An ECD and NMR/DP4+ Computational Pipeline for Structure Revision and Elucidation of Diphenazine-Based Natural Products.

Discovery and structure elucidation of natural products available in infinitesimally small quantities are recognized challenge. This challenge is epitomized by the diphenazine class of molecules that contain three bridged stereocenters, several conformations, ring fusions, and multiple spatially isolated phenols. Because empirical NMR and spatial analyses using ROESY/NOESY were unsuccessful in tackling these challenges, we developed a computational pipeline to determine the relative and absolute configurations and phenol positions of diphenazines as inhibitors of eukaryotic translation initiation factor 4E (eIF4E) protein-protein interactions. In this pipeline, we incorporated ECD and GIAO NMR calculations coupled with a DP4+ probability measure, enabling the structure revision of phenazinolin D (4), izumiphenazine A (5), and baraphenazine G (7) and the structure characterization of two new diphenazines, baraphenazine H (3) and izumiphenazine E (6). Importantly, through these efforts, we demonstrate the feasibility of NMR/DP4+ analysis for the determination of phenol positions in phenazine-based molecules, further expanding the limits of computational methods for the structure elucidation of complex natural products.

The role of olefin geometry in the activity of hydrocarbon stapled peptides targeting eukaryotic translation initiation factor 4E (eIF4E).

Hydrocarbon stapled (HCS) peptides are a class of cross-linked α-helix mimetics. The technology relies on the use of α,α'-disubstituted alkenyl amino acids, which fully contrain the helical region to typically yield peptides with enhanced structural ordering and biological activity. Recently, monosubstituted alkenyl amino acids were disclosed for peptide stapling; however, the impact that this tether has on HCS peptide structure and activity has not yet been fully explored. By applying this HCS to the disordered peptide eIF4E-binding protein 1 (4E-BP1), we discovered that this type of tethering has a dramatic effect on olefin geometry and activity of the resultant stapled peptides, where the putative trans isomer was found to exhibit enhanced in vitro and cellular inhibitory activity against eIF4E protein-protein interactions. We further demonstrated that the metathesis catalyst used for ring-closing metathesis can influence monosubstituted HCS peptide activity, presumably through alteration of the cis/trans olefin ratio. This study represents one of the first in-depth analyses of olefin isomers of a stapled peptide and highlights an additional feature for medicinal chemistry optimization of this class of peptide-based probes.

Consideration of Binding Kinetics in the Design of Stapled Peptide Mimics of the Disordered Proteins Eukaryotic Translation Initiation Factor 4E-Binding Protein 1 and Eukaryotic Translation Initiation Factor 4G.

Protein disorder plays a crucial role in signal transduction and is key for many cellular processes including transcription, translation, and cell cycle. Within the intrinsically disordered protein interactome, the α-helix is commonly used for binding, which is induced via a disorder-to-order transition. Because the targeting of protein-protein interactions (PPIs) remains an important challenge in medicinal chemistry, efforts have been made to mimic this secondary structure for rational inhibitor design through the use of stapled peptides. Cap-dependent mRNA translation is regulated by two disordered proteins, 4E-BP1 and eIF4G, that inhibit or stimulate the activity of the m7G cap-binding translation initiation factor, eIF4E, respectively. Both use an α-helical motif for eIF4E binding, warranting the investigation of stapled peptide mimics for manipulating eIF4E PPIs. Herein, we describe our efforts toward this goal, resulting in the synthesis of a cell-active stapled peptide for further development in manipulating aberrant cap-dependent translation in human diseases.

High-Throughput Chemical Probing of Full-Length Protein-Protein Interactions.

Human biology is regulated by a complex network of protein-protein interactions (PPIs), and disruption of this network has been implicated in many diseases. However, the targeting of PPIs remains a challenging area for chemical probe and drug discovery. Although many methodologies have been put forth to facilitate these efforts, new technologies are still needed. Current biochemical assays for PPIs are typically limited to motif-domain and domain-domain interactions, and assays that will enable the screening of full-length protein systems, which are more biologically relevant, are sparse. To overcome this barrier, we have developed a new assay technology, "PPI catalytic enzyme-linked click chemistry assay" or PPI cat-ELCCA, which utilizes click chemistry to afford catalytic signal amplification. To validate this approach, we have applied PPI cat-ELCCA to the eIF4E-4E-BP1  and eIF4E-eIF4G PPIs, key regulators of cap-dependent mRNA translation. Using these examples, we have demonstrated that PPI cat-ELCCA is amenable to full-length proteins, large (>200 kDa) and small (∼12 kDa), and is readily adaptable to automated high-throughput screening. Thus, PPI cat-ELCCA represents a powerful new tool in the toolbox of assays available to scientists interested in the targeting of disease-relevant PPIs.

High-throughput platform assay technology for the discovery of pre-microrna-selective small molecule probes.

MicroRNAs (miRNA) play critical roles in human development and disease. As such, the targeting of miRNAs is considered attractive as a novel therapeutic strategy. A major bottleneck toward this goal, however, has been the identification of small molecule probes that are specific for select RNAs and methods that will facilitate such discovery efforts. Using pre-microRNAs as proof-of-concept, herein we report a conceptually new and innovative approach for assaying RNA-small molecule interactions. Through this platform assay technology, which we term catalytic enzyme-linked click chemistry assay or cat-ELCCA, we have designed a method that can be implemented in high throughput, is virtually free of false readouts, and is general for all nucleic acids. Through cat-ELCCA, we envision the discovery of selective small molecule ligands for disease-relevant miRNAs to promote the field of RNA-targeted drug discovery and further our understanding of the role of miRNAs in cellular biology.

Chemoenzymatic reversible immobilization and labeling of proteins without prior purification.

Site-specific chemical modification of proteins is important for many applications in biology and biotechnology. Recently, our laboratory and others have exploited the high specificity of the enzyme protein farnesyltransferase (PFTase) to site-specifically modify proteins through the use of alternative substrates that incorporate bioorthogonal functionality including azides and alkynes. In this study, we evaluate two aldehyde-containing molecules as substrates for PFTase and as reactants in both oxime and hydrazone formation. Using green fluorescent protein (GFP) as a model system, we demonstrate that the purified protein can be enzymatically modified with either analogue to yield aldehyde-functionalized proteins. Oxime or hydrazone formation was then employed to immobilize, fluorescently label, or PEGylate the resulting aldehyde-containing proteins. Immobilization via hydrazone formation was also shown to be reversible via transoximization with a fluorescent alkoxyamine. After characterizing this labeling strategy using pure protein, the specificity of the enzymatic process was used to selectively label GFP present in crude E. coli extract followed by capture of the aldehyde-modified protein using hydrazide-agarose. Subsequent incubation of the immobilized protein using a fluorescently labeled or PEGylated alkoxyamine resulted in the release of pure GFP containing the desired site-specific covalent modifications. This procedure was also employed to produce PEGylated glucose-dependent insulinotropic polypeptide (GIP), a protein with potential therapeutic activity for diabetes. Given the specificity of the PFTase-catalyzed reaction coupled with the ability to introduce a CAAX-box recognition sequence onto almost any protein, this method shows great potential as a general approach for selective immobilization and labeling of recombinant proteins present in crude cellular extract without prior purification. Beyond generating site-specifically modified proteins, this approach for polypeptide modification could be particularly useful for large-scale production of protein conjugates for therapeutic or industrial applications.