Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

Proteolysis in plant immunity.

Compared with transcription and translation, protein degradation machineries can act faster and be targeted to different subcellular compartments, enabling immediate regulation of signaling events. It is therefore not surprising that proteolysis has been used extensively to control homeostasis of key regulators in different biological processes and pathways. Over the past decades, numerous studies have shown that proteolysis, where proteins are broken down to peptides or amino acids through ubiquitin-mediated degradation systems and proteases, is a key regulatory mechanism to control plant immunity output. Here, we briefly summarize the roles various proteases play during defense activation, focusing on recent findings. We also update the latest progress of ubiquitin-mediated degradation systems in modulating immunity by targeting plant membrane-localized pattern recognition receptors (PRRs), intracellular nucleotide-binding domain leucine-rich repeat receptors (NLRs), and downstream signaling components. Additionally, we highlight recent studies showcasing the importance of proteolysis in maintaining broad-spectrum resistance without obvious yield reduction, opening new directions for engineering elite crops that are resistant to a wide range of pathogens with high yield.

Effects of Amino Acid Supplementation to a Low-Protein Diet on the Growth Performance and Protein Metabolism-related Factors in Broiler Chicks.

A low-protein (LP) diet may alleviate the environmental impact of chicken meat production by reducing nitrogen excretion and ammonia emissions. Thus, this study investigated the effect of a 15% reduced protein diet with or without amino acid (AA) supplementation on the growth performance of broiler chicks from 10 to 35 days of age and the underlying mechanism for loss of skeletal muscle mass. Thirty-six male broiler chicks were allocated to three experimental groups based on body weight: control, LP, and essential AA-supplemented LP (LP+AA). The body weight gain, feed conversion ratio, and weight of breast muscles and legs significantly decreased only in the LP group at the end of the feeding period. Plasma uric acid levels were significantly lower in the LP+AA group than those of the other groups. In the LP group, mRNA levels of microtubule-associated protein 1 light chain 3 isoform B were significantly higher in the pectoralis major, whereas those of atrogin-1, muscle RING-finger protein-1, and myoblast determination protein 1 were significantly higher in the biceps femoris compared to those in the control group. There were no significant differences in insulin-like growth factor 1 mRNA levels in the liver or skeletal muscle between groups. These findings suggested that supplementation with essential AAs ameliorated the impaired effects of an LP diet on growth performance in broiler chicks, and that the transcriptional changes in proteolytic genes in skeletal muscles might be related to the impaired effects of the LP diet.

Extracellular proteolysis in cancer: Proteases, substrates, and mechanisms in tumor progression and metastasis.

A vast ensemble of extracellular proteins influences the development and progression of cancer, shaped and reshaped by a complex network of extracellular proteases. These proteases, belonging to the distinct classes of metalloproteases, serine proteases, cysteine proteases, and aspartic proteases, play a critical role in cancer. They often become dysregulated in cancer, with increases in pathological protease activity frequently driven by loss of normal latency controls, diminished regulation by endogenous protease inhibitors, and changes in localization. Dysregulated proteases accelerate tumor progression and metastasis by degrading protein barriers within the extracellular matrix (ECM), stimulating tumor growth, reactivating dormant tumor cells, facilitating tumor cell escape from immune surveillance, and shifting stromal cells toward cancer-promoting behaviors through the precise proteolysis of specific substrates to alter their functions. These crucial substrates include ECM proteins and proteoglycans, soluble proteins secreted by tumor and stromal cells, and extracellular domains of cell surface proteins, including membrane receptors and adhesion proteins. The complexity of the extracellular protease web presents a significant challenge to untangle. Nevertheless, technological strides in proteomics, chemical biology, and the development of new probes and reagents are enabling progress and advancing our understanding of the pivotal importance of extracellular proteolysis in cancer.

Secreted Aspartic Proteinases: Key Factors in Candida Infections and Host-Pathogen Interactions.

Extracellular proteases are key factors contributing to the virulence of pathogenic fungi from the genus Candida. Their proteolytic activities are crucial for extracting nutrients from the external environment, degrading host defenses, and destabilizing the internal balance of the human organism. Currently, the enzymes most frequently described in this context are secreted aspartic proteases (Saps). This review comprehensively explores the multifaceted roles of Saps, highlighting their importance in biofilm formation, tissue invasion through the degradation of extracellular matrix proteins and components of the coagulation cascade, modulation of host immune responses via impairment of neutrophil and monocyte/macrophage functions, and their contribution to antifungal resistance. Additionally, the diagnostic challenges associated with Candida infections and the potential of Saps as biomarkers were discussed. Furthermore, we examined the prospects of developing vaccines based on Saps and the use of protease inhibitors as adjunctive therapies for candidiasis. Given the complex biology of Saps and their central role in Candida pathogenicity, a multidisciplinary approach may pave the way for innovative diagnostic strategies and open new opportunities for innovative clinical interventions against candidiasis.

Proteomics reveals the mechanism of protein degradation and its relationship to sensorial and texture characteristics in dry-cured squid during processing.

Proteolysis in dry-cured squid contributes to the development of sensory and textural attributes. In this study, label-free quantitative proteomics was conducted to study the mechanism of proteolysis and its correlation with quality changes. The results showed that the protein profile of dry-cured squid changed markedly during processing, which was confirmed by the quantification of myofibrillar protein, amino nitrogen and total free acids, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Thirty-two key differentially abundant proteins were found to be correlated with sensory and texture characteristics, including myofibrillar protein, tubulin beta chain, collagens, heat shock proteins and cytochrome c. The correlation analysis indicated that myosin regulatory light chain and tubulin beta chain played the most important role in the development of texture and sensory attributes in squid samples during the dry-curing process. The results offered novel insights into proteolysis in dry-cured squid and its relationship to quality changes.

Reduced sympathetic activity is associated with the development of pain and muscle atrophy in a female rat model of fibromyalgia.

Fibromyalgia (FM) is characterized by chronic widespread musculoskeletal pain accompanied by fatigue and muscle atrophy. Although its etiology is not known, studies have shown that FM patients exhibit altered function of the sympathetic nervous system (SNS), which regulates nociception and muscle plasticity. Nevertheless, the precise SNS-mediated mechanisms governing hyperalgesia and skeletal muscle atrophy in FM remain unclear. Thus, we employed two distinct FM-like pain models, involving intramuscular injections of acidic saline (pH 4.0) or carrageenan in prepubertal female rats, and evaluated the catecholamine content, adrenergic signaling and overall muscle proteolysis. Subsequently, we assessed the contribution of the SNS to the development of hyperalgesia and muscle atrophy in acidic saline-injected rats treated with clenbuterol (a selective ß2-adrenergic receptor agonist) and in animals maintained under baseline conditions and subjected to epinephrine depletion through adrenodemedullation (ADM). Seven days after inducing an FM-like model with acidic saline or carrageenan, we observed widespread mechanical hyperalgesia along with loss of strength and/or muscle mass. These changes were associated with reduced catecholamine content, suggesting a common underlying mechanism. Notably, treatment with a ß2-agonist alleviated hyperalgesia and prevented muscle atrophy in acidic saline-induced FM-like pain, while epinephrine depletion induced mechanical hyperalgesia and increased muscle proteolysis in animals under baseline conditions. Together, the results suggest that reduced sympathetic activity is involved in the development of pain and muscle atrophy in the murine model of FM analyzed.

Systematic identification of structure-specific protein-protein interactions.

The physical interactome of a protein can be altered upon perturbation, modulating cell physiology and contributing to disease. Identifying interactome differences of normal and disease states of proteins could help understand disease mechanisms, but current methods do not pinpoint structure-specific PPIs and interaction interfaces proteome-wide. We used limited proteolysis-mass spectrometry (LiP-MS) to screen for structure-specific PPIs by probing for protease susceptibility changes of proteins in cellular extracts upon treatment with specific structural states of a protein. We first demonstrated that LiP-MS detects well-characterized PPIs, including antibody-target protein interactions and interactions with membrane proteins, and that it pinpoints interfaces, including epitopes. We then applied the approach to study conformation-specific interactors of the Parkinson's disease hallmark protein alpha-synuclein (aSyn). We identified known interactors of aSyn monomer and amyloid fibrils and provide a resource of novel putative conformation-specific aSyn interactors for validation in further studies. We also used our approach on GDP- and GTP-bound forms of two Rab GTPases, showing detection of differential candidate interactors of conformationally similar proteins. This approach is applicable to screen for structure-specific interactomes of any protein, including posttranslationally modified and unmodified, or metabolite-bound and unbound protein states.

Seneca valley virus 3C protease blocks EphA2-Mediated mTOR activation to facilitate viral replication.

The Seneca Valley virus (SVV) is a recently discovered porcine pathogen that causes vesicular diseases and poses a significant threat to the pig industry worldwide. Erythropoietin-producing hepatoma receptor A2 (EphA2) is involved in the activation of the AKT/mTOR signaling pathway, which is involved in autophagy. However, the regulatory relationship between SVV and EphA2 remains unclear. In this study, we demonstrated that EphA2 is proteolysed in SVV-infected BHK-21 and PK-15 cells. Overexpression of EphA2 significantly inhibited SVV replication, as evidenced by decreased viral protein expression, viral titers, and viral load, suggesting an antiviral function of EphA2. Subsequently, viral proteins involved in the proteolysis of EphA2 were screened, and the SVV 3C protease (3Cpro) was found to be responsible for this cleavage, depending on its protease activity. However, the protease activity sites of 3Cpro did not affect the interactions between 3Cpro and EphA2. We further determined that EphA2 overexpression inhibited autophagy by activating the mTOR pathway and suppressing SVV replication. Taken together, these results indicate that SVV 3Cpro targets EphA2 for cleavage to impair its EphA2-mediated antiviral activity and emphasize the potential of the molecular interactions involved in developing antiviral strategies against SVV infection.

Effects of Pulsed Electric Field on Meat Tenderization and Microbial Decontamination: A Review.

This review sought to categorize studies on meat tenderization and safety through pulsed electric field (PEF) treatment, with a particular focus on reconciling conflicting findings regarding the tenderization effect (i.e., the primary outcome of PEF treatment) and to discuss the underlying mechanisms of these effects. While the tenderization effect may vary depending on the homogeneity of PEF treatment and variations in the conditions of texture measurements, the protein associated with tenderization was degraded by PEF treatment in most studies. PEF technology enables the delivery of a high voltage for a brief duration, typically in the microsecond range, making it a non-thermal technology. One of the distinct advantages of PEF is its ability to preserve the freshness of meat due to its exceptionally short treatment time. While PEF studies have traditionally centered on pasteurizing liquid foods, research on its application to meat is steadily expanding. Therefore, this review aims to elucidate the mechanisms of PEF and provide current insights into the applications of this technology for meat tenderization and microbial inactivation.

Mechanistic insights into CrCEP1: A dual-function cysteine protease with endo- and transpeptidase activity.

Proteases, essential regulators of plant stress responses, remain enigmatic in their precise functional roles. By employing activity-based probes for real-time monitoring, this study aimed to delve into protease activities in Chlamydomonas reinhardtii exposed to oxidative stress induced by hydrogen peroxide. However, our work revealed that the activity-based probes strongly labelled three non-proteolytic proteins-PsbO, PsbP, and PsbQ-integral components of photosystem II's oxygen-evolving complex. Subsequent biochemical assays and mass spectrometry experiments revealed the involvement of CrCEP1, a previously uncharacterized papain-like cysteine protease, as the catalyst of this labelling reaction. Further experiments with recombinant CrCEP1 and PsbO proteins replicated the reaction in vitro. Our data unveiled that endopeptidase CrCEP1 also has transpeptidase activity, ligating probes and peptides to the N-termini of Psb proteins, thereby expanding the repertoire of its enzymatic activities. The hitherto unknown transpeptidase activity of CrCEP1, working in conjunction with its proteolytic activity, unveils putative complex and versatile roles for proteases in cellular processes during stress responses.

SPRING is a Dedicated Licensing Factor for SREBP-Specific Activation by S1P.

SREBP transcription factors are central regulators of lipid metabolism. Their proteolytic activation requires ER to the Golgi translocation and subsequent cleavage by site-1-protease (S1P). Produced as a proprotein, S1P undergoes autocatalytic cleavage from its precursor S1PA to mature S1PC form. Here, we report that SPRING (previously C12ORF29) and S1P interact through their ectodomains, and that this facilitates the autocatalytic cleavage of S1PA into its mature S1PC form. Reciprocally, we identified a S1P recognition-motif in SPRING and demonstrate that S1P-mediated cleavage leads to secretion of the SPRING ectodomain in cells, and in liver-specific Spring knockout (LKO) mice transduced with AAV-mSpring. By reconstituting SPRING variants into SPRINGKO cells we show that the SPRING ectodomain supports proteolytic maturation of S1P and SREBP signaling, but that S1P-mediated SPRING cleavage is not essential for these processes. Absence of SPRING modestly diminishes proteolytic maturation of S1PA→C and trafficking of S1PC to the Golgi. However, despite reaching the Golgi in SPRINGKO cells, S1PC fails to rescue SREBP signaling. Remarkably, whereas SREBP signaling was severely attenuated in SPRINGKO cells and LKO mice, that of ATF6, another S1P substrate, was unaffected in these models. Collectively, our study positions SPRING as a dedicated licensing factor for SREBP-specific activation by S1P.

A review on structure-function mechanism and signaling pathway of serine/threonine protein PIM kinases as a therapeutic target.

The proviral integration for the Moloney murine leukemia virus (PIM) kinases, belonging to serine/threonine kinase family, have been found to be overexpressed in various types of cancers, such as prostate, breast, colon, endometrial, gastric, and pancreatic cancer. The three isoforms PIM kinases i.e., PIM1, PIM2, and PIM3 share a high degree of sequence and structural similarity and phosphorylate substrates controlling tumorigenic phenotypes like proliferation and cell survival. Targeting short-lived PIM kinases presents an intriguing strategy as in vivo knock-down studies result in non-lethal phenotypes, indicating that clinical inhibition of PIM might have fewer adverse effects. The ATP binding site (hinge region) possesses distinctive attributes, which led to the development of novel small molecule scaffolds that target either one or all three PIM isoforms. Machine learning and structure-based approaches have been at the forefront of developing novel and effective chemical therapeutics against PIM in preclinical and clinical settings, and none have yet received approval for cancer treatment. The stability of PIM isoforms is maintained by PIM kinase activity, which leads to resistance against PIM inhibitors and chemotherapy; thus, to overcome such effects, PIM proteolysis targeting chimeras (PROTACs) are now being developed that specifically degrade PIM proteins. In this review, we recapitulate an overview of the oncogenic functions of PIM kinases, their structure, function, and crucial signaling network in different types of cancer, and the potential of pharmacological small-molecule inhibitors. Further, our comprehensive review also provides valuable insights for developing novel antitumor drugs that specifically target PIM kinases in the future. In conclusion, we provide insights into the benefits of degrading PIM kinases as opposed to blocking their catalytic activity to address the oncogenic potential of PIM kinases.

Synthetic lethality: targeting the SMARCA2 bromodomain for degradation in SMARCA4-deficient tumors - a review of patent literature from 2019-June 2023.

INTRODUCTION: SMARCA2 and SMARCA4 are subunits of the SWI/SNF complex which is a chromatin remodeling complex and a key epigenetic regulator that facilitates gene expression. Tumors with loss of function mutations in SMARCA4 rely on SMARCA2 for cell survival and this synthetic lethality is a potential therapeutic strategy to treat cancer. AREAS COVERED: The current review focuses on patent applications that claim proteolysis-targeting chimeras (PROTAC) degraders that bind the bromodomain site of SMARCA2 and are published between January 2019-June 2023. A total of 29 applications from 9 different applicants were evaluated. EXPERT OPINION: SMARCA2/4 bromodomain inhibitors do not lead to desired effects on cancer proliferation; however, companies have converted bromodomain binders into PROTACs to degrade the protein, with a preference for SMARCA2 over SMARCA4. Selective degradation of SMARCA2 is most likely required to be efficacious in the SMARCA4-deficient setting, while allowing for sufficient safety margin in normal tissues. With several patent applications disclosed recently, interest in targeting SMARCA2 should continue, especially with a selective SMARCA2 PROTAC now in the clinic from Prelude Therapeutics. The outcome of the clinical trials will influence the evolution of selective SMARCA2 PROTACs development.

Myostatin gene invalidation does not prevent skeletal muscle mass loss during experimental sepsis in mice.

Loss of muscle mass and function induced by sepsis contributes to physical inactivity and disability in intensive care unit patients. Limiting skeletal muscle deconditioning may thus be helpful in reducing the long-term effect of muscle wasting in patients. We tested the hypothesis that invalidation of the myostatin gene, which encodes a powerful negative regulator of skeletal muscle mass, could prevent or attenuate skeletal muscle wasting and improve survival of septic mice. Sepsis was induced by caecal ligature and puncture (CLP) in 13-week-old C57BL/6J wild-type and myostatin knock-out male mice. Survival rates were similar in wild-type and myostatin knock-out mice seven days after CLP. Loss in muscle mass was also similar in wild-type and myostatin knock-out mice 4 and 7 days after CLP. The loss in muscle mass was molecularly supported by an increase in the transcript level of E3-ubiquitin ligases and autophagy-lysosome markers. This transcriptional response was blunted in myostatin knock-out mice. No change was observed in the protein level of markers of the anabolic insulin/IGF1-Akt-mTOR pathway. Muscle strength was similarly decreased in wild-type and myostatin knock-out mice 4 and 7 days after CLP. This was associated with a modified expression of genes involved in ion homeostasis and excitation-contraction coupling, suggesting that a long-term functional recovery following experimental sepsis may be impaired by a dysregulated expression of molecular determinants of ion homeostasis and excitation-contraction coupling. In conclusion, myostatin gene invalidation does not provide any benefit in preventing skeletal muscle mass loss and strength in response to experimental sepsis. KEY POINTS: Survival rates are similar in wild-type and myostatin knock-out mice seven days after the induction of sepsis. Loss in muscle mass and muscle strength are similar in wild-type and myostatin knock-out mice 4 and 7 days after the induction of an experimental sepsis. Despite evidence of a transcriptional regulation, the protein level of markers of the anabolic insulin/IGF1-Akt-mTOR pathway remained unchanged. RT-qPCR analysis of autophagy-lysosome pathway markers indicates that activity of the pathway may be altered by experimental sepsis in wild-type and myostatin knock-out mice. Experimental sepsis induces greater variations in the mRNA levels of wild-type mice than those of myostatin knock-out mice, without providing any significant catabolic resistance or functional benefits.

An evaluation of the effectiveness of sumac and molasses as additives for alfalfa silage: Influence on nutrient composition, in vitro degradability and fermentation quality.

This study investigated the effects of sumac and molasses on nutrient composition, in vitro degradability and fermentation quality of alfalfa silage. Alfalfa was ensiled in quadruplicate in vacuum jars untreated group (A) or after the following treatments: sumac group at 10% (AS), molasses group at 5% (AM), and sumac (10%) and molasses (5%) group (ASM). Silos (n = 64) were stored for 0, 21, 45 or 60 days. The results showed that dry matter (DM) contents of the AS, AM and ASM groups were statistically higher than the control group (p < 0.001). Only on the 21st day of fermentation the crude ash content of the AS group was found to be significantly higher than the other groups (p < 0.05). In vitro, DM and organic matter degradation values of the AMS group increased significantly (p < 0.001). A significant decrease in alfalfa silage's pH values was determined with sumac and molasses additives (p < 0.001). The ammonia nitrogen (NH3-N) values of the control, AS, AM and ASM groups at Day 60 were determined as 9.08%, 7.22%, 7.00% and 6.81% respectively (p < 0.05). The water-soluble carbohydrate (WSC) values of all groups on the 60th day were significantly decreased compared to the 0th day (p < 0.001). When the groups were evaluated within themselves, there was a statistically significant difference between the 0th and 60th day lactic acid values. The acetic acid content of the A group on the 60th day was found to be significantly higher than the other groups (p < 0.01). There was a significant decrease in propionic acid levels on Days 21, 45 and 60 compared to Day 0 of fermentation (p < 0.001). The highest butyric acid (BA) level was determined in the A group on the 21st, 45th and 60th days of fermentation (p < 0.05). In conclusion, sumac prevents proteolysis depending on its tannin content. It improves silage fermentation positively thanks to its organic acid content, while the molasses additive is effective in silage fermentation, mainly depending on the WSC level. However, it was determined that neither additive could reduce the silage pH to the appropriate value ranges due to the low doses, and they could not mainly prevent the formation of BA.

MMP-9-dependent proteolysis of the histone H3 N-terminal tail: a critical epigenetic step in driving oncogenic transcription and colon tumorigenesis.

Matrix metalloproteinase 9 (MMP-9) is a member of the MMP family and has been recently identified as a nuclear protease capable of clipping histone H3 N-terminal tails (H3NT). This MMP-9-dependent H3NT proteolysis is critical for establishing an active state of gene transcription during osteoclast differentiation and melanoma development. However, whether H3NT cleavage by MMP-9 plays a similar role in other cellular events has not been explored. Here, we dissect the functional contribution of MMP-9-dependent H3NT clipping to colonic tumorigenesis by using a combination of genome-wide transcriptome data, ChIP/ChIPac-qPCR, CRISPR/dCas9 gene-targeting system, and in vivo xenograft models. We show that MMP-9 is overexpressed in colon cancer cells and catalyzes H3NT proteolysis to drive transcriptional activation of growth stimulatory genes. Our studies using knockdown and inhibition approaches clearly indicate that MMP-9 mediates transcriptional activation and promotes colonic tumorigenesis in a manner dependent on its protease activity toward H3NT. Remarkably, artificial H3NT proteolysis at target gene promoters with dCAS9-MMP-9 is sufficient for establishing their transcriptional competence in colon cancer cells, underscoring the importance of MMP-9-dependent H3NT proteolysis per se in the transactivation process. Our data establish new functions and mechanisms for MMP-9 in driving the oncogenic transcription program in colon cancer through H3NT proteolysis, and demonstrate how this epigenetic pathway can be exploited as a potential therapeutic target for cancer treatment.

EGFR degraders in non-small-cell lung cancer: Breakthrough and unresolved issue.

The epidermal growth factor receptor (EGFR) has been well validated as a therapeutic target for anticancer drug discovery. Osimertinib has become the first globally accessible third-generation EGFR inhibitor, representing one of the most advanced developments in non-small-cell lung cancer (NSCLC) therapy. However, a tertiary Cys797 to Ser797 (C797S) point mutation has hampered osimertinib treatment in patients with advanced EGFR-mutated NSCLC. Several classes of fourth-generation EGFR inhibitors were consequently discovered with the aim of overcoming the EGFRC797S mutation-mediated resistance. However, no clinical efficacy data of the fourth-generation EGFR inhibitors were reported to date, and EGFRC797S mutation-mediated resistance remains an "unmet clinical need." Proteolysis-targeting chimeric molecules (PROTACs) obtained from EGFR-TKIs have been developed to target drug resistance EGFR in NSCLC. Some PROTACs are from nature products. These degraders compared with EGFR inhibitors showed better efficiency in their cellular potency, inhibition, and toxicity profiles. In this review, we first introduce the structural properties of EGFR, the resistance, and mutations of EGFR, and then mainly focus on the recent advances of EGFR-targeting degraders along with its advantages and outstanding challenges.

A fully validated LC­MS/MS method for quantifying bevacizumab in plasma samples from patients with NSCLC and its implications in therapeutic drug monitoring.

Given the increasing use of bevacizumab in combinatorial drug therapy for a multitude of different cancer types, there is a need for therapeutic drug monitoring to analyze the possible correlation between drug trough concentration, and therapeutic effect and adverse reactions. An ultra-performance liquid chromatography tandem-mass spectrometry method was then developed and validated to determine bevacizumab levels in human plasma samples. Chromatographic separation was achieved on a Shimadzu InertSustainBio C18 HP column, whereas subsequent mass spectrometric analysis was performed using a Shimadzu 8050CL triple quadrupole mass spectrometer equipped with an electro-spray ionization source in the positive ion mode. In total, three multiple reaction monitoring transitions of each of the surrogate peptides were chosen with 'FTFSLDTSK' applied as the quantification peptide whereas 'VLIYFTSSLHSGVPSR' and 'STAYLQMNSLR' were designated as the verification peptides using the Skyline software. This analytical method was then fully validated, with specificity, linearity, lower limit of quantitation, accuracy, precision, stability, matrix effect and recovery calculated. The linearity of this method was developed to be within the concentration range 5-400 µg/ml for bevacizumab in human plasma. Subsequently, eight patients with non-small cell lung cancer (NSCLC) were recruited and injected with bevacizumab over three periods of treatment to analyze their steady-state trough concentration and differences. To conclude, the results of the present study suggest that bevacizumab can be monitored in a therapeutic setting in patients with NSCLC.