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Analytical Chemistry Oct 2021A stable molecular structure is important in the development of a protein candidate into a therapeutic product. A therapeutic protein often contains many different...
A stable molecular structure is important in the development of a protein candidate into a therapeutic product. A therapeutic protein often contains many different variants; some of them may have an impact on the conformational stability of the protein. Conventionally, to evaluate the impact of a variant on stability, the variant must be enriched to a reasonable purity, and then its stability characterized by chromatographic or biophysical techniques. However, it is often impractical to purify and characterize each variant in a therapeutic protein. A workflow, based on limited proteolysis followed by MS detection, was established to simultaneously assess the impact of a large number of variants on conformational stability without enrichment. Because a less stable domain is more susceptible to proteolytic degradation, conformational stability of the domain can be reported from the release rate of a proteolytic peptide. A kinetic model is established to quantitatively determine the extent of domain stabilization/destabilization of different variants. The methodology is demonstrated by examining variants known to affect the stability of immunoglobulin domains, such as different -glycoforms, methionine oxidations, and sequence variants. With this methodology, near 100 variants may be evaluated within 2 days in a single experiment. Insights into the sequence-stability relationship will be obtained by monitoring the large number of low-level sequence variants, facilitating engineering of more stable molecules.
Topics: Kinetics; Mass Spectrometry; Molecular Conformation; Protein Stability; Proteolysis
PubMed: 34637272
DOI: 10.1021/acs.analchem.1c03335 -
Drug Discovery Today Jan 2023The dawn of targeted degradation using proteolysis-targeting chimeras (PROTACs) against recalcitrant proteins has prompted numerous efforts to develop complementary... (Review)
Review
The dawn of targeted degradation using proteolysis-targeting chimeras (PROTACs) against recalcitrant proteins has prompted numerous efforts to develop complementary drugs. Although many of these are specifically directed against undruggable proteins, there is increasing interest in small molecule-based PROTACs that target intracellular pathways, and some have recently entered clinical trials. Concurrently, small molecule-based PROTACs that target protumorigenic pathways in cancer cells, the tumor microenvironment (TME), and angiogenesis have been found to have potent effects that synergize with the action of antibodies. This has led to the augmentation of PROTACs with variable substitution patterns. Several combinations with small molecules targeting undruggable proteins are now under clinical investigation. In this review, we discuss the recent milestones achieved as well as challenges encountered in this area of drug development, as well as our opinion on the best path forward.
Topics: Proteolysis; Proteins
PubMed: 36306996
DOI: 10.1016/j.drudis.2022.103417 -
Nature Biotechnology Apr 2024
Topics: Biodegradation, Environmental; Proteolysis
PubMed: 38448663
DOI: 10.1038/s41587-024-02164-9 -
Cancer Letters Mar 2023Proteolysis-targeting chimeras (PROTACs) are being developed as an effective method for degrading cancer-related proteins by modifying the endogenous... (Review)
Review
Proteolysis-targeting chimeras (PROTACs) are being developed as an effective method for degrading cancer-related proteins by modifying the endogenous ubiquitin-proteasome system. To investigate the dynamics between an E3 ligase and target protein, researchers have developed a wide variety of bifunctional PROTACs by combining small molecule ligands. These PROTACs employ numerous ligands, some of which are reversible, some of which are irreversible, some attach to orthosteric sites, while others bind to allosteric sites. Some are agonists, while others are antagonists, and the target protein may be activated in either a positive or negative manner. A variety of targeted ligand approaches can be used to enhance PROTAC properties, including tumor selectivity and drug delivery, and to overcome drug resistance. The processes and behaviors of small molecule-based PROTACs and targeted proteolysis approaches as anticancer therapeutic molecules have been introduced in this mini-review.
Topics: Humans; Ligands; Neoplasms; Proteasome Endopeptidase Complex; Proteins; Proteolysis; Ubiquitin-Protein Ligases
PubMed: 36642326
DOI: 10.1016/j.canlet.2023.216065 -
Food Chemistry Mar 2024Proteolysis prior to fibrillation can change the functional properties of protein fibrils. This study aimed to investigate the effects of proteolysis pretreatment by...
Proteolysis prior to fibrillation can change the functional properties of protein fibrils. This study aimed to investigate the effects of proteolysis pretreatment by alkaline protease on formation, structure, and foaming properties of rice protein fibrils. Thioflavin T fluorescence and conversion assays showed that proteolysis reduced the fibril formation capacity of rice protein. After 12 h of heating, the percent conversion of rice protein and rice protein hydrolysates to fibrils reached 60.32 ± 1.07% and 30.43 ± 2.01%, respectively. Transmission electron microscopy images showed that fibrils derived from rice protein hydrolysates had a longer contour length than rice protein fibrils. The foaming capacity and stability of rice protein hydrolysate fibrils increased by 16.70% and 11.27%, respectively, compared with those of rice protein fibrils. The addition of rice protein hydrolysate fibrils improved the texture of cakes. These results suggested that rice protein hydrolysate fibrils could be a promising plant-based foaming agent.
Topics: Proteolysis; Protein Hydrolysates; Oryza; Food
PubMed: 37907000
DOI: 10.1016/j.foodchem.2023.137765 -
Expert Opinion on Drug Discovery Dec 2019: Proteolysis - targeting chimeras (PROTACs) have emerged as a new modality with the potential to revolutionize drug discovery. PROTACs are heterobifunctional molecules... (Review)
Review
: Proteolysis - targeting chimeras (PROTACs) have emerged as a new modality with the potential to revolutionize drug discovery. PROTACs are heterobifunctional molecules comprising of a ligand targeting a protein of interest, a ligand targeting an E3 ligase and a connecting linker. The aim is instead of inhibiting the target to induce its proteasomal degradation. : PROTACs, due to their bifunctional design, possess properties that differentiate them from classical inhibitors. A structural analysis, based on published crystal aspects, kinetic features and aspects of selectivity are discussed. Specific types such as homoPROTACs, PROTACs targeting Tau protein and the first PROTACs recently entering clinical trials are examined. : PROTACs have shown remarkable biological responses in challenging targets, including an unprecedented selectivity over protein family members and even efficacy starting from weak or unspecific binders. Moreover, PROTACs are standing out from classical pharmacology by inducing the degradation of the target protein and not merely its inhibition. However, there are also challenges in the field, such as the rational structure optimization, the evolution of computational tools, limited structural data and the greatly anticipated clinical data. Despite the remaining hurdles, PROTACs are expected to soon become a new therapeutic category of drugs.
Topics: Animals; Drug Discovery; Humans; Ligands; Proteins; Proteolysis; Ubiquitin-Protein Ligases
PubMed: 31538491
DOI: 10.1080/17460441.2019.1659242 -
Cell Chemical Biology Jul 2021Molecular glues and proteolysis targeting chimeras (PROTACs) have emerged as small-molecule tools that selectively induce the degradation of a chosen protein and have... (Review)
Review
Molecular glues and proteolysis targeting chimeras (PROTACs) have emerged as small-molecule tools that selectively induce the degradation of a chosen protein and have shown therapeutic promise. Recently, several approaches employing light as an additional stimulus to control induced protein degradation have been reported. Here, we analyze the principles guiding the design of such systems, provide a survey of the literature published to date, and discuss opportunities for further development. Light-responsive degraders enable the precise temporal and spatial control of protein levels, making them useful research tools but also potential candidates for human precision medicine.
Topics: Humans; Precision Medicine; Proteins; Proteolysis; Small Molecule Libraries
PubMed: 34115971
DOI: 10.1016/j.chembiol.2021.05.010 -
Journal of Medicinal Chemistry Oct 2020The discovery and development of targeted protein degraders have become important areas of research in the field of medicinal chemistry. Inducing degradation of a target... (Review)
Review
The discovery and development of targeted protein degraders have become important areas of research in the field of medicinal chemistry. Inducing degradation of a target protein presents several advantages relative to simple inhibition including a potential for extended duration of action and more profound pharmacology. While engineered heterodimeric molecules have recently been a major focus within industry and academia, this Perspective highlights examples of targeted protein degradation observed for smaller, monomeric molecules. Methods and tools for evaluating protein degradation as well as a discussion of physical properties of monomeric vs engineered heterodimeric degraders are presented.
Topics: Binding Sites; Cell Line, Tumor; Drug Discovery; Humans; Molecular Structure; Protein Binding; Proteins; Proteolysis; Small Molecule Libraries; Ubiquitin-Protein Ligases
PubMed: 32352776
DOI: 10.1021/acs.jmedchem.0c00093 -
Methods in Molecular Biology (Clifton,... 2024Proteases are enzymes that catalyze the hydrolytic degradation of other proteins into peptides or amino acids through the digestion of the peptide bond. Promiscuous...
Proteases are enzymes that catalyze the hydrolytic degradation of other proteins into peptides or amino acids through the digestion of the peptide bond. Promiscuous proteases that target a wide range of proteins are distinguished from specific proteases that have a narrow range of substrates. In terms of activity, endoproteases cleave their substrates at specific residues within the target proteins, whereas exoproteases cleave from one extremity and may have processive activities. Proteases are therefore very useful tools to study proteins, notably their structure or conformation. In addition, proteases can be used to probe the topology of bacterial membrane proteins. Here, we describe limited protease accessibility assays to define inner membrane protein topology and conformational changes based on digestion profiles.
Topics: Proteolysis; Peptide Hydrolases; Endopeptidases; Membrane Proteins; Bacterial Proteins
PubMed: 37930525
DOI: 10.1007/978-1-0716-3445-5_8 -
Food Chemistry Apr 2017In dry fermented sausages, myofibrillar proteins undergo intense proteolysis generating small peptides and free amino acids that play a role in flavour generation. This...
In dry fermented sausages, myofibrillar proteins undergo intense proteolysis generating small peptides and free amino acids that play a role in flavour generation. This study aimed to identify small peptides arising from actin proteolysis, as influenced by the type of processing. Two acidification profiles were imposed, in order to mimic the pH normally obtained in southern-type and northern-type dry fermented sausages. The identification of peptides was done by liquid chromatography coupled to mass spectrometry in a data-independent positive mode of acquisition (LC-MS). During manufacturing of the dry fermented sausages, actin was highly proteolysed, especially in nine regions of the sequence. After fermentation, 52 and 42 actin-derived peptides were identified at high and low pH, respectively, which further increased to 66 and 144 peptides, respectively, at the end of ripening. Most peptides were released at the cleavage sites of cathepsins B and D, which thus play an important role.
Topics: Actins; Amino Acids; Animals; Fermentation; Humans; Hydrogen-Ion Concentration; Meat Products; Peptides; Proteolysis; Swine; Taste
PubMed: 27979096
DOI: 10.1016/j.foodchem.2016.11.023