclusion, our findings suggest that SIRT7 has a critical role in bone formation by regulating acylation of OSX.

Design and tuning of a cell-penetrating albumin derivative as a versatile nanovehicle for intracellular drug delivery.

Human serum albumin (HSA) is a superior carrier for delivering extracellular drugs. However, the development of a cell-penetrating HSA remains a great challenge due to its low membrane permeability. We report herein on the design of a series of palmitoyl-poly-arginine peptides (CPPs) and an evaluation of their cell-penetrating effects after forming a complex with HSA for use in intracellular drug delivery. The palmitoyl CPPs forms a stable complex with HSA by anchoring itself to the high affinity palmitate binding sites of HSA. Among the CPPs evaluated, a cyclic polypeptide composed of D-dodecaarginines, palmitoyl-cyclic-(D-Arg)12 was the most effective for facilitating the cellular uptake of HSA by HeLa cells. Such a superior cell-penetrating capability is primarily mediated by macropinocytosis. The effect of the CPP on pharmacological activity was examined using three drugs loaded in HSA via three different methods: a) an HSA-paclitaxel complex, b) an HSA-doxorubicin covalent conjugate and c) an HSA-thioredoxin fusion protein. The results showed that cell-penetrating efficiency was increased with a corresponding and significant enhancement in pharmacological activity. In conclusion, palmitoyl-cyclic-(D-Arg)12/HSA is a versatile cell-penetrating drug delivery system with great potential for use as a nano-carrier for a wide diversity of pharmaceutical applications.

Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion.

Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 °C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open.