Biopharmaceutical monoclonal antibodies (Mabs) show different chromatographic behaviors in the elution step on protein A chromatography, although Mabs have similar three-dimensional structures. It is well known that interactions of conventional protein A to the V_H3 subfamily variable region negatively affect Mabs elution properties. The mutation G29A is known to weaken this binding, although not always sufficiently. We designed novel protein A mutations, S33E and D36R, by a computer-aided evaluation based on the three-dimensional structure. These mutations are expected to not only eliminate protein A binding to the variable region of Mabs but also to maintain its alkaline stability, which is required for effective CIP (Clean in place) of the protein A affinity matrix. In view of the superior potential of C domain, an in vitro study was performed with the G29A mutant of C domain (C-G29A) as a model protein. Both pentameric C domain mutants (C-G29A/S33E.5d and C-G29A/D36R.5d) showed little binding ability to the V_H3 subfamily variable region of Mabs by BIACORE analysis. We used a C-G29A/S33E.5d-immobilized matrix to confirm that the elution profile of Mabs belonging to the V_H3 subfamily at pH 3.5 was significantly improved. This matrix also showed almost the same alkaline stability as did the C-G29A.5d-immobilized matrix. The engineered protein A ligand, whose binding ability to the variable region is completely eliminated, would enable the separation of Fab fragments in flow-through fractions from Mab digestions. Rational design by a computer-aided evaluation should enhance the efficiency of protein ligand engineering.

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In the design of anesthetics, the elucidation of molecular targets and their mechanisms of action are essential. The GABA_A receptor is known to be an important molecular target involved in producing loss of consciousness. However, the precise anesthetic target site and its characteristics are unclear. To elucidate the characteristics of the anesthetic binding site, we used the nicotinic acetylcholine (nACh) receptor as a model, as it is in the same superfamily as the GABA_A receptor, and the two receptors share a similar structure. In this study, we specifically examined the binding and molecular interactions of barbital enantiomers with the nACh receptor. We used docking simulation to study the binding mode (position, orientation, conformation) of amobarbital, and barbital enantiomers (isobarbital, pentobarbital) with the nACh receptor in its resting state. The nACh receptor structure was obtained from the Protein Data Bank. For flexible docking, the ASEDock 2005 program of the Molecular Operating Environment system was used. Amobarbital docked to the agonist binding site and channel pore of the nACh receptor. (R)- and (S)-isobarbital, and (R)- and (S)-pentobarbital docked to the agonist binding site, and R and S enantiomers docked to positions where the barbital rings were almost superimposed. In a situation where the dominant enantiomeric binding originates from its substructure without chiral point, the enantiomeric contribution to molecular discrimination turns out to be relatively small even if it contains chiral carbon. In this study, the major binding interactions between drug and the receptor were from barbital ring binding. Steric structural differences from chirality of the alkyl side chain did not produce large differences in drug binding forces. Similarly in the anesthetic action site, chiral carbon of the side chain of barbitals may not produce large differences in binding force, as these interactions are the nature of barbital structures. This implies that the barbital anesthetic binding site has no strict selectivity in discriminating between R and S enantiomers.

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In the development of the anti-allergic drug Suplatast Tosilate (IPD-1151T) we have reported the QSAR analysis using only the calculated π values, because the few logK values of sulfonium compounds had been measured till then. In this study, we measured the logK_TLC value of sulfonium compounds by using octylsililated silicagel plate (Merck HPTLC RP-8 F_254S). The logK_TLC values of the optimized compounds 52 and 67 (Suplatast Tosilate) of dimethylsulfonium p-toluenesulfonates derivatives were 0.07 and 0.06, respectively. Therefore, it was found that the desirable logK_TLC value of the sulfonium compound was approximately zero as the anti-allergic drug.

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Initial state-independent phenotypic diversification will be a powerful tool for directing cells to multiple phenotypes in practical situation, in which initial cellular states are unknown. In this study, we designed Symmetric Diversity Generator (SDG) for the initial state-independent phenotypic diversification, in which homogenous cells diversify into two phenotypes and the ratio of the phenotypes do not depend on the initial cellular state. The SDG consists of two mechanisms: an intracellular mutual inhibition by repressors and an intercellular activation of the repressor productions by intercellular activators that are expected to compensate imbalance of repressor concentrations and of intercellular activator concentrations. We computationally evaluated the initial state dependence of the SDG in terms of the ratio of the two phenotypes after the diversification, and found the SDG still has initial state dependence. For lower dependence, we designed two kinds of symmetric diversity generator focusing on degradation rate of activators and responsiveness of repressor productions to transcription factors, activators and repressors. Our computational evaluation suggests that the latter approach is much more promising than the former one because the intercellular activators can compensate the imbalance of the transcription factors in advance of response of repressor productions. The former approach would be used for improvement of robustness of other synthetic genetic circuits already designed.

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