Discovery of a first-in-class protein degrader for the c-ros oncogene 1 (ROS1).

The c-ros oncogene 1 (ROS1), an oncogenic driver, is known to induce non-small cell lung cancer (NSCLC) when overactivated, particularly through the formation of fusion proteins. Traditional targeted therapies focus on inhibiting ROS1 activity with ROS 1 inhibitors to manage cancer progression. However, a new strategy involving the design of protein degraders offers a more potent approach by completely degrading ROS1 fusion oncoproteins, thereby effectively blocking their kinase activity and enhancing anti-tumour potential. Utilizing PROteolysis-TArgeting Chimera (PROTAC) technology and informed by molecular docking and rational design, we report the first ROS1-specific PROTAC, SIAIS039. This degrader effectively targets multiple ROS1 fusion oncoproteins (CD74-ROS1, SDC4-ROS1 and SLC34A2-ROS1) in engineered Ba/F3 cells and HCC78 cells, demonstrating anti-tumour effects against ROS1 fusion-driven cancer cells. It suppresses cell proliferation, induces cell cycle arrest, and apoptosis, and inhibits clonogenicity. The anti-tumour efficacy of SIAIS039 surpasses two approved drugs, crizotinib and entrectinib, and matches that of the top inhibitors, including lorlatinib and taletrectinib. Mechanistic studies confirm that the degradation induced by 039 requires the participation of ROS1 ligands and E3 ubiquitin ligases, and involves the proteasome and ubiquitination. In addition, 039 exhibited excellent oral bioavailability in a mouse xenograft model, highlighting its potential for clinical application. In conclusion, our study presents a promising and novel therapeutic strategy for ROS1 fusion-positive NSCLC by targeting ROS1 fusion oncoproteins for degradation, laying the foundation for the development of further PROTAC and offering hope for patients with ROS1 fusion-positive NSCLC.

Identification of Proteolysis Targeting Chimeras (PROTACs) for Lysine Demethylase 5 and Their Neurite Outgrowth-Promoting Activity.

Lysine demethylase 5 (KDM5) proteins are involved in various neurological disorders, including Alzheimer's disease, and KDM5 inhibition is expected to be a therapeutic strategy for these diseases. However, the pharmacological effects of conventional KDM5 inhibitors are insufficient, as they only target the catalytic functionality of KDM5. To identify compounds that exhibit more potent pharmacological activity, we focused on proteolysis targeting chimeras (PROTACs), which degrade target proteins and thus inhibit their entire functionality. We designed and synthesized novel KDM5 PROTAC candidates based on previously identified KDM5 inhibitors. The results of cellular assays revealed that two compounds, 20b and 23b, exhibited significant neurite outgrowth-promoting activity through the degradation of KDM5A in neuroblastoma neuro 2a cells. These results suggest that KDM5 PROTACs are promising drug candidates for the treatment of neurological disorders.

Reproductive chemical database: a curated database of chemicals that modulate protein targets regulating important reproductive biological processes.

Recent studies have shifted the spotlight from adult disease to gametogenesis and embryo developmental events, and these are greatly affected by various environmental chemicals, such as drugs, metabolites, pollutants, and others. Growing research has highlighted the critical importance of identifying and understanding the roles of chemicals in reproductive biology. However, the functions and mechanisms of chemicals in reproductive processes remain incomplete. We developed a comprehensive database called the Reproductive Chemical Database (RCDB) ( https://yu.life.sjtu.edu.cn/ChenLab/RCDB ) to facilitate research on chemicals in reproductive biology. This resource is founded on rigorous manual literature extraction and precise protein target prediction methodologies. This database focuses on the delineation of chemicals associated with phenotypes, diseases, or endpoints intricately associated with four important reproductive processes: female and male gamete generation, fertilization, and embryo development in human and mouse. The RCDB encompasses 93 sub-GO processes, and it revealed 1447 intricate chemical-biological process interactions. To date, the RCDB has meticulously cataloged and annotated 830 distinct chemicals, while also predicting 614 target proteins from a selection of 3800 potential candidates. Additionally, the RCDB offers an online predictive tool that empowers researchers to ascertain whether specific chemicals play discernible functional roles in these reproductive processes. The RCDB is an exhaustive, cross-platform, manually curated database, which provides a user-friendly interface to search, browse, and use reproductive processes modulators and their comprehensive related information. The RCDB will help researchers to understand the whole reproductive process and related diseases and it has the potential to promote reproduction research in the pharmacological and pathophysiological areas.

Profibrotic Inflammatory Cytokines and Growth Factors Are Predicted as the Key Targets of Uncaria gambir (Hunter) Roxb. in Keloids: An Epistatic and Molecular Simulation Approach.

Keloid is characterized as the fibrotic tissue resulting from the increase of fibroblast activity. Uncaria gambir (Hunter) Roxb. possesses bioactive compounds that have potential as antifibrotic agents, while the mechanism of action in keloid has not yet been elucidated. The aim of this study was to investigate the interaction of gambir bioactive compounds with keloid target proteins using an epistatic and molecular simulation approach. The known bioactive compounds of gambir targets and keloid-related protein targets were screened using databases. The network was constructed and analyzed to obtain the core protein targets. The targets were enriched to describe the Gene Ontology (GO) and pathway related to the proteins. Eleven targets were defined as the main targets of gambir bioactive compounds related to keloid disease. Gambiriin C, Isogambirine, and Procyanidin B1 were identified as the most promising compounds with the highest binding energy to transforming growth factor beta 1 (TGFß1), AKT serine/threonine kinase 1 (AKT1), and matrix metallopeptidase 1 (MMP1) as the target proteins. GO enrichment and pathway analysis found that gambir bioactive compounds may act on keloid-related target proteins to regulate cell proliferation, migration, transcription, and signal transduction activity via profibrotic cytokine and growth factor signaling pathways. This study provides a reference for potential targets, compounds, and pathways to explain the mechanism of gambir against keloid.

Trauma diagnostic-related target proteins and their detection techniques.

Trauma is a significant health issue that not only leads to immediate death in many cases but also causes severe complications, such as sepsis, thrombosis, haemorrhage, acute respiratory distress syndrome and traumatic brain injury, among trauma patients. Target protein identification technology is a vital technique in the field of biomedical research, enabling the study of biomolecular interactions, drug discovery and disease treatment. It plays a crucial role in identifying key protein targets associated with specific diseases or biological processes, facilitating further research, drug design and the development of treatment strategies. The application of target protein technology in biomarker detection enables the timely identification of newly emerging infections and complications in trauma patients, facilitating expeditious medical interventions and leading to reduced post-trauma mortality rates and improved patient prognoses. This review provides an overview of the current applications of target protein identification technology in trauma-related complications and provides a brief overview of the current target protein identification technology, with the aim of reducing post-trauma mortality, improving diagnostic efficiency and prognostic outcomes for patients.

Deubiquitylase USP7 plays a crucial role in the lineage differentiation of preimplantation blastocysts.

Preimplantation embryos undergo a series of important biological events, including epigenetic reprogramming and lineage differentiation, and the key genes and specific mechanisms that regulate these events are critical to reproductive success. USP7 is a deubiquitinase involved in the regulation of a variety of cellular functions, yet its precise function and mechanism in preimplantation embryonic development remain unknown. Our results showed that RNAi-mediated silencing of USP7 in mouse embryos or treatment with P5091, a small molecule inhibitor of USP7, significantly reduced blastocyst rate and blastocyst quality, and decreased total and TE cell numbers per blastocyst, as well as destroying normal lineage differentiation. The results of single-cell RNA-seq, RT-qPCR, western blot, and immunofluorescence staining indicated that interference with USP7 caused failure of the morula-to-blastocyst transition and was accompanied by abnormal expression of key genes (Cdx2, Oct4, Nanog, Sox2) for lineage differentiation, decreased transcript levels, increased global DNA methylation, elevated repressive histone marks (H3K27me3), and decreased active histone marks (H3K4me3 and H3K27ac). Notably, USP7 may regulate the transition from the morula to blastocyst by stabilizing the target protein YAP through the ubiquitin-proteasome pathway. In conclusion, our results suggest that USP7 may play a crucial role in preimplantation embryonic development by regulating lineage differentiation and key epigenetic modifications.

Recently reported cell migration inhibitors: Opportunities and challenges for antimetastatic agents.

Antimetastatic agents are highly desirable for cancer treatment because of the severe medical challenges and high mortality resulting from tumor metastasis. Having demonstrated antimetastatic effects in numerous in vitro and in vivo studies, migration inhibitors present significant opportunities for developing a new class of anticancer drugs. To provide a useful overview on the latest research in migration inhibitors, this article first discusses their therapeutic significance, targetable proteins, and developmental avenues. Subsequently it reviews over 20 representative migration inhibitors reported in recent journals in terms of their inhibitory mechanism, potency, and potential clinical utility. The relevance of the target proteins to cellular migratory function is focused on as it is crucial for assessing the overall efficacy of the inhibitors.

Design, Synthesis, and Target Identification of Novel Phenylalanine Derivatives by Drug Affinity Responsive Target Stability (DARTS) in Xanthomonas oryzae pv Oryzae.

The increasing resistance displayed by plant phytopathogenic bacteria to conventional pesticides has heightened the urgency for the exploration of novel antibacterial agents possessing distinct modes of action (MOAs). In this study, a series of novel phenylalanine derivatives with the unique structure of acylhydrazone dithioether have been designed and synthesized. Bioassay results demonstrated that most target compounds exhibited excellent in vitro antibacterial activity against Xanthomonas oryzae pv oryzae (Xoo) and Xanthomonas axonopodis pv citri (Xac). Among them, the EC50 values of L3, L4, L6, L21, and L22 against Xoo were 7.4, 9.3, 6.7, 8.9, and 5.1 µg/mL, respectively, superior to that of bismerthiazol (BT) and thiodiazole copper (TC) (41.5 and >100 µg/mL); the EC50 values of L3, L4, L5, L6, L7, L8, L20, L21, and L22 against Xac were 5.6, 2.5, 6.2, 4.1, 4.2, 6.4, 6.3, 3.6, and 5.2 µg/mL, respectively, superior to that of BT and TC (43.3 and >100 µg/mL). An unmodified drug affinity responsive target stability (DARTS) technology was used to investigate the antibacterial MOAs of active compound L22, and the 50S ribosomal protein L2 (RL2) as an unprecedented target protein in Xoo cells was first discovered. The target protein RL2 was then expressed and purified. Furthermore, the in vitro interactions by microscale thermophoresis (Kd = 0.050 µM) and fluorescence titration (Ka = 1.4 × 105 M-1) experiments also demonstrated a strong binding force between compound L22 and RL2. Overall, these results not only facilitate the development of novel antibacterial agents but also establish a reliable method for exploring the targets of bactericides.

Depolymerization of actin filaments by Cucurbitacin I through binding G-actin.

Cucurbitacins have high economic value as they are a major source of food and have pharmacological properties. Cucurbitacin I (CuI) is a plant-derived natural tetracyclic triterpenoid compound that shows an anticancer effect via inhibiting the JAK2-STAT3 signaling pathway. The actin cytoskeleton is the most abundant protein in cells and regulates critical events through reorganization in cells. In this study, it is aimed at determining the direct effect of CuI on actin dynamics. The fluorescence profile of G-actin in the presence of CuI (1-200 nM) shifted to a higher temperature, suggesting that G-actin binds CuI and that G-actin-CuI is more thermally stable than the ligand-free form. CuI dose-dependently inhibited the polymerization of F-actin in vitro and disrupted actin filaments in endothelial cells. Docking and MD simulations suggested that CuI binds to the binding site formed by residues I136, I175, D154, and A138 that are at the interface of monomers in F-actin. The migration ability of cells treated with CuI for 24 h was significantly lower than the control group (p < .001). This study reveals the molecular mechanisms of CuI in the regulation of actin dynamics by binding G-actin. More importantly, this study indicates a novel role of CuI as an actin-targeting drug by binding directly to G-actin and may contribute to the mode of action of CuI on anticancer activities.

Targeted Protein Degradation Systems: Controlling Protein Stability Using E3 Ubiquitin Ligases in Eukaryotic Species.

This review explores various methods for modulating protein stability to achieve target protein degradation, which is a crucial aspect in the study of biological processes and drug design. Thirty years have passed since the introduction of heat-inducible degron cells utilizing the N-end rule, and methods for controlling protein stability using the ubiquitin-proteasome system have moved from academia to industry. This review covers protein stability control methods, from the early days to recent advancements, and discusses the evolution of techniques in this field. This review also addresses the challenges and future directions of protein stability control techniques by tracing their development from the inception of protein stability control methods to the present day.

Design, Synthesis, and Antitumor Activity Evaluation of Artemisinin Bivalent Ligands.

Five artemisinin bivalent ligands molecules 4a-4e were designed, synthesized, and confirmed by 1H NMR, 13C NMR, and low-resolution mass spectrometry, and the bioactivities of the target compounds were investigated against four human tumor cell lines in vitro, including BGC-823, HepG-2, MCF-7, and HCT-116. The results showed 4a, 4d, and 4e exhibited significantly tumor cell inhibitory activity compared with the artemisinin and dihydroartemisinin; compound 4e has good biological activity inhibiting BGC-823 with an IC50 value of 8.30 µmol/L. Then, the good correlations with biological results were validated by molecular docking through the established bivalent ligands multi-target model, which showed that 4e could bind well with the antitumor protein MMP-9.

Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Isorhamnetin Regulates Programmed Death Ligand-1 Expression by Suppressing the EGFR-STAT3 Signaling Pathway in Canine Mammary Tumors.

Programmed death ligand-1 (PD-L1) is highly expressed in a variety of cancer cells and suggests a poorer prognosis for patients. The natural compound isorhamnetin (ISO) shows promise in treating cancers and causing damage to canine mammary tumor (CMT) cells. We investigated the mechanism of ISO in reducing PD-L1 expression in CMT cells. Clustered, regularly interspaced short palindromic repeat-associated protein 9 (CRISPR/Cas9) was used to mediate CD274 knockout in U27 cells. Then, monoclonal cells were screened and cultured. Nucleotide sequencing and expression of PD-L1 were detected. Additionally, we examined cell migration, invasion, and damage. Immunofluorescent staining of PD-L1 was examined in U27 cells. The signaling pathways were measured by Western blotting. Murine xenotransplantation models and murine immunocompetent allograft mammary tumor models were established to evaluate the effect of ISO therapy. Expression of Ki-67, caspase3, and PD-L1 were analyzed by immunohistochemistry. A pull-down assay was used to explore which proteins could bind to ISO. Canine EGFR protein was purified and used to detect whether it directly binds to ISO using a surface plasmon resonance assay. ISO inhibited the EGFR-STAT3-PD-L1 signaling pathway and blocked cancer growth, significantly increasing the survival rate of healthy cells. The cell membrane receptor EGFR was identified as a direct target of ISO. ISO could be exploited as an antineoplastic treatment of CMT by targeting EGFR to suppress PD-L1 expression.

Unveiling Vacuolar H+-ATPase Subunit a as the Primary Target of the Pyridinylmethyl-Benzamide Fungicide, Fluopicolide.

An estimated 240 fungicides are presently in use, but the direct targets for the majority remain elusive, constraining fungicide development and efficient resistance monitoring. In this study, we found that Pcα-actinin knockout did not influence the sensitivity of Phytophthora capsici to fluopicolide, which is a notable oomycete inhibitor. Using a combination of Bulk Segregant Analysis Sequencing and Drug Affinity Responsive Target Stability (DARTS) assays, the vacuolar H+-ATPase subunit a (PcVHA-a) was pinpointed as the target protein of fluopicolide. We also confirmed four distinct point mutations in PcVHA-a responsible for fluopicolide resistance in P. capsici through site-directed mutagenesis. Molecular docking, ATPase activity assays, and a DARTS assay suggested a fluopicolide-PcVHA-a interaction. Sequence analysis and further molecular docking validated the specificity of fluopicolide for oomycetes or fish. These findings support the claim that PcVHA-a is the target of fluopicolide, proposing vacuolar H+-ATPase as a promising target for novel fungicide development.

Effect of Faeces Bombycis Extract on Cajal Interstitial Cells in Diabetic Gastroparesis Rats Based on PI3K/Akt/mTOR Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the therapeutic mechanism of Faeces Bombycis on diabetic gastroparesis （DGP） rats based on phosphatidylinositol 3-kinase/protein kinase B/mammalian rapamycin target protein （PI3K/Akt/mTOR） signaling pathway. MethodDGP rat model was prepared by random selection of 15 out of 105 rats as blank group. The rats successfully constructed were randomly divided into model group， high-，medium- and low- dose groups （3.2， 1.6， 0.8 g·kg-1） and moxapride group （1.5 mg·kg-1）， with 12 rats in each group， and were given gavage for 4 weeks. The gastric emptying rate and random blood glucose were measured. The morphological changes of gastric antrum were observed by hematoxylin-eosin （HE） staining， and the expression of the c-Kit gene was analyzed by immunohistochemistry. The apoptosis of Cajal interstitial cells was observed by in situ end labeling （TUNEL） staining， and the protein expressions of PI3K， phosphorylation（p）-PI3K， Akt， p-Akt， mTOR， and p-mTOR were detected by Western blot. ResultCompared with the blank group， the gastric emptying rate of the model group decreased significantly （P<0.01）， and the glandular structure of the gastric antrum was destroyed. The expression of c-Kit decreased （P<0.01）， and the apoptosis of Cajal interstitial cells （ICC） increased. Compared with the model group， the gastric emptying rate in the high， middle， and low-dose groups of Faeces Bombycis extract and mosapride group increased significantly （P<0.01）. The glandular structure of the gastric antrum became closer， and the apoptosis of ICC decreased. The expression of c-Kit in the high dose group of Faeces Bombycis extract increased significantly. After Western blot testing， compared with the blank group， the protein expression of p-Akt/Akt， p-PI3K/PI3K， and p-mTOR/mTOR in the model group increased. Compared with the model group， the protein expression of p-Akt/Akt in the high dose group of Faeces Bombycis extract decreased （P<0.01）， and the protein expression of p-PI3K/PI3K decreased in the middle and low dose groups of Faeces Bombycis extract and mosapride group decreased （P<0.05， P<0.01）. The protein expression of p-mTOR/mTOR decreased in the low dose group of Faeces Bombycis extract （P<0.05）. In terms of random blood glucose， compared with the blank group， the random blood glucose in the model group increased significantly （P<0.01）， and compared with the model group， the random blood glucose in the high and middle dose groups of Faeces Bombycis extract decreased significantly （P<0.05）. Compared with mosapride group， the protein expression of p-Akt/Akt decreased in the high dose group of Faeces Bombycis extract （P<0.05）， and the protein expression of p-PI3K/PI3K increased in the high， middle， and low dose groups of Faeces Bombycis extract （P<0.05， P<0.01）. ConclusionFaeces Bombycis extract can increase gastric emptying rate， reduce ICC apoptosis， and lower random blood glucose in DGP rats. The high dose group of Faeces Bombycis extract has a significant effect on inhibiting ICC apoptosis， and its mechanism may be related to the regulation of the PI3K/Akt/mTOR signaling pathway.

Unveiling poly(rC)-binding protein 2 as the target protein for curcusone C against prostate cancer: mechanism validation through click chemistry-activity based proteomics profiling approach.

BACKGROUND: Prostate cancer is a disease that seriously troubles men. However, there are some inevitable limitations in interventional therapy for prostate cancer patients at present, most of which are caused by low selectivity and high toxic side effects due to unclear drug targets. In this study, we identified the target protein of Curcusone C with anti-prostate cancer potential activity and verified its target and mechanism of action. METHODS: Click chemistry-activity based proteomics profiling (CC-ABPP) method was used to find target protein of Curcusone C against prostate cancer. Competitive CC-ABPP, drug affinity responsive target stability (DARTS) and surface plasmon resonance (SPR) methods were used to verifying the target protein. Moreover, potential mechanism was validated by western blot in vitro and by hematoxylin-eosin (HE) staining, detection of apoptosis in tumor tissue (TUNEL), and immunohistochemical (IHC) in vivo. RESULTS: We found that poly(rC)-binding protein 2 (PCBP2) was the target protein of Curcusone C. In addition, Curcusone C might disrupt the Bax/Bcl-2 balance in PC-3 cells by inhibiting the expression of the target protein PCBP2, thereby inducing mitochondrial damage and activation of the mitochondrial apoptosis pathway, and ultimately inducing apoptosis of prostate cancer cells. CONCLUSIONS: Curcusone C is a potential compound with anti-prostate cancer activity, and this effect occurs by targeting the PCBP2 protein, which in turn may affect the TGF/Smad signaling pathway and Bax/Bcl-2 balance. Our results laid a material and theoretical foundation for Curcusone C, to be widely used in anti-prostate cancer.

[Advances in the preclinical and clinical research of proteolysis targeting chimera].

Proteolysis targeting chimera (PROTAC) refers to heterobifunctional small molecules that can simultaneously bind an E3 ubiquitin ligase and a target protein, enabling specific degradation of the target protein with the aid of the ubiquitin proteasome system. At present, most PROTAC drugs are in the clinical trial stage, and the ligands are mainly non-covalent compounds. PROTAC drugs have the advantage of overcoming drug resistance and degrading "undruggable" target proteins, but non-covalent ligands could lead to the hook effect that undermines drug efficacy. With its own advantages, covalent ligands can avoid the occurrence of this phenomenon, which is of great help to the development of PROTAC. This review summarizes the progress in preclinical and clinical research and application of PROTAC molecules targeting three different classes of protein targets, including intranuclear, transmembrane, and cytosolic proteins. We also offer perspective discussions to provide research ideas and references for the future development of PROTAC.

Discovery of a SHP2 Degrader with In Vivo Anti-Tumor Activity.

Src homology 2 domain-containing phosphatase 2 (SHP2) is an attractive target for cancer therapy due to its multifaceted roles in both tumor and immune cells. Herein, we designed and synthesized a novel series of proteolysis targeting chimeras (PROTACs) using a SHP2 allosteric inhibitor as warhead, with the goal of achieving SHP2 degradation both inside the cell and in vivo. Among these molecules, compound P9 induces efficient degradation of SHP2 (DC50 = 35.2 ± 1.5 nM) in a concentration- and time-dependent manner. Mechanistic investigation illustrates that the P9-mediated SHP2 degradation requires the recruitment of the E3 ligase and is ubiquitination- and proteasome-dependent. P9 shows improved anti-tumor activity in a number of cancer cell lines over its parent allosteric inhibitor. Importantly, administration of P9 leads to a nearly complete tumor regression in a xenograft mouse model, as a result of robust SHP2 depletion and suppression of phospho-ERK1/2 in the tumor. Hence, P9 represents the first SHP2 PROTAC molecule with excellent in vivo efficacy. It is anticipated that P9 could serve not only as a new chemical tool to interrogate SHP2 biology but also as a starting point for the development of novel therapeutics targeting SHP2.

RIPTACs: A groundbreaking approach to drug discovery.

Regulated induced proximity targeting chimeras (RIPTACs), a new class of heterobifunctional molecules, show promise in specifically targeting and eliminating cancer cells while leaving healthy cells unharmed. As a groundbreaking drug discovery approach, RIPTACs work by forming a stable complex with two proteins, one specifically found in cancer cells (target protein, TP) and the other pan-essential for cell survival (effector protein, EP), selectively disrupting the function of the EP in cancer cells and causing cell death. Interestingly, the TPs need not be linked to disease progression, broadening the spectrum of potential drug targets. This review summarizes the discovery and recent advances of the RIPTAC strategy. Additionally, it discusses the associated opportunities and challenges as well as future perspectives in this field.

Identifying therapeutic monoclonal antibodies using target protein collision electrophoresis reflex assay to separate the wheat from the chaff.

Monoclonal gammopathies are characterized by the presence of monoclonal immunoglobulins, also known as M-proteins. Therapeutic monoclonal antibodies (t-mAbs) can interfere in laboratory assays used to monitor the state of disease, such as serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). To establish a correct interpretation of IFE, Target protein-Collision Immunofixation Electrophoresis Reflex Assay (T-CIERA) was developed to identify t-mAbs in IFE. Here we demonstrate that T-CIERA is applicable to a wide variety of t-mAbs for which the target protein is commercially available. Moreover, the shift observed was characteristic for each t-mAb, and T-CIERA enabled the identification of multiple t-mAbs sharing a common target protein. Additionally, the lower limit of detection (LLOD) was determined objectively, and T-CIERA demonstrated an adequate LLOD for all tested t-mAbs. Furthermore, T-CIERA was also successfully applied to serum samples obtained from patients receiving daratumumab, isatuximab, elotuzumab, and durvalumab treatment. In conclusion, T-CIERA is a suitable reflex assay for identifying a wide variety of t-mAbs, including those for which no commercial assay is available to deal with their interference. Moreover, CD38-CIERA could serve as an alternative or complementary test to the commercially available Hydrashift assay kits. T-CIERA would enable laboratories without mass spectrometry equipment and expertise in this area to distinguish between drug and disease to improve clinical response monitoring and diagnosis of monoclonal gammopathies.