A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate

A Cocktail of Thermally Stable, Chemically Synthesized Capture Agents for the Efficient Detection of Anti-Gp41 Antibodies from Human Sera

We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60°C

Transforming a Pair of Orthogonal tRNA-aminoacyl-tRNA Synthetase from Archaea to Function in Mammalian Cells

A previously engineered Methanocaldococcus jannaschii –tyrosyl-tRNA synthetase pair orthogonal to Escherichia coli was modified to become orthogonal in mammalian cells. The resulting -tyrosyl-tRNA synthetase pair was able to suppress an amber codon in the green fluorescent protein, GFP, and in a foldon protein in mammalian cells. The methodology reported here will allow rapid transformation of the much larger collection of existing tyrosyl-tRNA synthetases that were already evolved for the incorporation of an array of over 50 unnatural amino acids into proteins in Escherichia coli into proteins in mammalian cells. Thus we will be able to introduce a large array of possibilities for protein modifications in mammalian cells

A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands

We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies

Studies in pharmaceutical biotechnology : protein-protein interactions and beyond

textPharmaceutical biotechnology has been emerging as a defined, increasingly important area of science dedicated to the discovery and delivery of drugs and therapies for the treatment of various human diseases. In contrast to the advancement in pharmaceutical biotechnology, current drug discovery efforts are facing unprecedented challenges. Difficulties in identifying novel drug targets and developing effective and safe drugs are closely related to the complexity of the network of interacting human proteins. Protein-protein interactions mediate virtually all cellular processes. Therefore both identification and understanding of protein-protein interactions are essential to the process of deciphering disease mechanisms and developing treatments. Unfortunately, our current knowledge and understanding of the human interactome is largely incomplete. Most of the unknown protein-protein interactions are expected to be weak and/or transient, hence are not easily identified. These unknown or uncharacterized interactions could affect the efficacy and toxicity of drug candidates, contributing to the high rate of failure. In an attempt to facilitate the ongoing efforts in drug discovery, we describe herein a series of novel methods and their applications addressing the broad topic of protein-protein interactions. We have developed a highly efficient site-specific protein cross-linking technology mediated by the genetically incorporated non-canonical amino acid L-DOPA to facilitate the identification and characterization of weak protein-protein interactions. We also established a protocol to incorporate L-DOPA into proteins in mammalian cells to enable in vivo site-specific protein cross-kinking. We then applied the DOPA-mediated cross-linking methodology to design a protein probe which can potentially serve as a diagnostic tool or a modulator of protein-protein interactions in vivo. To deliver such engineered proteins or other bioanalytical reagents into single live cells, we established a laser-assisted cellular nano-surgery protocol which would enable detailed observations of cell-to-cell variability and communication. Finally we investigated a possible experimental scheme to genetically evolve a fluorescent peptide, which has tremendous potential as a tool in cellular imaging and dynamic observation of protein-protein interactions in vivo. We aim to contribute to the discovery and development of new drugs and eventually to the overall health of our society by adding the technology above to the array of currently available bioanalytical tools.Cellular and Molecular Biolog

Rapid discovery of peptidomimetics as antibody alternatives via epitope-targeted screening

We report on the development of peptide-based ligands as antibody alternatives for protein recognition. In the developing world, antibody(Ab)-based rapid diagnostic tests (RDTs) are the preferred method of disease detection for their ease-of-use and low cost relative to techniques such as microscopy and PCR. However, the performance and reliability of RDTs to detect biomarkers are severely hindered by the limitations of Abs. Abs are expensive biomols. that have batch-to-batch variability, exhibit cross-reactivity, and target a single site on a protein. As a result, Abs cannot always sensitively distinguish between disease biomarkers or detect highly polymorphic proteins. Peptide-based ligands are low-cost alternatives to Abs that can be engineered to bind specific protein targets with high affinity. They can be adapted to simultaneously target multiple epitopes, distinguish between homologous structures, and detect proteins with high sequence diversity. We employ a high throughput epitope-targeted screening protocol with combinatorial peptide libraries to rapidly discover ligands for protein recognition. We apply our peptide-based ligands as biosensors towards challenging protein targets for the development of Ab-free RDTs

The orthogonality of the <i>M. jannaschii</i> TyrRS- pair was verified on western blots probed with anti-V5 antibodies.

<p>Expression of full-length foldon was monitored when various tRNAs were introduced into the HEK 293T cells. Note that the tRNA mutants used in these experiments were slightly different from those depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011263#pone-0011263-g001" target="_blank">figure 1</a>.</p

Suppression of an amber codon inserted into the GFP-encoding gene.

<p>(A) Full-length GFP was expressed in HEK 293T cells only in the presence of a <i>M. jannaschii</i> TyrRS- pair designed to be orthogonal to mammalian cells. (B) Western blot analysis of full-length GFP probed with anti-His antibodies.</p