Structure-Based Design of a Macrocyclic PROTAC

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co-crystal structure of macroPROTAC-1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12-fold loss of binary binding affinity for Brd4, macroPROTAC-1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.     

Structure‐Based Design of a Macrocyclic PROTAC

Constraining a molecule in its bioactive conformation via macrocyclization represents an attractive strategy to rationally design functional chemical probes. While this approach has been applied to enzyme inhibitors or receptor antagonists, to date it remains unprecedented for bifunctional molecules that bring proteins together, such as PROTAC degraders. Herein, we report the design and synthesis of a macrocyclic PROTAC by adding a cyclizing linker to the BET degrader MZ1. A co‐crystal structure of macroPROTAC‐1 bound in a ternary complex with VHL and the second bromodomain of Brd4 validated the rational design. Biophysical studies revealed enhanced discrimination between the second and the first bromodomains of BET proteins. Despite a 12‐fold loss of binary binding affinity for Brd4, macroPROTAC‐1 exhibited cellular activity comparable to MZ1. Our findings support macrocyclization as an advantageous strategy to enhance PROTAC degradation potency and selectivity between homologous targets.     

Optogenetic Apoptosis: Light‐Triggered Cell Death

An optogenetic Bax has been designed that facilitates light‐induced apoptosis. We demonstrate that mitochondrial recruitment of a genetically encoded light‐responsive Bax results in the release of mitochondrial proteins, downstream caspase‐3 cleavage, changes in cellular morphology, and ultimately cell death. Mutagenesis of a key phosphorylatable residue or modification of the C‐terminus mitigates background (dark) levels of apoptosis that result from Bax overexpression. The mechanism of optogenetic Bax‐mediated apoptosis was explored using a series of small molecules known to interfere with various steps in programmed cell death. Optogenetic Bax appears to form a mitochondrial apoptosis‐induced channel analogous to that of endogenous Bax.     

New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo

Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded "aldehyde tag" for site-specific labeling with aminooxy- or hydrazide-functionalized probes. In this report, we screened FGEs from M. tuberculosis and S. coelicolor against synthetic peptide libraries and identified new substrate sequences that diverge from the canonical motif. We found that E. coli's FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins.     

Suzuki Cross-Coupling Reaction with Genetically Encoded Fluorosulfates for Fluorogenic Protein Labeling

A palladium-catalyzed cross-coupling reaction with aryl halide functionalities has recently emerged as a valuable tool for protein modification. Herein, a new fluorogenic modification methodology for proteins, with genetically encoded fluorosulfate-l -tyrosine, which exhibits high efficiency and biocompatibility in bacterial cells as well as in aqueous medium, is described. Furthermore, the cross-coupling of 4-cyanophenylboronic acid on green fluorescent protein was shown to possess a unique fluorogenic property, which could open up the possibility of a responsive “off/on” switch with great potential to enable spectroscopic imaging of proteins with minimal background noise. Taken together, a convenient and efficient catalytic system has been developed that may provide broad utilities in protein visualization and live-cell imaging.     

Genetically encoding protein oxidative damage

Posttranslational modification of tyrosine residues in proteins, to produce 3-nitrotyrosine (3-NT), is associated with over 50 disease states including transplant rejection, lung infection, central nervous system and ocular inflammation shock, cancer, and neurological disorders (for example, Alzheimer's disease, Parkinson's disease, and stroke). The levels of 3-NT increase in aging tissue, and levels of 3-NT in proteins are a predictor of disease risk. Here we report the evolution and characterization of an aminoacyl-tRNA synthetase/tRNA pair for the cotranslational, site-specific incorporation of 3-NT into proteins at genetically encoded sites. To demonstrate the utility of our approach for studying the effect on protein function of nitration on sites defined in vivo, we prepared manganese superoxide dismutase (MnSOD) that is homogeneously nitrated at a site known to be modified in disease-related inflammatory responses, and we measured the effect of this defined modification on protein function.     

A cellular FRET-based sensor for beta-O-GlcNAc, a dynamic carbohydrate modification involved in signaling

beta-O-N-Acetyl-d-glucosamine (O-GlcNAc) is a dynamic carbohydrate modification that is involved in cell signaling and has been implicated in a variety of disease states, including Alzheimer's and type-II diabetes. Despite the importance of this modification, little is known about the spatial and temporal localization of O-GlcNAc during signaling. This is due to the lack of methods for the study of O-GlcNAc in living cell systems. Herein we report the first genetically encoded FRET-based sensor for the detection of O-GlcNAc dynamics in live mammalian cells.     

Direct site-selective covalent protein immobilization catalyzed by a phosphopantetheinyl transferase

Immobilization of proteins onto solid supports is important in the preparation of functional protein microarrays and in the development of bead-based bioassays, biosensors, and industrial biocatalysts. In order to generate the stable, functional, and homogeneous materials required for these applications, attention has focused on methods that enable the efficient and site-specific covalent immobilization of recombinant proteins onto a wide range of platforms. To this end, the phosphopantetheinyl transferase Sfp was employed to catalyze the direct immobilization of recombinant proteins bearing the small, genetically encoded ybbR tag onto surfaces functionalized with CoA. Using mass spectrometry, it was shown that the Sfp catalyzes immobilization of a model acyl carrier protein (ACP) onto CoA-derivatized PEGA resin beads through specific covalent bond formation. Luciferase (Luc) and glutathione-S-transferase (GST) ybbR-fusion proteins were similarly immobilized onto PEGA resin retaining high levels of enzyme activity. This strategy was also successfully applied for the immobilization of the ACP, as well as ybbR-Luc, -GST, and -thioredoxin fusion proteins, on hydrogel microarray slides. Overall, the Sfp-catalyzed surface ligation is mild, quantitative, and rapid, occurring in a single step without prior chemical modification of the target protein. Immobilization of the target proteins directly from a cell lysate mixture was also demonstrated.     

A Versatile Approach for Site‐Specific Lysine Acylation in Proteins

Using amber suppression in coordination with a mutant pyrrolysyl‐tRNA synthetase‐tRNA^Pyl pair, azidonorleucine is genetically encoded in E. coli . Its genetic incorporation followed by traceless Staudinger ligation with a phosphinothioester allows the convenient synthesis of a protein with a site‐specifically installed lysine acylation. By simply changing the phosphinothioester identity, any lysine acylation type could be introduced. Using this approach, we demonstrated that both lysine acetylation and lysine succinylation can be installed selectively in ubiquitin and synthesized histone H3 with succinylation at its K4 position (H3K4su). Using an H3K4su‐H4 tetramer as a substrate, we further confirmed that Sirt5 is an active histone desuccinylase. Lysine succinylation is a recently identified post‐translational modification. The reported technique makes it possible to explicate regulatory functions of this modification in proteins.     

Residue choice defines efficiency and influence of bioorthogonal protein modification via genetically encoded strain promoted Click chemistry

GFP and a FRET compatible dye were used to assess the influence of genetically encoded aryl azide positioning on Click chemistry-based protein conjugation. While modification efficiency of the sampled mutants using a strain promoted reaction varied by as much as ～10 fold, there was no simple correlation with accessibility of the aryl azide on GFP's surface. One labeled GFP mutant (Gln204AzPhe) exhibited high efficiency FRET (～90%) and an unprecedented pseudo-Stokes shift of 126 nm.     

Constructing Photoactivatable Protein with Genetically Encoded Photocaged Glutamic Acid

Genetically replacing an essential residue with the corresponding photocaged analogues via genetic code expansion (GCE) constitutes a useful and unique strategy to directly and effectively generate photoactivatable proteins. However, the application of this strategy is severely hampered by the limited number of encoded photocaged proteinogenic amino acids. Herein, we report the genetic incorporation of photocaged glutamic acid analogues in E. coli and mammalian cells and demonstrate their use in constructing photoactivatable variants of various fluorescent proteins and SpyCatcher. We believe genetically encoded photocaged Glu would significantly promote the design and application of photoactivatable proteins in many areas.     

Optogenetic Engineering: Light‐Directed Cell Motility

Genetically encoded, light‐activatable proteins provide the means to probe biochemical pathways at specific subcellular locations with exquisite temporal control. However, engineering these systems in order to provide a dramatic jump in localized activity, while retaining a low dark‐state background remains a significant challenge. When placed within the framework of a genetically encodable, light‐activatable heterodimerizer system, the actin‐remodelling protein cofilin induces dramatic changes in the F‐actin network and consequent cell motility upon illumination. We demonstrate that the use of a partially impaired mutant of cofilin is critical for maintaining low background activity in the dark. We also show that light‐directed recruitment of the reduced activity cofilin mutants to the cytoskeleton is sufficient to induce F‐actin remodeling, formation of filopodia, and directed cell motility.     

A Cysteine-Directed Proximity-Driven Crosslinking Method for Native Peptide Bicyclization

Efficient and site-specific modification of native peptides and proteins is desirable for synthesizing antibody-drug conjugates as well as for constructing chemically modified peptide libraries using genetically encoded platforms such as phage display. In particular, there is much interest in efficient multicyclization of native peptides due to the appeals of multicyclic peptides as therapeutics. However, conventional approaches for multicyclic peptide synthesis require orthogonal protecting groups or non-proteinogenic clickable handles. Herein, we report a cysteine-directed proximity-driven strategy for the constructing bicyclic peptides from simple natural peptide precursors. This linear to bicycle transformation initiates with rapid cysteine labeling, which then triggers proximity-driven amine-selective cyclization. This bicyclization proceeds rapidly under physiologic conditions, yielding bicyclic peptides with a Cys-Lys-Cys, Lys-Cys-Lys or N-terminus-Cys-Cys stapling pattern. We demonstrate the utility and power of this strategy by constructing bicyclic peptides fused to proteins as well as to the M13 phage, paving the way to phage display of novel bicyclic peptide libraries.     

Halomethyl-Triazoles for Rapid,Site-Selective Protein Modification

Post-translational modifications (PTMs) are used by organisms to control protein structure and function after protein translation, but their study is complicated and their roles are not often well understood as PTMs are difficult to introduce onto proteins selectively. Designing reagents that are both good mimics of PTMs, but also only modify select amino acid residues in proteins is challenging. Frequently, both a chemical warhead and linker are used, creating a product that is a misrepresentation of the natural modification. We have previously shown that biotin-chloromethyl-triazole is an effective reagent for cysteine modification to give S-Lys derivatives where the triazole is a good mimic of natural lysine acylation. Here, we demonstrate both how the reactivity of the alkylating reagents can be increased and how the range of triazole PTM mimics can be expanded. These new iodomethyl-triazole reagents are able to modify a cysteine residue on a histone protein with excellent selectivity in 30 min to give PTM mimics of acylated lysine side-chains. Studies on the more complicated, folded protein SCP-2L showed promising reactivity, but also suggested the halomethyl-triazoles are potent alkylators of methionine residues.     

Scope of amino acid recognition by cucurbit[8]uril

This paper describes the molecular recognition of amino acids by cucurbit[8]uril (Q8) and by the 1:1 complex between Q8 and methyl viologen (MV) in purely aqueous solution. These hosts are known to bind aromatic peptides with high affinity and sequence specificity, but prior work has focused on only a small subset of amino acids. In an effort to elucidate the scope and limitations of amino acid recognition by Q8 and Q8?MV, a comprehensive examination of the 20 genetically encoded amino acids was carried out by ¹H NMR spectroscopy and isothermal titration calorimetry. We find that both Q8 and Q8?MV bind measurably to only tryptophan, phenylalanine, and tyrosine. These results demonstrate that Q8 and Q8?MV are highly selective in the context of all genetically encoded amino acids and are therefore promising for the development of recognition-intensive applications involving peptides, proteins, and proteomes.     

Generation of a Genetically Encoded,Photoactivatable Intein for the Controlled Production of Cyclic Peptides

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split‐intein‐mediated SICLOPPS technology provides a generic access to fully genetically encoded head‐to‐tail cyclized peptides and large libraries thereof (SICLOPPS=split‐intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light‐dependent intein using the photocaged tyrosine derivative ortho‐nitrobenzyltyrosine incorporated at an internal, non‐catalytic position is now reported. Stable intein precursors were purified from the E. coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.     

Generation of a Genetically Encoded,Photoactivatable Intein for the Controlled Production of Cyclic Peptides

Cyclic peptides are important natural products and hold great promise for the identification of new bioactive molecules. The split‐intein‐mediated SICLOPPS technology provides a generic access to fully genetically encoded head‐to‐tail cyclized peptides and large libraries thereof (SICLOPPS=split‐intein circular ligation of peptides and proteins). However, owing to the spontaneous protein splicing reaction, product formation occurs inside cells, making peptide isolation inconvenient and precluding traditional in vitro assays for inhibitor discovery. The design of a genetically encoded, light‐dependent intein using the photocaged tyrosine derivative ortho‐nitrobenzyltyrosine incorporated at an internal, non‐catalytic position is now reported. Stable intein precursors were purified from the E. coli expression host and subsequently subjected to light activation in vitro for both the regular protein splicing format and cyclic peptide production, including the natural product segetalin H as an example. The activity of the intein could also be triggered in living cells.     

A photoactivatable prenylated cysteine designed to study isoprenoid recognition.

Protein prenylation, involving the alkylation of a specific C-terminal cysteine with a C(15) or C(20) isoprenoid unit, is an essential posttranslational modification required by most GTP-binding proteins for normal biological activity. Despite the ubiquitous nature of this modification and numerous efforts aimed at inhibiting prenylating enzymes for therapeutic purposes, the function of prenylation remains unclear. To explore the role the isoprenoid plays in mediating protein-protein recognition, we have synthesized a photoactivatable, isoprenoid-containing cysteine analogue (2) designed to act as a mimic of the C-terminus of prenylated proteins. Photolysis experiments with 2 and RhoGDI (GDI), a protein which interacts with prenylated Rho proteins, suggest that the GDI is in direct contact with the isoprenoid moiety. These results, obtained using purified GDI as well as Escherichia coli (E. coli) crude extract containing GDI, suggest that this analogue will be an effective and versatile tool for the investigation of putative isoprenoid binding sites in a variety of systems. Incorporation of this analogue into peptides or proteins should allow for even more specific interactions between the photoactivatable isoprenoid and any number of isoprenoid binding proteins.     

Traceless and site-specific ubiquitination of recombinant proteins

Protein ubiquitination is a post-translational modification that regulates almost all aspects of eukaryotic biology. Here we discover the first routes for the efficient site-specific incorporation of δ-thiol-L-lysine (7) and δ-hydroxy-L-lysine (8) into recombinant proteins, via evolution of a pyrrolysyl-tRNA synthetase/tRNA(CUA) pair. We combine the genetically directed incorporation of 7 with native chemical ligation and desulfurization to yield an entirely native isopeptide bond between substrate proteins and ubiquitin. We exemplify this approach by demonstrating the synthesis of a ubiquitin dimer and the first synthesis of ubiquitinated SUMO.     

Molecular design of glycoprotein mimetics: glycoblotting by engineered proteins with an oxylamino-functionalized amino acid residue

The general and efficient method for the site-directed glycosylation of proteins is a key step in order to understand the biological importance of the carbohydrate chains of proteins and to control functional roles of the engineered glycoproteins in terms of the development of improved glycoprotein therapeutics. We have developed a novel method for site-directed glycosylation of proteins based on chemoselective blotting of common reducing sugars by genetically encoded proteins. The oxylamino-functionalized L-homoserine residues, 2-amino-4-O-(N-methylaminooxy) butanoic acid and 2-amino-4-aminooxy butanoic acid, were efficiently incorporated into proteins by using the four-base codon/anticodon pair strategy in Escherichia coli in vitro translation. Direct and chemoselective coupling between unmodified simple sugars and N-methylaminooxy group displayed on the engineered streptavidin allowed for the combinatorial synthesis of novel glycoprotein mimetics.