PubMed · 7629355
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J E Newman. 1995. Bonding amalgams.. https://doi.org/10.14219/jada.archive.1995.0291
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Stimuli-responsive polymers exhibit reversible phase changes in response to changes in environmental factors such as pH or temperature. Conjugating such polymers to antibodies and proteins provides molecular systems for applications such as affinity separations, immunoassays and enzyme recovery and recycling. Here we show that conjugating a temperature-sensitive polymer to a genetically engineered site on a protein allows the protein's ligand binding affinity to be controlled. We synthesized a mutant of the protein streptavidin to enable site-specific conjugation of the responsive polymer near the protein's binding site. Normal binding of biotin to the modified protein occurs below 32 degrees C, whereas above this temperature the polymer collapses and blocks binding. The collapse of the polymer and thus the enabling and disabling of binding, is reversible. Such environmentally triggered control of binding may find many applications in biotechnology and biomedicine, such as the control of enzyme reaction rates and of biosensor activity, and the controlled release of drugs.
Four analogs of cyclosporin A (CsA) were synthesized to determine if the biological activities of CsA analogs generated by multiple amino acid replacements are predictable from the effects on biological activity of analogs with single residue changes. CsA analogs [Phe7]CsA (8a), [D-MeAla3,Phe7]CsA (8b), [D-Ser8,Phe7]CsA (8c), and [D-MeAla3,Phe7,D-Ser8]CsA (8d) were designed by modification of positions 3, 7, and 8, which are adjacent to one effector region of the cyclophilin-bound CsA complex. The syntheses of CsA analogs 8a-d were carried out by suitable modifications of the reported strategy. Each analog was characterized by NMR in deuterated chloroform and DMSO solutions, and their biological activities as inhibitors of cis-trans-peptidyl prolyl isomerase (PPIase), inhibitors of proliferation in BDF1 mouse spleen cells stimulated with concanavalin A (Con A), and inhibitors of IL-2 release stimulated with PMA/ionomycin by Jurkat cells were determined. Incorporation of the phenylalanine residue in position 7 diminished activities 5-8-fold. Substitution at position 3 decreased activity nearly 2-fold, and substitution at position 8 did not lower activities. However, when all three modifications (D-MeAla3,Phe7, and D-Ser8) were incorporated into one molecule, the resulting analog, 8d, was found to bind more tightly to cyclophilin than CsA (Ki = 3 +/- 1.5 vs 6 +/- 2 nM) and to produce the full immunosuppressive effect in the other assay systems. Our structure-activity results show that combinations of substitutions that individually lower PPIase or immunosuppressive activity produce fully active analogs when combined in a single compound. These results suggest that other, multimodified CsA derivatives may be discovered that possess excellent or improved immunosuppressive activities even though they contain a substitution that otherwise reduces immunosuppressive activity.
We have previously shown that alpha-2-O-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2Me) has a higher affinity to bromelain-treated hemagglutinin (HA) of influenza A virus than sialic acid from natural sources (Machytka et al., 1993, FEBS Lett. 334, 117-120). We have now compared the inhibitory effects of alpha-Neu5thioAc2Me and other sialic acid analogs on receptor binding and plaque formation of intact influenza A viruses. When alpha-Neu5thioAc2Me was polymerized by conjugation to polyacrylamide, its affinity to HA increased 10(3)-fold. When analyzed by plaque reduction, the alpha-Neu5thioAc2 polymer was about 10 times more efficient as an inhibitor of virus replication than the alpha-Neu5Ac2 polymer, stressing the importance of sulfur at C5. The S-glycoside alpha-2-S-methyl-5-N-thioacetylneuraminic acid (alpha-Neu5thioAc2SMe) had the same affinity to HA as alpha-Neu5thioAc2Me, but was resistant to neuraminidase. The alpha-Neu5thioAc2S polymer interfered with the replication of a wider spectrum of influenza A virus subtypes than the alpha-Neu5thioAc2 polymer. The results indicate that the alpha-Neu5thioAc2S polymer has the potential to be used as an inhibitor of influenza virus infection.