Prey for the Proteasome: Targeted Protein Degradation—A Medicinal Chemist's Perspective

Targeted protein degradation (TPD), the ability to control a proteins fate by triggering its degradation in a highly selective and effective manner, has created tremendous excitement in chemical biology and drug discovery within the past decades. The TPD field is spearheaded by small molecule induced protein degradation with molecular glues and proteolysis targeting chimeras (PROTACs) paving the way to expand the druggable space and to create a new paradigm in drug discovery. However, besides the therapeutic angle of TPD a plethora of novel techniques to modulate and control protein levels have been developed. This enables chemical biologists to better understand protein function and to discover and verify new therapeutic targets. This Review gives a comprehensive overview of chemical biology techniques inducing TPD. It explains the strengths and weaknesses of these methods in the context of drug discovery and discusses their future potential from a medicinal chemist's perspective.     

A Systematic Investigation of Proteoforms with N-Terminal Glycine and Their Dynamics Reveals Its Impacts on Protein Stability

The N-termini of proteins can regulate their degradation, and the same protein with different N-termini may have distinct dynamics. Recently, it was found that N-terminal glycine can serve as a degron recognized by two E3 ligases, but N-terminal glycine was also reported to stabilize proteins. Here we developed a chemoenzymatic method for selective enrichment of proteoforms with N-terminal glycine and integrated dual protease cleavage to further improve the enrichment specificity. Over 2000 unique peptides with protein N-terminal glycine were analyzed from >1000 proteins, and most of them are previously unknown, indicating the effectiveness of the current method to capture low-abundance proteoforms with N-terminal glycine. The degradation rates of proteoforms with N-terminal glycine were quantified along with those of proteins from the whole proteome. Bioinformatic analyses reveal that proteoforms with N-terminal glycine with the fastest and slowest degradation rates have different functions and localizations. Membrane proteins with N-terminal glycine and proteins with N-terminal glycine from the N-terminal methionine excision degrade more rapidly. Furthermore, the secondary structures, adjacent amino acid residues, and protease specificities for N-terminal glycine are also vital for protein degradation. The results advance our understanding of the effects of N-terminal glycine on protein properties and functions.     

Module Assembly for Designing Multivalent Mid‐Sized Inhibitors of Protein‐Protein Interactions

Developing clinically relevant synthetic agents that are capable of disrupting protein‐protein interactions (PPIs) is now a major goal of scientific research. In an effort to explore new methodologies that are applicable to the design of synthetic PPI inhibitors, we examined a strategy based on the assembly of small module compounds to create multivalent mid‐sized agents. This personal account describes three particular approaches based on module assembly: metal‐chelating‐based ligand assembly, covalent chemical ligation templated by a targeted protein, and bivalent inhibitor design for simultaneous targeting of the active pocket and protein surface. These strategies were shown to be useful for synthesizing minimally sized synthetic agents for targeting PPIs and may enable development of agents that are applicable to inhibition of intracellular PPIs.     

Affinity and chemical enrichment strategies for mapping low‐abundance protein modifications and protein‐interaction networks

Protein post‐translational modifications and protein interactions are the central research areas in mass‐spectrometry‐based proteomics. Protein post‐translational modifications affect protein structures, stabilities, activities, and all cellular processes are achieved by interactions among proteins and protein complexes. With the continuing advancements of mass spectrometry instrumentations of better sensitivity, speed, and performance, selective enrichment of modifications/interactions of interest from complex cellular matrices during the sample preparation has become the overwhelming bottleneck in the proteomics workflow. Therefore, many strategies have been developed to address this issue by targeting specific modifications/interactions based on their physical properties or chemical reactivities, but only a few have been successfully applied for systematic proteome‐wide study. In this review, we summarized the highlights of recent developments in the affinity enrichment methods focusing mainly on low stoichiometric protein lipidations. Besides, to identify potential glyoxal modified arginines, a small part was added for profiling reactive arginine sites using an enrichment reagent. A detailed section was provided for the enrichment of protein interactions by affinity purification and chemical cross‐linking, to shed light on the potentials of different enrichment strategies, along with the unique challenges in investigating individual protein post‐translational modification or protein interaction network.     

Synthesis of clustered glycoside-antigen conjugates by two one-pot, orthogonal, chemoselective ligation reactions: scope and limitations

Major histocompatibility class II antigens have been bound to clustered glycosides for selective targeting of the dendritic cell mannose receptor. Di-, tetra-, and octavalent glycoside-antigen conjugates have been obtained after two, orthogonal, hydrazone/thioether ligations, performed by using thio derivatives of D-mannose, D-galactose, or D(-)-quinic acid, glyoxylyl (or hydrazino)-N-chloroacetylated lysinyl trees, and N-terminal hydrazino (or glyoxylyl) peptide antigens. Successful one-pot condensations have been developed to account for the nature of the antigens and the valency of the trees.     

Modification of N-terminal α-amino groups of peptides and proteins using ketenes

A method of highly selective N-terminal modification of proteins as well as peptides by an isolated ketene was developed. Modification of a library of unprotected peptides XSKFR (X varies over 20 natural amino acids) by an alkyne-functionalized ketene (1) at room temperature at pH 6.3 resulted in excellent N-terminal selectivity (modified α-amino group/modified ε-amino group = >99:1) for 13 out of the 20 peptides and moderate-to-high N-terminal selectivity (4:1 to 48:1) for 6 of the 7 remaining peptides. Using an alkyne-functionalized N-hydroxysuccinimide (NHS) ester (2) instead of 1, the modification of peptides XSKFR gave internal lysine-modified peptides for 5 out of the 20 peptides and moderate-to-low N-terminal selectivity (5:1 to 1:4) for 13 out of the 20 peptides. Proteins including insulin, lysozyme, RNaseA, and a therapeutic protein BCArg were selectively N-terminally modified at room temperature using ketene 1, in contrast to the formation of significant or major amounts of di-, tri-, or tetra-modified proteins in the modification by NHS ester 2. The 1-modified proteins were further functionalized by a dansyl azide compound through click chemistry without the need for prior treatment.     

Site‐Specific Protection and Dual Labeling of Human Epidermal Growth Factor (hEGF) for Targeting,Imaging, and Cargo Delivery

Well‐defined human epidermal growth factor (hEGF) constructs featuring selectively addressable labels are urgently needed to address outstanding questions regarding hEGF biology. A protein‐engineering approach was developed to provide access to hEGF constructs that carry two cysteine‐based site‐specific orthogonal labeling sites in multi‐milligram quantities. Also, a site‐selective (de)protection and labeling approach was devised, which allows selective modification of these hEGF constructs. The hEGF, featuring three native disulfide bonds, was expressed featuring additional sulfhydryl groups, in the form of cysteine residues, as orthogonal ligation sites at both the N and C termini. Temporary protection of the N‐terminal cysteine unit, in the form of a thiazolidine ring, avoids interference with protein folding and enables sequential labeling in conjunction with the cysteine residue at the C terminus. Based on thus‐generated hEGF constructs, sequential and site‐specific labeling with a variety of molecular probes could be demonstrated, thus leading to a biological fully functional hEGF with specifically incorporated fluorophores or protein cargo and native cellular targeting and uptake profiles. Thus, this novel strategy provides a robust entry to high‐yielding access of hEGF and rapid and easy site‐specific and multifunctional dual labeling of this growth factor.     

Selective N-terminal modification of peptides and proteins: Recent progresses and applications

Numerous strategies for linking desired chemical probes with target peptides and proteins have been developed and applied in the field of biological chemistry. Approaches for site-specific modification of native amino acid residues in test tubes and biological contexts represent novel biological tools for understanding the role of peptides and proteins. Selective N-terminal modification strategies have been broadly studied especially in the last 10 years, as N-terminal positions are typically so...     

A method for rapidly confirming protein N-terminal sequences by matrix-assisted laser desorption/ionization mass spectrometry

The N-terminal sequence is important for the identification of a protein and the confirmation of its N-terminal processing. Although mass spectrometry (MS) is a sensitive and high-throughput method to sequence and identify peptides and proteins, N-terminal peptides, diluted among most of the peptides that do not originate at the N-termini, are not easy to identify directly with MS. To develop a simple and rapid method to identify and sequence the N-terminal peptide of a protein, a new strategy based on specific sulfonation of terminal amino groups and selective monitoring of the sulfonated peptide was introduced. After a protein had been guanidinated, 2-sulfobenzoylated, and reduced, it was digested with trypsin and analyzed by MS. Because of the strong acidity of sulfonic groups and the specific sulfonation of alpha-amino groups, the sulfonated N-terminal peptide dominated as base peak in the negative mode peptide mass fingerprint (PMF) and was easy to identify. The N-terminal peptide was then selected as precursor ion for tandem mass spectrometric (MS/MS) analysis. Four proteins were tested with this method and their N-terminal peptides were successfully recognized and sequenced. The results suggest that the addition of a sulfonic acid group facilitates the identification and de novo sequencing of N-terminal peptides.     

Rational Heme Protein Design: All Roads Lead to Rome

Heme proteins are among the most abundant and important metalloproteins, exerting diverse biological functions including oxygen transport, small molecule sensing, selective C? H bond activation, nitrite reduction, and electron transfer. Rational heme protein designs focus on the modification of the heme‐binding active site and the heme group, protein hybridization and domain swapping, and de novo design. These strategies not only provide us with unique advantages for illustrating the structure–property–reactivity–function (SPRF) relationship of heme proteins in nature but also endow us with the ability to create novel biocatalysts and biosensors.     

Small‐Molecule PROTACS: New Approaches to Protein Degradation

The current inhibitor‐based approach to therapeutics has inherent limitations owing to its occupancy‐based model: 1) there is a need to maintain high systemic exposure to ensure sufficient in vivo inhibition, 2) high in vivo concentrations bring potential for off‐target side effects, and 3) there is a need to bind to an active site, thus limiting the drug target space. As an alternative, induced protein degradation lacks these limitations. Based on an event‐driven model, this approach offers a novel catalytic mechanism to irreversibly inhibit protein function by targeting protein destruction through recruitment to the cellular quality control machinery. Prior protein degrading strategies have lacked therapeutic potential. However, recent reports of small‐molecule‐based proteolysis‐targeting chimeras (PROTACs) have demonstrated that this technology can effectively decrease the cellular levels of several protein classes.     

Small‐Molecule Control of Intracellular Protein Levels through Modulation of the Ubiquitin Proteasome System

Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be readily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small‐molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activity, can be targeted instead, thus increasing the number of druggable targets. Whereas targeting of the proteasome itself can lead to a global increase in protein levels, the targeting of other components of the UPS (e.g., the E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small‐molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small‐molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets, such as enzymes and receptors.     

A method for N-terminal de novo sequencing of Nα-blocked proteins by mass spectrometry

A method for de novo sequencing of N(α)-blocked proteins by mass spectrometry (MS) is presented. The approach consists of enzymatic digestion of N(α)-blocked protein, recovery of N-terminal peptide by depletion of non-N-terminal peptides from the digest pool, and selective derivatization of a C-terminal α-carboxyl group of isolated N-terminal peptide. The C-terminal α-carboxyl group of the N-terminal peptide was selectively derivatized with 3-aminopropyl-tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-propylamine), according to oxazolone chemistry. The reagent TMPP-propylamine was designed to facilitate sequence analysis with MALDI-MS by mass- and charge-tagging. All of the identities and N-terminal sequences of two N(α)-acetylated proteins (rabbit phosphorylase b and bovine calmodulin) and human orexin A, which has pyroglutamic acid at the N-terminus, were successfully analyzed by allowing for the y-type ions almost exclusively.     

Samarium(II) dibromide-promoted selective deprotection of a benzoyl protective group

The selective deprotection of a benzoyl group was very important methodology in the field of organic synthesis. Various methods for debenzoylation were investigated and developed in the past six decades, but more useful and selective strategies are now being strongly desired. In response to this strong demand, we developed the novel and selective deprotection of a benzoyl group by use of samarium(II) dibromide and a proton source. This deprotective reaction proceeded smoothly and the desired compound was obtained in good to excellent yields. In this paper, we will report the details of this deprotective reaction.     

Four‐fold Channel‐Nicked Human Ferritin Nanocages for Active Drug Loading and pH‐Responsive Drug Release

Human ferritins are emerging platforms for non‐toxic protein‐based drug delivery, owing to their intrinsic or acquirable targeting abilities to cancer cells and hollow cage structures for drug loading. However, reliable strategies for high‐level drug encapsulation within ferritin cavities and prompt cellular drug release are still lacking. Ferritin nanocages were developed with partially opened hydrophobic channels, which provide stable routes for spontaneous and highly accumulated loading of Fe^II‐conjugated drugs as well as pH‐responsive rapid drug release at endoplasmic pH. Multiple cancer‐related compounds, such as doxorubicin, curcumin, and quercetin, were actively and heavily loaded onto the prepared nicked ferritin. Drugs on these minimally modified ferritins were effectively delivered inside cancer cells with high toxicity.     

An advance in proline ligation

Native chemical ligation (NCL) is widely applicable for building proteins in the laboratory. Since the discovery of this method, many strategies have been developed to enhance its capability and efficiency. Because of the poor reactivity of proline thioesters, ligation at a C-terminal proline site is not readily accomplished. Here, we demonstrate that ligation at an N-terminal protein is feasible using the combined logic of NCL and metal-free dethiylation (MFD).     

不同的引导肽对丝状噬菌体基因8蛋白展示外源多肽的影响

为了研究不同的引导肽对在基因8蛋白上展示外源多肽的影响,利用基因工程方法将一短肽插入基因8蛋白的N端,并将引导多肽去除或用基因3蛋白的引导肽序列替代基因8的引导肽序列.采用放射性脉冲追踪技术检测不同的引导肽对在基因8蛋白上展示外源多肽的作用.结果表明,无引导肽序列的引导,蛋白前体无法向成熟蛋白转化.基因3蛋白的引导肽可以引导展示有外源多肽的基因8蛋白,完成从前体向成熟蛋白的转化,但转化率明显下降.研究结果对阐明噬菌体外壳蛋白在大肠杆菌中的跨膜机制具有重要的意义.     

Eine gentherapeutische Strategie zur Behandlung von Energiestoffwechsel-Erkrankungen

Genetic variations of the mitochondrial genome lead to severe neuromuscular diseases in man. A treatment of these utilizing a somatic gene therapy approach is invariably linked to a mitochondrial transfection system. A novel technique of targeting nucleic acids to mitochondria has been developed that takes advantage of the protein import pathway. The system is based on chimerical molecules that are composed of a DNA and a protein moiety, harboring the information for mitochondrial targeting. Upon recognition of these molecules by a receptor on the outer mitochondrial membrane, the molecules are able to cross the membrane system and are released into the matrix of the organelle. The further development of this technique will give to rise to strategies for the treatment of mitochondrial DNA diseases by a somatic gene therapy approach.     

Selective patterning of Si-based biosensor surfaces using isotropic silicon etchants

Ultra-sensitive, label-free biosensors have the potential to have a tremendous impact on fields like medical diagnostics. For the majority of these Si-based integrated devices, it is necessary to functionalize the surface with a targeting ligand in order to perform specific biodetection. To do this, silane coupling agents are commonly used to immobilize the targeting ligand. However, this method typically results in the bioconjugation of the entire device surface, which is undesirable. To compensate for this effect, researchers have developed complex blocking strategies that result in selective patterning of the sensor surface. Recently, silane coupling agents were used to attach biomolecules to the surface of silica toroidal biosensors integrated on a silicon wafer. Interestingly, only the silica biosensor surface was conjugated. Here, we hypothesize why this selective patterning occurred. Specifically, the silicon etchant (xenon difluoride), which is used in the fabrication of the biosensor, appears to reduce the efficiency of the silane coupling attachment to the underlying silicon wafer. These results will enable future researchers to more easily control the bioconjugation of their sensor surfaces, thus improving biosensor device performance.