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Adachi, Motoyasu; Arai, Shigeki; Matsumoto, Fumiko; Kuroki, Ryota; Hatanaka, Takaaki*; Ito, Yuji*; Hidaka, Koshi*; Tsuda, Yuko*; Kiso, Yoshiaki*
no journal, ,
HIV protease is known as a drug target protein. To compare interactions between HIVPR and inhibitors, single-chained HIVPR linked by a disulfide bond was designed as N98C mutant. Both WT N98C and A17 N98C mutants were expressed as inclusion body and refolded by dilution method. The yield of the purified enzymes was similar to that of WT. We will report the results of interactions between inhibitors and WT N98C and A17 N98C mutants by physicochemical and crystal structure analyses.
Hidaka, Koshi*; Adachi, Motoyasu; Kuroki, Ryota; Tokai, Satoko*; Akaji, Kenichi*; Tsuda, Yuko*; Kiso, Yoshiaki*
no journal, ,
In the HIV protease inhibitor study, we faced on a problem of much difference in activity of a compound with 2,6-dimethylphenoxyacetyl moiety against the enzyme and the virus. The protease inhibitory potency was plateau because of the limited structural modifications. Therefore, we shifted to modify the property such as reduction of the hydrophobicity. During the struggles, amino substitution succeeded in improving the water solubility to enhance the anti-HIV activity and with the sustained protease inhibitory activity. This result stimulated us to modify the Apns-containing inhibitors for the development of therapeutic drug and to utilize them as aspartic proteases probes.
Hidaka, Koshi*; Toda, Yuki*; Adachi, Motoyasu; Kuroki, Ryota; Kiso, Yoshiaki*
no journal, ,
Molecular dynamic simulations of the inhibitor suggested existence of additional stable bridging water molecules to support the binding with mutated proteases. To increase the numbers of bridging water molecules, we replaced amide with sulfonyl and oxamide structures at both terminals of pseudo-symmetric peptides based on HMC. In summary, oxamide modifications resulted in a moderate activity loss against lopinavir-resistant mutations compared to inhibitors without oxamide. This method to accompany multiple bridging water molecules could be applicable to design protease inhibitors to defeat the drug resistance from amino acid mutations.
Adachi, Motoyasu; Hatanaka, Takaaki*; Ito, Yuji*; Hidaka, Koshi*; Tsuda, Yuko*; Kiso, Yoshiaki*; Kuroki, Ryota
no journal, ,
HIV protease is known as a drug target protein. It is important to clear relationship between structural data and kinetic and physicochemical parameters for drug design. We prepared single-chained enzyme linked by two amino acids and cross-liked enzyme bridged by disulfide bond. The two single-chained enzymes were expressed as inclusion body and refolded by dilution method. The purified enzyme complexed with inhibitor KNI-272 was crystallized, and solved the crystal structures. The designed sc- and cl-HIV-PR will be useful for evaluate the affinity of newly designed inhibitors from kinetic and thermodynamic point of view. Finally, we also report the results of analysis in affinity of inhibitors by surface plasmon resonance.