Synthesis, binding affinities and metabolic stability of dimeric dermorphin analogs modified withβ3-homo-amino acids

2016 ◽  
Vol 22 (4) ◽  
pp. 222-227 ◽  
Author(s):  
Oliwia Frączak ◽  
Anika Lasota ◽  
Dagmara Tymecka ◽  
Piotr Kosson ◽  
Adriana Muchowska ◽  
...  
Keyword(s):  
Amino Acids ◽  
Metabolic Stability ◽  
Binding Affinities

The synthesis and opioid receptor binding affinities of dermorphin and deltorphin analogs with dibasic amino acids in position 2

1993 ◽  
pp. 455-457
Author(s):  
Andrzej W. Lipkowski ◽  
Aleksandra Misicka ◽  
Jirina Slaninova ◽  
Robert Horvath ◽  
Henry I. Yamamura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Opioid Receptor ◽  
Receptor Binding ◽  
Binding Affinities ◽  
Dibasic Amino Acids ◽  
Opioid Receptor Binding

scholarly journals Chemical Diversification of Simple Synthetic Antibodies

2020 ◽  
Author(s):  
Mariha Islam ◽  
Haixing P. Kehoe ◽  
Jacob B. Lissoos ◽  
Manjie Huang ◽  
Christopher E. Ghadban ◽  
...  
Keyword(s):  
Amino Acids ◽  
Chemical Reactivity ◽  
Basic Research ◽  
Single Species ◽  
Soluble Antigen ◽  
Binding Affinities ◽  
Yeast Display ◽  
Binding Function ◽  
Yeast Surface ◽  
Synthetic Antibodies

<p>Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in the canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality to augment antibody binding properties. Using yeast displayed antibodies, we explored the presentation of noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs) and evaluated the properties of the resulting constructs. To enable systematic characterization of ncAA incorporation sites, we first investigated whether diversification of a single antibody loop would support isolation of binding clones. We constructed a billion-member library containing canonical amino acid diversity and loop length diversity only within the 3rd complementarity determining region of the heavy chain (CDR-H3). Screens against a series of immunoglobulins from three species resulted in the isolation of antibodies exhibiting moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity. These findings confirmed that antibody specificity can be mediated by a single CDR. With this constrained diversity, we were able to utilize additional CDRs for the installation of chemically reactive and photo-crosslinkable ncAAs. Apparent binding affinities of ncAA-substituted synthetic antibodies on the yeast surface revealed that ncAA incorporation is generally well tolerated. However, changes in binding affinities did occur upon substitution, and varied based on factors including ncAA side chain identity, location of ncAA incorporation, and the ncAA incorporation machinery used. We further investigated chemical modifications facilitated by ncAA installation. Multiple azide-containing ncAAs supported both <a>copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition </a>(SPAAC) without abrogation of binding function following the installation of bulky probes. Similarly, several alkyne substitutions facilitated CuAAC without apparent disruption of binding function. Finally, antibodies substituted with a photo-crosslinkable ncAA were evaluated for ultraviolet-mediated crosslinking on the yeast surface. Competition-based assays revealed position-dependent linkages that could not be displaced by excess soluble antigen, strongly suggesting successful crosslinking. Key findings regarding CuAAC reactions and photo-crosslinking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. These consistent behaviors between the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with “chemically augmented” binding functions. More specifically, our findings provide the means to productively integrate antibodies with ncAAs by leveraging simple synthetic antibodies. The efficient preparation and chemical diversification of antibodies on the yeast surface opens up new possibilities for discovering “drug-like” protein leads in high throughput.</p>

Induction of tyrosine phosphorylation by the erythropoietin receptor correlates with mitogenesis

1991 ◽  
Vol 11 (10) ◽  
pp. 4895-4902
Author(s):  
O Miura ◽  
A D'Andrea ◽  
D Kabat ◽  
J N Ihle
Keyword(s):  
Amino Acids ◽  
Tyrosine Phosphorylation ◽  
Erythropoietin Receptor ◽  
Carboxyl Terminus ◽  
Binding Affinities ◽  
Two Dimensional ◽  
Hematopoietic Growth Factors ◽  
Dependent Cell ◽  
Receptor Beta ◽  
Responsive Cell

A role for tyrosine phosphorylation in the signal-transducing mechanisms of several hematopoietic growth factors has been hypothesized. To extend these observations, we have examined the effects of erythropoietin (Epo) on tyrosine phosphorylation in an Epo-responsive cell that was obtained by transfecting the murine erythropoietin receptor (EpoR) into an interleukin-3 (IL-3)-dependent cell line. By two-dimensional analysis of phosphotyrosine-containing proteins isolated with a monoclonal antibody (1G2) against phosphotyrosine, Epo and IL-3 were found to rapidly induce tyrosine phosphorylation of comparable substrates of 92, 70, and 56 kDa. In addition, Epo uniquely induced phosphorylation of a 72-kDa substrate while IL-3 uniquely induced phosphorylation of a 140-kDa substrate. Immunoprecipitation and mixing experiments indicated that the 72-kDa substrate may represent a small fraction of the EpoR. To explore the significance of tyrosine phosphorylation, we generated two mutants of the EpoR that lacked 108 or 146 amino acids at their carboxyl termini. In addition we constructed an internally deleted mutant that lacked 20 amino acids in a region of sequence homology with the IL-2 receptor beta chain. Although all mutants were expressed at comparable levels and had comparable binding affinities for Epo, only the mutant lacking 108 amino acids at the carboxyl terminus retained significant mitogenic activity or the ability to induce tyrosine phosphorylation.

scholarly journals Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

2012 ◽  
Vol 33 (1) ◽  
Author(s):  
Yuki Fujii ◽  
Shiho Tanaka ◽  
Manami Otsuki ◽  
Yasushi Hoshino ◽  
Chinatsu Morimoto ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Bacillus Thuringiensis ◽  
No Reduction ◽  
Amino Acid Sequences ◽  
Binding Affinities ◽  
Binding Mechanism ◽  
Wild Type ◽  
Critical Amino Acid ◽  
Cry Toxin

Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein.

Synthesis and erythropoietin receptor binding affinities of N,N-disubstituted amino acids

2000 ◽  
Vol 10 (17) ◽  
pp. 1995-1999 ◽  
Author(s):  
Peter J Connolly ◽  
Steven K Wetter ◽  
William V Murray ◽  
Dana L Johnson ◽  
Frank J McMahon ◽  
...  
Keyword(s):  
Amino Acids ◽  
Receptor Binding ◽  
Erythropoietin Receptor ◽  
Binding Affinities ◽  
Disubstituted Amino Acids

The Study of Specificities of Interaction between Peptides and MHC Molecules

2010 ◽  
Vol 143-144 ◽  
pp. 1254-1258 ◽  
Author(s):  
Tao Liu ◽  
Zhan Xin Zhang ◽  
Huan Wei ◽  
Hong Kui Hu ◽  
Feng Ming Wang
Keyword(s):  
Amino Acids ◽  
Mhc Class I ◽  
Class I ◽  
Support Vector ◽  
Binding Affinities ◽  
Mhc Molecules ◽  
Mhc Class I Molecule ◽  
Antigen Peptide ◽  
Amino Acid Descriptors ◽  
Epitope Vaccines

Determining which peptides bind to a specific major histocompatibility complex (MHC) class I molecule is not only helpful to understand the mechanism of immunity, but also to develop effective anti-tumor epitope vaccines. In order to further study the specificity of MHC class I molecule binding antigen peptide, the support vector regression (SVR) and four amino acid descriptors were used to build four models of predicting binding affinities between peptides and MHC class I molecules. Comparison among performances of the four models indicated that the model based on physicochemical properties of amino acids is more satisfying (AC=75.0%, CC=0.499). Furthermore, the specificities of MHC class I molecule binding antigen peptide were obtained through analysis based on the contribution of the amino acids to peptide-MHC class I molecule binding affinities in the predictive model.

Determination of relative binding affinities of labeling molecules with amino acids by using scanning tunneling microscopy

2011 ◽  
Vol 47 (38) ◽  
pp. 10638 ◽  
Author(s):  
Chenxuan Wang ◽  
Xiaobo Mao ◽  
Aihua Yang ◽  
Lin Niu ◽  
Shengnan Wang ◽  
...  
Keyword(s):  
Amino Acids ◽  
Scanning Tunneling Microscopy ◽  
Scanning Tunneling ◽  
Binding Affinities ◽  
Tunneling Microscopy ◽  
Relative Binding

scholarly journals Chemical Diversification of Simple Synthetic Antibodies

2020 ◽  
Author(s):  
Mariha Islam ◽  
Haixing P. Kehoe ◽  
Jacob B. Lissoos ◽  
Manjie Huang ◽  
Christopher E. Ghadban ◽  
...  
Keyword(s):  
Amino Acids ◽  
Chemical Reactivity ◽  
Basic Research ◽  
Single Species ◽  
Soluble Antigen ◽  
Binding Affinities ◽  
Yeast Display ◽  
Binding Function ◽  
Yeast Surface ◽  
Synthetic Antibodies

<p>Antibodies possess properties that make them valuable as therapeutics, diagnostics, and basic research tools. However, antibody chemical reactivity and covalent antigen binding are constrained, or even prevented, by the narrow range of chemistries encoded in the canonical amino acids. In this work, we investigate strategies for leveraging an expanded range of chemical functionality to augment antibody binding properties. Using yeast displayed antibodies, we explored the presentation of noncanonical amino acids (ncAAs) in or near antibody complementarity determining regions (CDRs) and evaluated the properties of the resulting constructs. To enable systematic characterization of ncAA incorporation sites, we first investigated whether diversification of a single antibody loop would support isolation of binding clones. We constructed a billion-member library containing canonical amino acid diversity and loop length diversity only within the 3rd complementarity determining region of the heavy chain (CDR-H3). Screens against a series of immunoglobulins from three species resulted in the isolation of antibodies exhibiting moderate affinities (double- to triple-digit nanomolar affinities) and, in several cases, single-species specificity. These findings confirmed that antibody specificity can be mediated by a single CDR. With this constrained diversity, we were able to utilize additional CDRs for the installation of chemically reactive and photo-crosslinkable ncAAs. Apparent binding affinities of ncAA-substituted synthetic antibodies on the yeast surface revealed that ncAA incorporation is generally well tolerated. However, changes in binding affinities did occur upon substitution, and varied based on factors including ncAA side chain identity, location of ncAA incorporation, and the ncAA incorporation machinery used. We further investigated chemical modifications facilitated by ncAA installation. Multiple azide-containing ncAAs supported both <a>copper-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition </a>(SPAAC) without abrogation of binding function following the installation of bulky probes. Similarly, several alkyne substitutions facilitated CuAAC without apparent disruption of binding function. Finally, antibodies substituted with a photo-crosslinkable ncAA were evaluated for ultraviolet-mediated crosslinking on the yeast surface. Competition-based assays revealed position-dependent linkages that could not be displaced by excess soluble antigen, strongly suggesting successful crosslinking. Key findings regarding CuAAC reactions and photo-crosslinking on the yeast surface were confirmed using soluble forms of ncAA-substituted clones. These consistent behaviors between the yeast surface and in solution suggest that chemical diversification can be incorporated into yeast display screening approaches. Taken together, our results highlight the power of integrating the use of yeast display and ncAAs in search of proteins with “chemically augmented” binding functions. More specifically, our findings provide the means to productively integrate antibodies with ncAAs by leveraging simple synthetic antibodies. The efficient preparation and chemical diversification of antibodies on the yeast surface opens up new possibilities for discovering “drug-like” protein leads in high throughput.</p>

scholarly journals ‘Carbon-Monoxide-Releasing Molecule-2 (CORM-2)’ Is a Misnomer: Ruthenium Toxicity, Not CO Release, Accounts for Its Antimicrobial Effects

Antioxidants ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 915
Author(s):  
Hannah M. Southam ◽  
Michael P. Williamson ◽  
Jonathan A. Chapman ◽  
Rhiannon L. Lyon ◽  
Clare R. Trevitt ◽  
...  
Keyword(s):  
Amino Acids ◽  
Carbon Monoxide ◽  
Mammalian Cells ◽  
Reduced Glutathione ◽  
Biological Effects ◽  
Significant Loss ◽  
Binding Affinities ◽  
Strong Negative Correlation ◽  
Inhibition Of Growth ◽  
Intracellular Target

Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological ‘gasotransmitter’, in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (<0.1 mol CO per mol CORM-2). A strong negative correlation between viability and cellular ruthenium accumulation implies that ruthenium toxicity underlies biocidal activity. Exogenous amino acids and thiols (especially cysteine, glutathione and N-acetyl cysteine) protected bacteria against inhibition of growth by CORM-2. Bacteria treated with 30 μM CORM-2, with added cysteine and histidine, exhibited no significant loss of viability, but were killed in the absence of these amino acids. Their prevention of toxicity correlates with their CORM-2-binding affinities (Cys, Kd 3 μM; His, Kd 130 μM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 μM; GSSG, Kd 25,000 μM). The toxicity of low, but potent, levels (15 μM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data.

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