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1.
ACS Chem Biol ; 16(9): 1757-1769, 2021 09 17.
Article in English | MEDLINE | ID: mdl-34406751

ABSTRACT

Cysteine-rich knob domains found in the ultralong complementarity determining regions of a subset of bovine antibodies are capable of functioning autonomously as 3-6 kDa peptides. While they can be expressed recombinantly in cellular systems, in this paper we show that knob domains are also readily amenable to a chemical synthesis, with a co-crystal structure of a chemically synthesized knob domain in complex with an antigen showing structural equivalence to the biological product. For drug discovery, following the immunization of cattle, knob domain peptides can be synthesized directly from antibody sequence data, combining the power and diversity of the bovine immune repertoire with the ability to rapidly incorporate nonbiological modifications. We demonstrate that, through rational design with non-natural amino acids, a paratope diversity can be massively expanded, in this case improving the efficacy of an allosteric peptide. As a potential route to further improve stability, we also performed head-to-tail cyclizations, exploiting the proximity of the N and C termini to synthesize functional, fully cyclic antibody fragments. Lastly, we highlight the stability of knob domains in plasma and, through pharmacokinetic studies, use palmitoylation as a route to extend the plasma half-life of knob domains in vivo. This study presents an antibody-derived medicinal chemistry platform, with protocols for solid-phase synthesis of knob domains, together with the characterization of their molecular structures, in vitro pharmacology, and pharmacokinetics.


Subject(s)
Complementarity Determining Regions/chemistry , Immunoglobulin Fragments/chemistry , Peptides, Cyclic/chemical synthesis , Amino Acid Sequence , Animals , Cattle , Immunoglobulin Fragments/blood , Immunoglobulin Fragments/pharmacology , Male , Models, Molecular , Peptides, Cyclic/blood , Peptides, Cyclic/pharmacokinetics , Protein Binding , Protein Domains , Protein Folding , Rats, Sprague-Dawley , Solid-Phase Synthesis Techniques , Tandem Mass Spectrometry , Thermodynamics
2.
Open Biol ; 5(9): 150105, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26400472

ABSTRACT

Wild-type and variant forms of transthyretin (TTR), a normal plasma protein, are amyloidogenic and can be deposited in the tissues as amyloid fibrils causing acquired and hereditary systemic TTR amyloidosis, a debilitating and usually fatal disease. Reduction in the abundance of amyloid fibril precursor proteins arrests amyloid deposition and halts disease progression in all forms of amyloidosis including TTR type. Our previous demonstration that circulating serum amyloid P component (SAP) is efficiently depleted by administration of a specific small molecule ligand compound, that non-covalently crosslinks pairs of SAP molecules, suggested that TTR may be also amenable to this approach. We first confirmed that chemically crosslinked human TTR is rapidly cleared from the circulation in mice. In order to crosslink pairs of TTR molecules, promote their accelerated clearance and thus therapeutically deplete plasma TTR, we prepared a range of bivalent specific ligands for the thyroxine binding sites of TTR. Non-covalently bound human TTR-ligand complexes were formed that were stable in vitro and in vivo, but they were not cleared from the plasma of mice in vivo more rapidly than native uncomplexed TTR. Therapeutic depletion of circulating TTR will require additional mechanisms.


Subject(s)
Cross-Linking Reagents/chemistry , Ligands , Prealbumin/metabolism , Animals , Binding Sites , Humans , Mice , Mice, Inbred C57BL , Models, Molecular , Piperidines/chemistry , Prealbumin/chemistry , Protein Structure, Quaternary , Thyroxine/chemistry , Thyroxine/metabolism
3.
Eur J Biochem ; 270(11): 2369-76, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12755691

ABSTRACT

We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T-cell receptor zeta chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the zeta chain. Preliminary 1H-NMR studies of recombinant zeta chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six zeta chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino-acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T-cell receptor zeta chain. After in vitro phosphorylation by Lck, the partially phosphorylated zeta chain was digested with trypsin. Separation and identification of the zeta chain fragments using LC-MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino-acid context of the six zeta chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.


Subject(s)
Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/metabolism , Membrane Proteins/chemistry , Receptors, Antigen, T-Cell/chemistry , Tyrosine/metabolism , Amino Acid Motifs , Amino Acid Sequence , Animals , Chromatography, Liquid , Cloning, Molecular , Glutathione Transferase/metabolism , Humans , Kinetics , Magnetic Resonance Spectroscopy , Mass Spectrometry , Membrane Proteins/metabolism , Mice , Molecular Sequence Data , Peptide Biosynthesis , Peptides/chemistry , Phosphorylation , Receptors, Antigen, T-Cell/metabolism , Transfection , Tumor Cells, Cultured , Tyrosine/chemistry
4.
J Pept Sci ; 9(4): 221-8, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12725243

ABSTRACT

The process of native chemical ligation (NCL) is well described in the literature. An N-terminal cysteine-containing peptide reacts with a C-terminal thioester-containing peptide to yield a native amide bond after transesterification and acyl transfer. An N-terminal cysteine is required as both the N-terminal amino function and the sidechain thiol participate in the ligation reaction. In certain circumstances it is desirable, or even imperative, that the N-terminal region of a peptidic reaction partner remain unmodified, for Instance for the retention of biological activity after ligation. This work discusses the synthesis of a pseudo-N-terminal cysteine building block for incorporation into peptides using standard methods of solid phase synthesis. Upon deprotection, this building block affords a de facto N-terminal cysteine positioned on an amino acid sidechain. which is capable of undergoing native chemical ligation with a thioester. The syntheses of several peptides and structures containing this motif are detailed, their reactions discussed. and further applications of this technology proposed.


Subject(s)
Cysteine/analogs & derivatives , Peptides/chemical synthesis , Amino Acid Sequence , Cysteine/chemical synthesis , Green Fluorescent Proteins , Luminescent Proteins/chemistry
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