Target For Drug Development Research Articles

Discovery of First-in-Class PROTAC Degraders of SARS-CoV-2 Main Protease.

We have witnessed three coronavirus (CoV) outbreaks in the past two decades, including the COVID-19 pandemic caused by SARS-CoV-2. Main protease (MPro), a highly conserved protease among various CoVs, is essential for viral replication and pathogenesis, making it a prime target for antiviral drug development. Here, we leverage proteolysis targeting chimera (PROTAC) technology to develop a new class of small-molecule antivirals that induce the degradation of SARS-CoV-2 MPro. Among them, MPD2 was demonstrated to effectively reduce MPro protein levels in 293T cells, relying on a time-dependent, CRBN-mediated, and proteasome-driven mechanism. Furthermore, MPD2 exhibited remarkable efficacy in diminishing MPro protein levels in SARS-CoV-2-infected A549-ACE2 cells. MPD2 also displayed potent antiviral activity against various SARS-CoV-2 strains and exhibited enhanced potency against nirmatrelvir-resistant viruses. Overall, this proof-of-concept study highlights the potential of targeted protein degradation of MPro as an innovative approach for developing antivirals that could fight against drug-resistant viral variants.

Targeted Therapy for Highly Desmoplastic and Immunosuppressive Tumor Microenvironment of Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with a very poor prognosis. Despite advancements in treatment strategies, PDAC remains recalcitrant to therapies because patients are often diagnosed at an advanced stage. The advanced stage of PDAC is characterized by metastasis, which typically renders it unresectable by surgery or untreatable by chemotherapy. The tumor microenvironment (TME) of PDAC comprises highly proliferative myofibroblast-like cells and hosts the intense deposition of a extracellular matrix component that forms dense fibrous connective tissue, a process called the desmoplastic reaction. In desmoplastic TMEs, the incessant aberration of signaling pathways contributes to immunosuppression by suppressing antitumor immunity. This feature offers a protective barrier that impedes the targeted delivery of drugs. In addition, the efficacy of immunotherapy is compromised because of the immune cold TME of PDAC. Targeted therapy approaches towards stromal and immunosuppressive TMEs are challenging. In this review, we discuss cellular and non-cellular TME components that contain actionable targets for drug development. We also highlight findings from preclinical studies and provide updates about the efficacies of new investigational drugs in clinical trials.

A comprehensive survey on deep learning-based identification and predicting the interaction mechanism of long non-coding RNAs.

Long noncoding RNAs (lncRNAs) have been discovered to be extensively involved in eukaryotic epigenetic, transcriptional, and post-transcriptional regulatory processes with the advancements in sequencing technology and genomics research. Therefore, they play crucial roles in the body's normal physiology and various disease outcomes. Presently, numerous unknown lncRNA sequencing data require exploration. Establishing deep learning-based prediction models for lncRNAs provides valuable insights for researchers, substantially reducing time and costs associated with trial and error and facilitating the disease-relevant lncRNA identification for prognosis analysis and targeted drug development as the era of artificial intelligence progresses. However, most lncRNA-related researchers lack awareness of the latest advancements in deep learning models and model selection and application in functional research on lncRNAs. Thus, we elucidate the concept of deep learning models, explore several prevalent deep learning algorithms and their data preferences, conduct a comprehensive review of recent literature studies with exemplary predictive performance over the past 5years in conjunction with diverse prediction functions, critically analyze and discuss the merits and limitations of current deep learning models and solutions, while also proposing prospects based on cutting-edge advancements in lncRNA research.

The Anti-proliferative Effects of Ficus carica Latex on cancer and normal cell

The stems of unripe fig fruits or leaves contain a white liquid called fig latex. Fig latex contains compounds that are effective against cancer cells. It is used for various purposes in folk medicine and has been proven to have superior pharmacological properties in modern studies. The industrial value of latex from a cosmetic perspective is increasing as well. In the current study, the cytotoxic effect of fig latex obtained from Ficus carica was evaluated by MTT analysis on lung cancer (A549), breast cancer (MCF-7), triple-negative breast cancer (MDA-MB-231), and non-tumorogenic lung (MRC-5) cell lines. All cell lines were treated with various dilutions of fig latex ranging from 1/1500 to 1/12 for 48 hours. In cancer cells, it induced cell death in a concentration-dependent manner, and high doses caused a more serious cytotoxic effect. On the other hand, it showed less cytotoxicity on healthy cells compared to cancer cells. Fig latex IC50 values were found to be 1/26, 1/40, 1/45, and 1/7 for A549, MCF-7, MDA-MB-231, and MRC-5 cells, respectively. Based on our findings, fig latex showed selective cytotoxicity against cancer cells, making it a possible target for future cancer drug development. However, further research is needed to fully understand the potential use of fig latex and its bioactive compounds in cancer treatment in terms of their functional properties.

Therapeutic Advances and Challenges for the Management of HPV-Associated Oropharyngeal Cancer.

The use of conventional chemotherapy in conjunction with targeted and immunotherapy drugs has emerged as an option to limit the severity of side effects in patients diagnosed with head and neck cancer (HNC), particularly oropharyngeal cancer (OPC). OPC prevalence has increased exponentially in the past 30 years due to the prevalence of human papillomavirus (HPV) infection. This study reports a comprehensive review of clinical trials registered in public databases and reported in the literature (PubMed/Medline, Scopus, and ISI web of science databases). Of the 55 clinical trials identified, the majority (83.3%) were conducted after 2015, of which 77.7% were performed in the United States alone. Eight drugs have been approved by the FDA for HNC, including both generic and commercial forms: bleomycin sulfate, cetuximab (Erbitux), docetaxel (Taxotere), hydroxyurea (Hydrea), pembrolizumab (Keytruda), loqtorzi (Toripalimab-tpzi), methotrexate sodium (Trexall), and nivolumab (Opdivo). The most common drugs to treat HPV-associated OPC under these clinical trials and implemented as well for HPV-negative HNC include cisplatin, nivolumab, cetuximab, paclitaxel, pembrolizumab, 5-fluorouracil, and docetaxel. Few studies have highlighted the necessity for new drugs specifically tailored to patients with HPV-associated OPC, where molecular mechanisms and clinical prognosis are distinct from HPV-negative tumors. In this context, we identified most mutated genes found in HPV-associated OPC that can represent potential targets for drug development. These include TP53, PIK3CA, PTEN, NOTCH1, RB1, FAT1, FBXW7, HRAS, KRAS, and CDKN2A.

A Retrospective Review on Dysregulated Autophagy in Polycystic Ovary Syndrome: From Pathogenesis to Therapeutic Strategies.

The main purpose of this article is to explore the relationship between autophagy and the pathological mechanism of PCOS, and to find potential therapeutic methods that can alleviate the pathological mechanism of PCOS by targeting autophagy. Relevant literatures were searched in the following databases, including: PubMed, MEDLINE, Web of Science, Scopus. The search terms were "autophagy", "PCOS", "polycystic ovary syndrome", "ovulation", "hyperandrogenemia", "insulin resistance", "inflammatory state", "circadian rhythm" and "treatment", which were combined according to the retrieval methods of different databases. Through analysis, we uncovered that abnormal levels of autophagy were closely related to abnormal ovulation, insulin resistance, hyperandrogenemia, and low-grade inflammation in patients with PCOS. Lifestyle intervention, melatonin, vitamin D, and probiotics, etc. were able to improve the pathological mechanism of PCOS via targeting autophagy. In conclusion, autophagy disorder is a key pathological mechanism in PCOS and is also a potential target for drug development and design.

Integrated bioinformatics analysis identified leucine rich repeat containing 15 and secreted phosphoprotein 1 as hub genes for calcific aortic valve disease and osteoarthritis.

Calcific aortic valve disease (CAVD) and osteoarthritis (OA) are common diseases in the ageing population and share similar pathogenesis, especially in inflammation. This study aims to discover potential diagnostic and therapeutic targets in patients with CAVD and OA. Three CAVD datasets and one OA dataset were obtained from the Gene Expression Omnibus database. We used bioinformatics methods to search for key genes and immune infiltration, and established a ceRNA network. Immunohistochemical staining was performed to verify the expression of candidate genes in human and mice aortic valve tissues. Two key genes obtained, leucine rich repeat containing 15 (LRRC15) and secreted phosphoprotein 1 (SPP1), were further screened using machine learning and verified in human and mice aortic valve tissues. Compared to normal tissues, the infiltration of immune cells in CAVD tissues was significantly higher, and the expressions of LRRC15 and SPP1 were positively correlated with immune cells infiltration. Moreover, the ceRNA network showed extensive regulatory interactions based on LRRC15 and SPP1. The authors' findings identified LRRC15 and SPP1 as hub genes in immunological mechanisms during CAVD and OA initiation and progression, as well as potential targets for drug development.

Nephro-protective effects of alpha-lipoic acid in type I diabetes.

Type 1 diabetes (T1D) is an insulin-dependent autoimmune condition. Short chain fatty acids (SCFAs) are volatile fatty acids with 1-6 carbon atoms that influence glucose storage in the body and can reduce appetite, potentially decreasing T1D risk. Alpha-lipoic acid (α-LA), a type of SCFA, has previously been used to treat diabetic neuropathy and inflammation due to its antioxidant properties. This study aims to assess α-LA's protective effects against T1D and associated kidney damage in rats induced with streptozotocin. Diabetic rats were treated with α-LA orally for 15 days, resulting in improved blood glucose (56% decrease) and kidney function markers like blood urea nitrogen, creatinine and uric acid. α-LA also showed significant antioxidant effects by decreasing LPO as well as improving activities of antioxidant enzymes like superoxide dismutase, catalase and glutathione-S transferase and alleviated kidney damage caused by diabetes. Docking experiments suggest that α-LA may regulate diabetes-related changes at the epigenetic level through interactions with the SIRT1 protein, indicating its potential as a target for future antidiabetic drug development.

Targeting EFHD2 inhibits interferon-γ signaling and ameliorates non-alcoholic steatohepatitis

The precise pathomechanisms underlying the development of non-alcoholic steatohepatitis (NASH, also known as metabolic dysfunction-associated steatohepatitis [MASH]) remain incompletely understood. In this study, we investigated the potential role of EF-hand domain family member D2 (EFHD2), a novel molecule specific to immune cells, in the pathogenesis of NASH. Hepatic EFHD2 expression was characterized in patients with NASH and two diet-induced NASH mouse models. Single-cell RNA sequencing (scRNA-seq) and double-immunohistochemistry were employed to explore EFHD2 expression patterns in NASH livers. The effects of global and myeloid-specific EFHD2 deletion on NASH and NASH-related hepatocellular carcinoma were assessed. Molecular mechanisms underlying EFHD2 function were investigated, while chemical and genetic investigations were performed to assess its potential as a therapeutic target. EFHD2 expression was significantly elevated in hepatic macrophages/monocytes in both patients with NASH and mice. Deletion of EFHD2, either globally or specifically in myeloid cells, improved hepatic steatosis, reduced immune cell infiltration, inhibited lipid peroxidation-induced ferroptosis, and attenuated fibrosis in NASH. Additionally, it hindered the development of NASH-related hepatocellular carcinoma. Specifically, deletion of myeloid EFHD2 prevented the replacement of TIM4+ resident Kupffer cells by infiltrated monocytes and reversed the decreases in patrolling monocytes and CD4+/CD8+ T cell ratio in NASH. Mechanistically, our investigation revealed that EFHD2 in myeloid cells interacts with cytosolic YWHAZ (14-3-3ζ), facilitating the translocation of IFNγR2 (interferon-γ receptor-2) onto the plasma membrane. This interaction mediates interferon-γ signaling, which triggers immune and inflammatory responses in macrophages during NASH. Finally, a novel stapled α-helical peptide targeting EFHD2 was shown to be effective in protecting against NASH pathology in mice. Our study reveals a pivotal immunomodulatory and inflammatory role of EFHD2 in NASH, underscoring EFHD2 as a promising druggable target for NASH treatment. Non-alcoholic steatohepatitis (NASH) represents an advanced stage of non-alcoholic fatty liver disease (NAFLD); however, not all patients with NAFLD progress to NASH. A key challenge is identifying the factors that trigger inflammation, which propels the transition from simple fatty liver to NASH. Our research pinpointed EFHD2 as a pivotal driver of NASH, orchestrating the over-activation of interferon-γ signaling within the liver during NASH progression. A stapled peptide designed to target EFHD2 exhibited therapeutic promise in NASH mice. These findings support the potential of EFHD2 as a therapeutic target in NASH.

A Comprehensive Review on Potential Molecular Drug Targets for the Management of Alzheimer's Disease.

Alzheimer's disease (AD) is an onset and incurable neurodegenerative disorder that has been linked to various genetic, environmental, and lifestyle factors. Recent research has revealed several potential targets for drug development, such as the prevention of Aβ production and removal, prevention of tau hyperphosphorylation, and keeping neurons alive. Drugs that target numerous ADrelated variables have been developed, and early results are encouraging. This review provides a concise map of the different receptor signaling pathways associated with Alzheimer's Disease, as well as insight into drug design based on these pathways. It discusses the molecular mechanisms of AD pathogenesis, such as oxidative stress, aging, Aβ turnover, thiol groups, and mitochondrial activities, and their role in the disease. It also reviews the potential drug targets, in vivo active agents, and docking studies done in AD and provides prospects for future drug development. This review intends to provide more clarity on the molecular processes that occur in Alzheimer's patient's brains, which can be of use in diagnosing and preventing the condition.

A novel antibiotic 3-isopropylhexahydro-4H-pyrido[1,2-α]pyrazine-1,4(6H)-dione isolated from the thermophilic bacterium Bacillus licheniformis QX928

A microbial strain designated Bacillus licheniformis QX928 was screened from hot springs in Sichuan Province, China, and a compound generated in the culture of this strain clearly inhibited Pseudomonas aeruginosa ATCC27853. The measured minimum inhibitory and lowest bactericidal concentrations were (13±0.17) and (22±0.72) mg L-1, respectively. The compound was identified as 3-isopropylhexahydro-4H-pyrido[1,2-α]pyrazine-1,4(6H)-dione (IPHPPD). A SciFinder search revealed that IPHPPD could be the first compound synthesized by microorganisms that had both antibacterial and anti-quorum sensing properties. At low concentrations, IPHPPD interfered with the signaling factors and population effects of P. aeruginosa, thereby altering the biofilm morphology and structure. IPHPPD more strongly inhibited P. aeruginosa at high concentrations, primarily by reducing its virulence factors, cell membrane permeability and energy metabolism. A transcriptome analysis highlighted the role of IPHPPD in the transcriptional regulation of cellular metabolism and quorum sensing. Thus, the results of this study provide critical evidence that IPHPPD is a potential target for drug development to prevent and treat diseases in animals.

Targeting Moonlighting Enzymes in Cancer.

Moonlighting enzymes are multifunctional proteins that perform multiple functions beyond their primary role as catalytic enzymes. Extensive research and clinical practice have demonstrated their pivotal roles in the development and progression of cancer, making them promising targets for drug development. This article delves into multiple notable moonlighting enzymes, including GSK-3, GAPDH, and ENO1, and with a particular emphasis on an enigmatic phosphatase, PTP4A3. We scrutinize their distinct roles in cancer and the mechanisms that dictate their ability to switch roles. Lastly, we discuss the potential of an innovative approach to develop drugs targeting these moonlighting enzymes: target protein degradation. This strategy holds promise for effectively tackling moonlighting enzymes in the context of cancer therapy.

Centrosomes and associated proteins in pathogenesis and treatment of breast cancer.

Breast cancer is the most prevalent malignancy among women worldwide. Despite significant advances in treatment, it remains one of the leading causes of female mortality. The inability to effectively treat advanced and/or treatment-resistant breast cancer demonstrates the need to develop novel treatment strategies and targeted therapies. Centrosomes and their associated proteins have been shown to play key roles in the pathogenesis of breast cancer and thus represent promising targets for drug and biomarker development. Centrosomes are fundamental cellular structures in the mammalian cell that are responsible for error-free execution of cell division. Centrosome amplification and aberrant expression of its associated proteins such as Polo-like kinases (PLKs), Aurora kinases (AURKs) and Cyclin-dependent kinases (CDKs) have been observed in various cancers, including breast cancer. These aberrations in breast cancer are thought to cause improper chromosomal segregation during mitosis, leading to chromosomal instability and uncontrolled cell division, allowing cancer cells to acquire new genetic changes that result in evasion of cell death and the promotion of tumor formation. Various chemical compounds developed against PLKs and AURKs have shown meaningful antitumorigenic effects in breast cancer cells in vitro and in vivo. The mechanism of action of these inhibitors is likely related to exacerbation of numerical genomic instability, such as aneuploidy or polyploidy. Furthermore, growing evidence demonstrates enhanced antitumorigenic effects when inhibitors specific to centrosome-associated proteins are used in combination with either radiation or chemotherapy drugs in breast cancer. This review focuses on the current knowledge regarding the roles of centrosome and centrosome-associated proteins in breast cancer pathogenesis and their utility as novel targets for breast cancer treatment.

TRPM5 activation depends on a synergistic effect of calcium and PKC phosphorylation

Transient receptor potential melastatin 5 (TRPM5) is a calcium-activated monovalent-specific ion channel involved in insulin secretion and taste transduction, making it an attractive target for drug development in various pathologies. While TRPM5 activation involves ligand binding to Gq/G-protein coupled receptors (GPCR) and subsequent elevation of intracellular calcium levels, recent reports suggest the need for additional molecular determinants. Hence, the mechanism of TRPM5 activation remains to be elucidated. Here, we show that PKC phosphorylation and the elevation of intracellular Ca2+ levels are required for TRPM5 activation, with PKC phosphorylation being crucial for channel-evoked currents, primarily at physiological membrane potentials. In contrast, physiological relevant calcium levels alone only induce TRPM5 activation at positive voltages. Our findings highlight the necessity of coordinated intracellular calcium release and PKC phosphorylation for TRPM5 activation. Thus, our results suggest that regulation of PKC activity could be a promising therapeutic target for diseases associated with TRPM5 modulation.

Integrated analysis of scRNA-seq and bulk RNA-seq identifies FBXO2 as a candidate biomarker associated with chemoresistance in HGSOC

BackgroundHigh-grade serous ovarian carcinoma (HGSOC) is the most prevalent and aggressive histological subtype of epithelial ovarian cancer. Around 80% of individuals will experience a recurrence within five years because of resistance to chemotherapy, despite initially responding well to platinum-based treatment. Biomarkers associated with chemoresistance are desperately needed in clinical practice. MethodsWe jointly analyzed the transcriptomic profiles of single-cell and bulk datasets of HGSOC to identify cell types associated with chemoresistance. Copy number variation (CNV) inference was performed to identify malignant cells. We subsequently analyzed the expression of candidate biomarkers and their relationship with patients' prognosis. The enrichment analysis and potential biological function of candidate biomarkers were explored. Then, we validated the candidate biomarker using in vitro experiments. ResultsWe identified 8871 malignant epithelial cells in a single-cell RNA sequencing dataset, of which 861 cells were associated with chemoresistance. Among these malignant epithelial cells, FBXO2 (F-box protein 2) is highly expressed in cells related to chemoresistance. Moreover, FBXO2 expression was found to be higher in epithelial cells from chemoresistance samples compared to those from chemosensitivity samples in a separate single-cell RNA sequencing dataset. Patients exhibiting elevated levels of FBXO2 experienced poorer outcomes in terms of both overall survival (OS) and progression-free survival (PFS). FBXO2 could impact chemoresistance by influencing the PI3K-Akt signaling pathway, focal adhesion, and ECM-receptor interactions and regulating tumorigenesis. The 50% maximum inhibitory concentration (IC50) of cisplatin decreased in A2780 and SKOV3 ovarian carcinoma cell lines with silenced FBXO2 during an in vitro experiment. ConclusionsWe determined that FBXO2 is a potential biomarker linked to chemoresistance in HGSOC by combining single-cell RNA-seq and bulk RNA-seq dataset. Our results suggest that FBXO2 could serve as a valuable prognostic marker and potential target for drug development in HGSOC.

Unraveling the peculiarities and development of novel inhibitors of leishmanial arginyl-tRNA synthetase.

Aminoacylation by tRNA synthetase is a crucial part of protein synthesis and is widely recognized as a therapeutic target for drug development. Unlike the arginyl-tRNA synthetases (ArgRSs) reported previously, here, we report an ArgRS of Leishmania donovani (LdArgRS) that can follow the canonical two-step aminoacylation process. Since a previously uncharacterized insertion region is present within its catalytic domain, we implemented the splicing by overlap extension PCR (SOE-PCR) method to create a deletion mutant (ΔIns-LdArgRS) devoid of this region to investigate its function. Notably, the purified LdArgRS and ΔIns-LdArgRS exhibited different oligomeric states along with variations in their enzymatic activity. The full-length protein showed better catalytic efficiency than ΔIns-LdArgRS, and the insertion region was identified as the tRNA binding domain. In addition, a benzothiazolo-coumarin derivative (Comp-7j) possessing high pharmacokinetic properties was recognized as a competitive and more specific inhibitor of LdArgRS than its human counterpart. Removal of the insertion region altered the mode of inhibition for ΔIns-LdArgRS and caused a reduction in the inhibitor's binding affinity. Both purified proteins depicted variances in the secondary structural content upon ligand binding and thus, thermostability. Apart from the trypanosomatid-specific insertion and Rossmann fold motif, LdArgRS revealed typical structural characteristics of ArgRSs, and Comp-7j was found to bind within the ATP binding pocket. Furthermore, the placement of tRNAArg near the insertion region enhanced the stability and compactness of LdArgRS compared to other ligands. This study thus reports a unique ArgRS with respect to catalytic as well as structural properties, which can be considered a plausible drug target for the derivation of novel anti-leishmanial agents.

Unlocking potential diabetes therapeutics: Insights into alpha-glucosidase inhibition

Diabetes mellitus (DM) stands as a significant global health challenge, ranking third among causes of mortality worldwide. Despite extensive endeavors, effective treatments for diabetes remain elusive. Alpha-glucosidase, a pivotal enzyme in glycoprotein metabolism regulation, emerges as a promising therapeutic target for mitigating postprandial hyperglycemia, a hallmark of diabetes. In this study, we employed advanced computational methodologies to identify inhibitors of lysosomal α-glucosidase. Leveraging pharmacophore-based virtual screening of a vast library of drug-like molecules, we unearthed 161 potential inhibitors capable of binding within the enzyme's active pocket. Subsequent molecular docking simulations yielded five lead molecules exhibiting robust binding affinities and favorable interaction profiles with critical catalytic residues. Further analysis through molecular dynamics simulations provided crucial insights into the stability and conformational dynamics of the inhibitor-enzyme complexes. Notably, certain residues emerged as pivotal for the retention of inhibitors within the active site. Our findings underscore the potential of LIG1-LIG5 as promising inhibitors against lysosomal α-glucosidase, presenting novel avenues for the development of effective diabetes therapeutics. This research enriches our understanding of the molecular underpinnings of diabetes treatment, facilitating targeted drug development strategies in the quest for improved patient outcomes.

Computational design and in vitro assay of lantadene-based novel inhibitors of NS3 protease of dengue virus

Abstract Dengue virus (DENV) infection is one of the diseases for which no drug is available for the treatment. The DENV NS2B-NS3 protease is considered to be the prime target for anti-dengue drug development because of its importance in the development of new virus subunits via DENV poly-protein breakdown. Pentacyclic triterpenoids (Lantadenes) from the weed Lantana camara L. and its semi-synthetic congeners have shown a wide array of biological activities in the last two decades. The virtual screening strategy was used on the library of 78 natural and semi-synthetic lantadenes to predict the potent antagonists for the NS2B-NS3 protease enzyme of DENV and their experimental validation by in vitro assay of lead molecules. In the in silico analysis of 78 triterpenoids, two lead molecules (−10.60 and −9.93 kcal/mol) were predicted to be inhibitors of protease (viral) when compared to its reference ligand 1,8-dihydroxy-4,5-dinitroanthraquinone (−5.377 kcal/mol). At the same time, binding affinity, pharmacokinetic, and toxicity profiling, along with molecular dynamics simulations, were studied. The in vitro viral infection inhibition assay inferred that lead molecule 62 exhibited a 60% and 45% reduction in DENV titers at 10 and 5 µM concentrations, respectively. The lead molecule 62 can further be optimized for its pharmacophore and has the potential to be developed as a drug-like molecule.

Molecular mechanism of muscarinic acetylcholine receptor M3 interaction with Gq

Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets due to their involvement in physiopathological processes. Although the structure of the M3-miniGq complex was recently published, the lack of information on the intracellular loop 3 (ICL3) of M3 and extensive modification of Gαq impedes the elucidation of the molecular mechanism of M3-Gq coupling under more physiological condition. Here, we describe the molecular mechanism underlying the dynamic interactions between full-length wild-type M3 and Gq using hydrogen-deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cell systems. We propose a detailed analysis of M3-Gq coupling through examination of previously well-defined binding interfaces and neglected regions. Our findings suggest potential binding interfaces between M3 and Gq in pre-assembled and functionally active complexes. Furthermore, M3 ICL3 negatively affected M3-Gq coupling, and the Gαq AHD underwent unique conformational changes during M3-Gq coupling.

Abstract 22: Development and characterization of human tumor specific cytotoxic lymphocytes for preclinical drug development

Abstract Heterogeneity within a tumor allows it to better adapt to changing conditions and can have therefore a negative impact on a patient’s prognosis, response to treatment and overall health. Breast cancer is a highly heterogeneous disease caused by distinct genetic mutations in mammary epithelial cells, resulting in significantly different forms of the disease and a need for highly personalized treatment schemes for patients. Hence, it is important to develop and improve (immuno)therapies for breast cancer patients. Adoptive T-cell therapy with autologous CD8+ cytotoxic T cell lymphocytes (CTL) is a promising method that has already shown promising effects in melanoma patients. Unfortunately, there is almost no information about the use in breast cancer patients. We developed an in vitro protocol to generate tumor-specific CTLs by priming on HER2+ breast cancer cell lines JIMT-1 and SKBR-3. Subsequently, these primed CTLs were tested in 2D and 3D immune cell killing assays in a life cell imaging device in combination with an endpoint viability assay. The activity was compared with unspecific CD8+T cells activated by phytohemagglutinin (PHA) or αCD2/αCD3/αCD28, HER2-peptide specific T cells and HER2-CAR T cells serving as a positive control. We investigated the feasibility to freeze CTLs for use in subsequent studies and validated the relevance of HER2 as the main druggable target in the tumor lines using an siRNA approach. The CTLs were most effective on JIMT-1 in a 3D setting, whereas the SKBR-3 CTLs displayed similar activity in 2D and in 3D. The respective CTLs were tumor line specific, as SKBR-3 CTLs did not kill efficiently the JIMT-1 cells and vice versa. The same CTLs displayed the same efficacy pattern after thawing but less pronounced. Thus, within one experimental set-up it is recommended to use either one. The Her2 knockdown in the target cells reduced the killing activity of the CTLs slightly. However, this effect was not statistically significant proving the importance of CTLs as treatment option as well as neoantigens as targets in drug development. In summary, the CTLs proved to capture the respective tumor heterogeneity showing high killing rates in both SK-BR3 and JIMT-1 being more active than HER2-peptide specific T cells. The availability of CTLs in a preclinical setting facilitates screening approaches for combinational studies. Beyond that they can serve as starting material to develop cancer vaccines and identify highly immunogenic neoantigens. Citation Format: Ina Rohleff, Lea Gröne, Kanstantsin Lashuk, Julia B. Schueler. Development and characterization of human tumor specific cytotoxic lymphocytes for preclinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 22.