A General Route for Post-Translational Cyclization of mRNA Display Libraries

Cyclic peptides are attractive scaffolds for the design of conformationally constrained molecular therapeutics. Previously, biological display libraries could only be cyclized via disulfide bonds, which are labile and can be reduced in an intracellular environment. In this paper, we construct high diversity, covalently cyclized mRNA display libraries (>10^(13) sequences) and analyze the cyclization reaction using MALDI-TOF MS and unnatural amino acid incorporation. Our route allows the extent of cyclization to be evaluated quantitatively and is broadly applicable to a variety of cyclization chemistries

Synthesis and Purification of Cyclic Peptide Autophagy Inhibitor 4B1W and Its Target LC3a

https://openworks.mdanderson.org/sumexp21/1167/thumbnail.jp

Critical behavior of gravitating sphalerons

We examine the gravitational collapse of sphaleron type configurations in Einstein--Yang--Mills--Higgs theory. Working in spherical symmetry, we investigate the critical behavior in this model. We provide evidence that for various initial configurations, there can be three different critical transitions between possible endstates with different critical solutions sitting on the threshold between these outcomes. In addition, we show that within the dispersive and black hole regimes, there are new possible endstates, namely a stable, regular sphaleron and a stable, hairy black hole.Comment: Latex, 14 pages, 8 figure

Apoptosis detection in retinal ganglion cells using quantitative changes in multichannel fluorescence colocalization

KcapTR488 is a dual-fluorophore peptide sensor for the real-time reporting of programmed cell death by fluorescence imaging. KcapTR488 contains a nuclear localization sequence (NLS) conjugated with Texas Red, a caspase-cleavable sequence (DEVD), and a C-terminus conjugated to Alexa Fluor 488 (AF488). The synthesis and preliminary evaluation in cellulo of KcapTR488 for monitoring cell death by fluorescence imaging has been previously reported, but its utility in vivo has yet to be tested or validated. Herein, in vitro solution experiments verified the intramolecular fluorescence resonance energy transfer (FRET) between the two fluorophores and enabled a quantitative analysis of enzyme rates and selectivity. The sensor delivery kinetics in live rat models were quantified by ex vivo fluorescence microscopy. Studies in healthy control retinas demonstrated that KcapTR488 concentrated in the nucleus of retinal ganglion cells (RGC), with a strong colocalization of red and green fluorescence signals producing robust FRET signals, indicating an intact reporter. By contrast, using an acute but mild NMDA-induced retinal injury model, dual-color confocal ex vivo microscopy of cleaved KcapTR488 identified sensor activation as early as 2 h after injection. Quantitative changes in fluorescence colocalization were superior to changes in FRET for monitoring injury progression. Longitudinal monitoring revealed that the NLS-Texas Red fragment of the cleaved sensor moved out of the cell body, down the axon, and exited the retina, consistent with anterograde axonal transport. Thus, KcapTR488 may be a powerful tool to study RGC death pathways in live preclinical models of glaucoma

Iterative in Situ Click Chemistry Assembles a Branched Capture Agent and Allosteric Inhibitor for Akt1

We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to preinhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties

In situ click chemistry: from small molecule discovery to synthetic antibodies

Advances in the fields of proteomics, molecular imaging, and therapeutics are closely linked to the availability of affinity reagents that selectively recognize their biological targets. Here we present a review of Iterative Peptide In Situ Click Chemistry (IPISC), a novel screening technology for designing peptide multiligands with high affinity and specificity. This technology builds upon in situ click chemistry, a kinetic target-guided synthesis approach where the protein target catalyzes the conjugation of two small molecules, typically through the azide–alkyne Huisgen cycloaddition. Integrating this methodology with solid phase peptide libraries enables the assembly of linear and branched peptide multiligands we refer to as Protein Catalyzed Capture Agents (PCC Agents). The resulting structures can be thought of as analogous to the antigen recognition site of antibodies and serve as antibody replacements in biochemical and cell-based applications. In this review, we discuss the recent progress in ligand design through IPISC and related approaches, focusing on the improvements in affinity and specificity as multiligands are assembled by target-catalyzed peptide conjugation. We compare the IPISC process to small molecule in situ click chemistry with particular emphasis on the advantages and technical challenges of constructing antibody-like PCC Agents

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 Chemical Epitope-Targeting Strategy for Protein Capture Agents: The Serine 474 Epitope of the Kinase Akt2

Target and click: Peptide ligands targeted to the C-terminal motif of the kinase Akt2 were obtained by combining phosphate recognition of a dinuclear zinc(II) complex with in situ click chemistry to target this epitope. The peptide ligands (shown as XXXXX) selectively bind the C-terminal polypeptide of Akt2, and are selective for Akt2 relative to the Akt1 and Akt3 isoforms. The ligands differentially modulate Akt2 activity

Iterative in situ click chemistry creates antibody-like protein-capture agents

Iterative in situ click chemistry (see scheme for the tertiary ligand screen) and the one-bead-one-compound method for the creation of a peptide library enable the fragment-based assembly of selective high-affinity protein-capture agents. The resulting ligands are water-soluble and stable chemically, biochemically, and thermally. They can be produced in gram quantities through copper (I)-catalyzed cycloaddition

Relativistic MHD and black hole excision: Formulation and initial tests

A new algorithm for solving the general relativistic MHD equations is described in this paper. We design our scheme to incorporate black hole excision with smooth boundaries, and to simplify solving the combined Einstein and MHD equations with AMR. The fluid equations are solved using a finite difference Convex ENO method. Excision is implemented using overlapping grids. Elliptic and hyperbolic divergence cleaning techniques allow for maximum flexibility in choosing coordinate systems, and we compare both methods for a standard problem. Numerical results of standard test problems are presented in two-dimensional flat space using excision, overlapping grids, and elliptic and hyperbolic divergence cleaning.Comment: 22 pages, 8 figure