Human Target Research Articles

Addressing Cardiovascular Toxicity Risk of Electronic Nicotine Delivery Systems in the Twenty-First Century: "What Are the Tools Needed for the Job?" and "Do We Have Them?"

Cigarette smoking is positively and robustly associated with cardiovascular disease (CVD), including hypertension, atherosclerosis, cardiac arrhythmias, stroke, thromboembolism, myocardial infarctions, and heart failure. However, after more than a decade of ENDS presence in the U.S. marketplace, uncertainty persists regarding the long-term health consequences of ENDS use for CVD. New approach methods (NAMs) in the field of toxicology are being developed to enhance rapid prediction of human health hazards. Recent technical advances can now consider impact of biological factors such as sex and race/ethnicity, permitting application of NAMs findings to health equity and environmental justice issues. This has been the case for hazard assessments of drugs and environmental chemicals in areas such as cardiovascular, respiratory, and developmental toxicity. Despite these advances, a shortage of widely accepted methodologies to predict the impact of ENDS use on human health slows the application of regulatory oversight and the protection of public health. Minimizing the time between the emergence of risk (e.g., ENDS use) and the administration of well-founded regulatory policy requires thoughtful consideration of the currently available sources of data, their applicability to the prediction of health outcomes, and whether these available data streams are enough to support an actionable decision. This challenge forms the basis of this white paper on how best to reveal potential toxicities of ENDS use in the human cardiovascular system-a primary target of conventional tobacco smoking. We identify current approaches used to evaluate the impacts of tobacco on cardiovascular health, in particular emerging techniques that replace, reduce, and refine slower and more costly animal models with NAMs platforms that can be applied to tobacco regulatory science. The limitations of these emerging platforms are addressed, and systems biology approaches to close the knowledge gap between traditional models and NAMs are proposed. It is hoped that these suggestions and their adoption within the greater scientific community will result in fresh data streams that will support and enhance the scientific evaluation and subsequent decision-making of tobacco regulatory agencies worldwide.

Toxicological Effects of Naturally Occurring Endocrine Disruptors on Various Human Health Targets: A Rapid Review.

Endocrine-disrupting compounds are chemicals that alter the normal functioning of the endocrine system of living organisms. They can be natural (N-EDCs) or synthetic compounds (S-EDCs). N-EDCs can belong to different groups, such as phytoestrogens (PEs), including flavonoids, or mycotoxins originating from plants or fungi, and cyanotoxins, derived from bacteria. Humans encounter these substances in their daily lives. The aim of this rapid review (RR) is to provide a fine mapping of N-EDCs and their toxicological effects on human health in terms of various medical conditions or adverse consequences. This work is based on an extensive literature search and follows a rigorous step-by-step approach (search strategy, analysis strategy and data extraction), to select eligible papers published between 2019 and 2023 in the PubMed database, and to define a set of aspects characterizing N-EDCs and the different human target systems. Of the N-EDCs identified in this RR, flavonoids are the most representative class. Male and female reproductive systems were the targets most affected by N-EDCs, followed by the endocrine, nervous, bone and cardiovascular systems. In addition, the perinatal, pubertal and pregnancy periods were found to be particularly susceptible to natural endocrine disruptors. Considering their current daily use, more toxicological research on N-EDCs is required.

Abstract 1892: KK2260: A fully human tetravalent bispecific antibody targeting TfR1 and EGFR for the treatment of EGFR-expressing tumors

Abstract Background: TfR1 is a transmembrane glycoprotein involved in iron uptake and regulation of cell growth. High TfR1 expression has been reported in different types of cancers, therefore recognized as a target molecule for cancer therapy. Despite the promise of potent efficacy of TfR1-targeted agents, there is a risk of side effects (i.e., severe anemia), since TfR1 is also highly expressed in certain normal cells (e.g., reticulocytes). KK2260 is a fully human tetravalent bispecific antibody (bsAb) based on REGULGENTTM technology targeting TfR1 and EGFR. This bsAb was designed for EGFR-expressing cell selective anti-cancer drug which could not be achieved by conventional monospecific Ab. In this study, we evaluated the mechanism of action (MOA), anti-cancer effect, and toxicity of KK2260. Methods: Binding activity of KK2260 to TfR1 and EGFR was evaluated by surface plasmon resonance method. Ligand blocking activity was evaluated by binding competition on esophageal squamous cell carcinoma (ESCC) OE21 cells. Growth inhibitory activity of KK2260 on cancer cells or hematopoietic progenitor cells was evaluated by proliferation inhibition assay. In some studies, excessive iron or anti-EGFR parent Ab was added. Effect on the cell surface target expression levels was evaluated by flow cytometry. The intracellular iron level was evaluated by cell imaging. Effect on EGF induced EGFR phosphorylation was evaluated by Western blotting. The in vivo efficacy of KK2260 was evaluated in cell line-derived and patient-derived xenograft (PDX) models of ESCC selected for high EGFR expression level. The toxicity of KK2260 was evaluated in cynomolgus monkeys. Results: KK2260 bound to TfR1 and EGFR with no ligand binding inhibition. KK2260 inhibited cell growth of moderate to high EGFR-expressing cancer cells, but not against EGFR-negative cancer cells or hematopoietic progenitor cells. KK2260 downregulated TfR1 expression on OE21 cells and the intracellular iron level decreased, but no effect was observed on EGFR phosphorylation. The growth inhibitory activity of KK2260 was abolished by adding excessive amounts of iron or reduced by inhibiting the binding to EGFR. Potent anti-tumor activity was observed in the ESCC PDX models with tumor regression. In the intermittent-dose toxicity study of cynomolgus monkeys, KK2260 was well-tolerated. Conclusions: KK2260 demonstrated potent anti-cancer effect both in vitro and in vivo. Intriguingly, KK2260 did not inhibit EGFR phosphorylation while EGFR-binding dependent TfR1 downregulation resulted in intracellular iron deficiency, which was considered as the MOA of KK2260. KK2260 was well tolerated in non-human primates. These preclinical data suggest the unique property of KK2260 is expected to provide significant benefit to patients with advanced ESCC and other EGFR-expressing tumors and Phase 1 study is currently on going in Japan (jRCT2031230372). Citation Format: Keisuke Mitamura, Hironori Otsuki, Keiko Nakahashi, Ayaka Kitagawa, Masayo Kamigaki, Yohei Kurio, Miho Inoue, Yasunori Uemura. KK2260: A fully human tetravalent bispecific antibody targeting TfR1 and EGFR for the treatment of EGFR-expressing tumors [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 1892.

Abstract 7495: Validation of microfluidic isolation of circulating pancreatic tumor cells from blood

Abstract Background. Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis and is the fourth leading cause of cancer-related deaths in the US. PDAC is diagnosed in the late stages, which limits treatment options and yields poor clinical outcomes. Recurrence is a significant issue for patients undergoing surgery for PDAC. Circulating tumor cells (CTCs) are thought to be a potential mediator of recurrence, facilitating the formation of secondary tumor sites after surgery. Thus, there is a need to isolate CTCs to improve understanding of disease recurrence, to ultimately improve outcomes. Herein, we aim to validate our inertial microfluidic (iMF) system for isolating pancreatic CTCs from blood. We also benchmark our iMF system to a commercially available immunomagnetic negative selection system, EasySep™, and quantify target cell recovery and cell enrichment. Methods. Our iMF system employs the differential cell sizes of CTCs from blood cells and inertial migration of such cells in a straight microchannel (150 µm × 50 µm × 24 mm) to enrich CTCs from blood. The iMF system sample flow rate used is 100 µL/min, with a 200 µL/min PBS buffer flow rate. The EasySep™ Direct Human CTC Enrichment kit targets CD2, CD14, CD16, CD19, CD45, CD61, CD66b, and Glycophorin to remove blood cells from the sample using magnetic particles, thereby separating them from CTCs. We spiked low cell concentrations of 50, 100, and 500 Hoechst-stained pancreatic carcinoma cells, PANC1, in 1 mL of healthy blood then captured using either EasySep™ or iMF. Cell enumeration was performed after centrifugation or Cytospin. Results. PANC1 cells and WBCs have average sizes of 18 µm and 8.4 µm, respectively. We characterized our iMF device to capture cells ≥ 12 µm, which yields capture of most PANC1 cells. Indeed, our iMF system achieved 82.8±19.2%, 72.5±16.3%, and 59.3±3.8% recovery for 50, 100, and 500 cell-spikes in lysed blood, respectively. For the same 50, 100, and 500 cell-spikes, the total iMF CTC yields after Cytospin were 46.3±11.8, 44.3±9.1, and 32.7±2.1%, while the total CTC yields from EasySep™ were 4.6±3, 5.3±2.1, 8.3±1%, respectively. When we processed whole blood directly, EasySep™ had lower cell recovery, 3.8±0.2, 4.6±1, and 3.3±0.4%, respectively. Our system also demonstrated 8 × higher target cell enrichment compared to EasySep™. Blood lysis before iMF separation did not drastically affect the recovery of target cells. Conclusions. We determined that our system has improved performance over the commercial isolation system, EasySep™, thereby validating the effectiveness of microfluidic isolation for PDAC. In future studies, we can then use our iMF system in CTC isolation from routine blood draws from PDAC patients undergoing surgical resection. We may then isolate real patient samples and study relevant phenotypes of PDAC CTCs which may therefore be used for identifying certain mechanisms of PDAC progression. Citation Format: Celine Macaraniag, Ifra Khan, Jian Zhou, Pier Cristoforo Giulianotti, Alain Borgeat, Gina Votta-Velis, Ian Papautsky. Validation of microfluidic isolation of circulating pancreatic tumor cells from blood [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 7495.

Abstract 410: ASXL3 is a potential biomarker of response of SCLC to targeted therapies

Abstract Small Cell Lung Cancer (SCLC) has been designated a recalcitrant cancer and a priority by National Cancer Institute. Despite recent insights regarding mechanisms mediating stemness, heterogeneity, and metastatic potential, these advances have yet to be fully translated to effective regimens for the treatment of SCLC. Previously, we identified Additional Sex Combs Like-3 (ASXL3) as a novel mediator of pluripotency in human respiratory epithelia and potential therapeutic target in SCLC. ASXL3 is one of three members of the ASXL family, whereas the functions of ASXL1 and ASXL2 as components of the PR-DUB complex have been extensively studied, the functions and significance of ASXL3 during malignant transformation remain elusive. To further investigate the relevance of ASXL3 activation in SCLC, we initially studied ASXL3 expression levels in various SCLC subtypes by qRT-PCR, digital RT-PCR, RNA-seq, and western blotting analysis. These studies demonstrated that ASCL (A) and ASCL and NeuroD1 (A+N) subtypes of SCLC cells contain thousand-folds more ASXL3 (ASXL3high) than Pou2f3 (P) and YAP1 (Y) SCLC subtypes (ASXL3low). ASXL3 levels were elevated when cancer cells were cultured under stemness-enriched conditions. Under normal culture conditions, ASXL3high SCLC cells showed higher intrinsic DNA damage, evidenced by a high percentage of γ-H2AX positive cells compared to ASXL3low SCLC cells. Additionally, ASXL3high SCLC cells displayed 38% γ-H2AX positive metaphase cells, and 42% of abnormal chromosome segregations during mitosis, including lagging chromosomes, DNA bridges, and unequal chromosome segregation indicative of profound genome instability. One reason for this extensive genomic instability is that ATM pathway activation in response to DNA replication stress is impaired in ASXL3high SCLC cells. Following exposures to 1mM hydroxyurea (HU) for 4, 8, and 24 hours, ATM autophosphorylation levels in ASXL3high SCLC cells were much lower than in ASXL3low SCLC cells. This insufficiency significantly affected the activation of downstream cascade targets including CHK2, KAP-1 and EZH2. Consistent with these findings, ASXL3high SCLC cells showed reduced sensitivities to ATM inhibitors. However, ASXL3high SCLC cells exhibited higher activity of PLK1 and were more sensitive to hydroxyurea. Importantly, low dose HU reduced the IC50 values of the PLK1 inhibitors, BI2536 or NMS-P937 by 80-100-fold in ASXL3high but not ASXL3low SCLC cells, suggesting a strong synergy between these two drugs in ASXL3high SCLC cells. Collectively, these findings suggest that ASXL3 functions to protect cells from lethality induced by genomic instability through monitoring ATM activation. Experiments are underway using SCLC PDX models to demonstrate the efficacy of this combined treatment in-vivo as a prelude to possible evaluation of this drug combination in SCLC patients. Citation Format: Ruihong Wang, Haitao Wang, Xinwei Wu, Keerti Mishra, Stephanie Shiffka, Frank Hernandez, Mary Zhang, Umay Tuana Tolunay, Sudheer Gara, Sichuan Xi, Shamus R. Carr, Choung D. Hoang, David S. Schrump. ASXL3 is a potential biomarker of response of SCLC to targeted therapies [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 410.

Abstract 3545: Atlas of therapeutic targets: Evolution of druggable proteome

Abstract Selection of therapeutic targets and modalities is of fundamental importance in drug discovery, as it can have a direct impact on the economics of drug discovery as well as its success rate. With the advancement of chem-bio and proteomics methods together with advent of artificial intelligence, the scope of the druggable proteome is expanding. The choice of drug targets gains further importance as we link it to potential clinical efficacy and safety, genetic variation and patient populations. Drug targets are often selected based on inadequate understanding of target biology, tractability by existing toolbox of therapeutic modalities and insufficient insight into possible clinical outcome. We have developed an “Atlas of Therapeutic Targets”, an interactive resource that provides classification of drug target families with linked information on clinical and investigational drugs. The atlas also provides key insights and score for tractability of targets with various therapeutic modalities. Particularly, our scoring system grades the targets on dimensions such as ligandability, involvement in protein-protein complexes, structural disorder, hotspot analysis and cross-referencing with known targets, thus yielding a total tractability score of 1-10. Dissection of targets based on such dimensions, enables critical evaluation therapeutic modalities as well tools and methodologies needed for their conversion into successful drug discovery programs. We curate 900+ human drug targets, analysis of which indicates the continued dominance of privileged target families across disease areas, but also the growth of novel first-in-class mechanisms. This atlas will be available as a resource for the wide drug discovery community. Citation Format: Ivan Babic, Elmar Nurmemmedov. Atlas of therapeutic targets: Evolution of druggable proteome [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 3545.

Abstract 5766: Fungal genomes in drug discovery: Application of CETSA to study the mechanism of action of brefeldin A

Abstract Fungal genomes encode a vast collection of chemical diversity that has evolved over millions of years to engage specific targets and modulate diverse biological processes. These Genetically Encoded small Molecules (GEMs) constitute a largely untapped source of potential therapeutics. Although fungal GEMs such as cyclosporine, lovastatin, and penicillin have revolutionized modern medicine, these GEMs were discovered serendipitously through bioactivity-guided fractionation of fungal extracts. LifeMine is redefining GEM discovery through the deployment of proprietary genomic search algorithms that enable GEM target prediction a priori. Our strategy facilitates focused search-and-retrieval campaigns to rationally discover molecules selected by Nature to modulate the desired target protein. These GEM drug leads often exhibit unique structural and mechanistic features, necessitating comprehensive deconvolution of the human target profile and mechanism of action during the development process from GEM to clinical candidate. As a proof-of-concept study, we leveraged CETSA Explore to study the biological mechanism of the fungal polyketide lactone Brefeldin A (BFA) in human THP-1 cells. This GEM, originally isolated from the filamentous fungus Penicillium brefeldianis, inhibits protein trafficking between the endoplasmic reticulum and the Golgi complex, eliciting antiviral, antifungal, and anticancer activity. Our CETSA profiling study confirmed the Golgi-associated guanine nucleotide exchange factor GBF1 as the primary target of BFA, validating the established mechanism of this GEM as an inhibitor of GBF1-mediated activation of the small GTPase ARF1. These results demonstrate the utility of integrating the LifeMine GEM discovery engine with downstream proteomic profiling technologies to facilitate the mechanistic characterization of fungal GEMs en route to novel therapeutics. Citation Format: Tomas Friman, Benjamin M. Fontaine, Alexey Chernobrovkin, Daniel Martinez Molina, Victoria Brehmer, Stina Lundgren, Paola M. Castaldi, Gregory L. Verdine. Fungal genomes in drug discovery: Application of CETSA to study the mechanism of action of brefeldin A [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 5766.

Abstract 6658: TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy

Abstract Background: Oncolytic viruses, such as the chimeric vesicular stomatitis virus variant VSV-GP have dual modes of action, direct tumor-cell killing, and activation of tumor-specific immunity. While tumor cells deficient in type I interferon (IFN) response are permissive to VSV-GP replication and killing, IFN competent tumor cells are protected from VSV-GP-mediated oncolysis. Our previous data demonstrated that VSV-GP therapy turns immunologically ‘cold’ tumors ‘hot’ by increasing infiltration of T cells into the tumor. The aim of this study was to use the VSV-GP platform to locally express a T cell engager (TcE) molecule to redirect these T cells to destroy non-permissive IFN competent tumor cells expressing the target of the TcE on their cell surface. Methods: We selected EpCAM as the target antigen and used a bispecific EpCAM-TcE molecule consisting of two variable single chain fragments against EpCAM and CD3, respectively. VSV-GP encoding EpCAM-TcE (VSV-GP-EpCAM-TcE) or an invariant control TcE (VSV-GP-TNP-TcE) were generated as previously described. T cells were co-cultured with human target cells expressing EpCAM on their cell surface, either infected with the VSV-GP-EpCAM-TcE virus or with supernatant from VSV-GP-EpCAM-TcE infected cells. T cell activation, proliferation, degranulation and cytokine secretion were analyzed by flow cytometry and multiplex cytokine assays as a measure of TcE mediated cross-linking of T cells to their target on cancer cells. Patient-derived head and neck squamous cell carcinoma (HNSCC) tumors were sectioned into approximately 200 µM slices and infected with the virus. T cell activation was measured by cytokine secretion. The therapeutic potential of VSV-GP-EpCAM-TcE was tested in vivo in cell-line derived xenograft (CDX) models implanted in immunodeficient mice humanized with donor-derived PBMCs or CD34+ cells. Results: Virus fitness was maintained for VSV-GP encoding the TcE molecule. Using co-culture assays, we demonstrated that EpCAM-TcE molecule is successfully expressed by VSV-GP and can activate both CD4+ and CD8+ T cells resulting in proliferation, degranulation, and cytokine secretion. Furthermore, we validated the concept using patient-derived HNSCC tumor slices which are representative of complex human tumor ecosystems. We could confirm that cancer cells infected by VSV-GP-EpCAM-TcE virus express functional TcE molecule and activates T cells to secrete IFNγ and TNFα. Finally, we tested the concept in two different CDX models where VSV-GP-EpCAM-TcE showed improved tumor growth inhibition due to a combined effect of oncolysis and T cell activation by EpCAM-CD3 TcE. Summary: The oncolytic virus VSV-GP can be successfully armed with a TcE to redirect tumor-infiltrating T cells to destroy tumor cells which are not permissive to viral replication. This resulted in an enhanced therapeutic efficacy compared to just the oncolytic effect. Citation Format: Aparna Ponnurangam, Theresa Schwaiger, Sophie Walter, Simone Bartl, Melissa Mayr, Monika Petersson, Tanja Schoenberger, Fabian Heinemann, Peter Gross, Wolfgang Rist, Srinath Kasturirangan, Charlotte Lempp, Jutta Petschenka, Krishna Das, Nolden Tobias. TcE armed oncolytic virus VSV-GP combines oncolysis and T-cell retargeting for improved efficacy [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 6658.

Abstract 5337: Profiling of human CD34+ donors in a myeloid-supportive HIS model provides increased predictability and facilitates study design for evaluation of immuno-oncology therapeutics

Abstract Background: Humanized immune system (HIS) mice are critical immuno-oncology tools for in vivo evaluation of therapeutic target engagement and efficacy. HIS mice require a human donor source of CD34+ hematopoietic stem cells to provide the human immune cell system. However, variance across donors complicates study design and hypothesis testing. To determine if parameters critical to drug discovery are consistent across batches of HIS mice from the same donor, we evaluated two independent cohorts of human tumor xenograft-bearing mice made using a common set of five different CD34+ cell donors. Our study included evaluation of tumor growth kinetics, human immune reconstitution and phenotype, and the pharmacokinetics and anti-tumor efficacy of a therapeutic monoclonal antibody directed against a human target expressed on tumor-associated macrophages (TAMs). Method: HIS NOG-EXL (huNOG-EXL) mice were created using CD34+ cells from five donors in two independent cohorts (~12 weeks apart). Baseline immune reconstitution in blood was assessed at 10 weeks post engraftment. MDA-MB-231 cells were inoculated bilaterally and mice were randomized into groups for immune profiling, pharmacokinetics, and efficacy studies. Body weight, clinical observations, endogenous hIgG levels, and tumor growth were measured. For efficacy, tumors grew to ~100 mm3 and mice were dosed intraperitoneally every five days (6 doses total). Separate cohorts of mice were used for immune phenotyping and the single-dose PK study. Results: Donor consistency was seen for some of the parameters assessed but not all. The overall health outcomes for a given donor appeared to have an impact on the consistency of important parameters (e.g. efficacy). Donors that had poor health outcomes (body condition scores [BCS] <3) showed less consistency than those that experienced good (BCS=3) outcomes. The health trajectory, peripheral human immune cell engraftment, and PK profiles were similar across experiments for a given donor. Total human immune cell infiltration in tumors was similar across experiments, but there were notable shifts in immune cell composition. Whereas mice in the first cohort showed a dominant TAM phenotype within the tumor (~>50% of all hCD45+ cells), mice across donors in the second cohort had reduced TAMs and corresponding increases in several T cell subsets. Efficacy was consistent in donors with good health outcomes (Donors 1 and 4) and was inconsistent in those with poor outcomes (Donors 3 and 5). Conclusion: Our study demonstrates that a return-to-donor approach can provide information that is reproducible across cohorts to help build a donor profile. An understanding of this profile should afford greater predictability and facilitate study design for drug discovery applications. Citation Format: Janell R. Richardson, Megan M. MacBride, Esther Andino, Emily Sack, Nicholas Smith, Po Y. Ho, Fang Xiao, Katelynn A. McEachin, David T. Omstead, Rachel Grgich, Michael N. Alonso, Justin A. Kenkel, Shelley E. Ackerman. Profiling of human CD34+ donors in a myeloid-supportive HIS model provides increased predictability and facilitates study design for evaluation of immuno-oncology therapeutics [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 5337.

Abstract 4183: Utility of the highly immunodeficient SRG rat for combined drug efficacy, pharmacokinetics, and toxicology studies in tumor-bearing animals

Abstract Immunodeficient rodents are vital pre-clinical disease models. Historically, immunodeficient mice have been standard for hosting human cell and tissue xenografts, enabling cancer drug efficacy and tolerability testing. However, an immunodeficient rat that supports a variety of human cancer types provides a larger model for easier surgical manipulation and serial blood and tumor tissue sampling, allowing for efficacy, pharmacokinetics, and toxicology testing in the same animal. We created a Sprague Dawley Rag2 -/-, Il2rg -/- rat (SRG Rat®; SRG) that is highly supportive of human cell and tissue engraftment. The SRG rat lacks B, T, and NK cells and is more immunodeficient than the Nude rat. Here we show comparative pathology between the SRG rat and its parental strain, the CRL Sprague Dawley (CD). We also present data from an efficacy study in tumor bearing SRG rats, demonstrating that serial blood draws and tumor biopsies can be performed in tumor bearing animals to assess drug pharmacokinetics and pharmacodynamics within a single study. First, we assessed 10 SRG (5/sex) and 10 CD (5/sex) at 8-10 weeks of age. Body weight, hematology, and clinical chemistry parameters were measured. Microscopic examinations were performed on routine H&E slide preparations. Male and female SRG displayed lower mean body weight when compared to sex- and age-matched CD. Compared to CD, SRG had reduced lymphoid tissue and circulating monocytes, and highly reduced circulating lymphocytes, neutrophils, eosinophils, and basophils; expected phenotypes for this severely immunodeficient rat. Thymus was not present in SRG rats, spleens were grossly smaller, and bone marrow cellularity was decreased compared to CD. Microscopically, mandibular lymph nodes and lymphoid tissue in spleen, lung, and intestine were not observed in SRG rats, consistent with the 92% decrease in circulating lymphocytes. To demonstrate the utility of the SRG for tumor inhibition, pharmacokinetics, and pharmacodynamics, we inoculated rats with human VCaP prostate cancer cell-derived tumors, then treated these animals and collected serial blood samples and tumor biopsies to assess tumor biomarkers. Treatment led to decreased PSA and AR, two key proteins expressed in prostate cancer, correlating to decreased tumor growth. These data demonstrate that the SRG rat is suitable for toxicology studies and has comparable pathology to the CD rat, except for reduced lymphoid tissue and WBCs, which are expected phenotypes. In addition, the SRG rat supports the growth of human cancer tissue for treatment studies. The large size of the SRG rat and comparative pathology to the CD rat support its value as an immunodeficient model for assessing toxicity during efficacy testing for de-risking safety concerns in the presence of the human target tissue. Citation Format: Diane Begemann, Cynthia Dunn, Darrell Robertson, Lauren Keach, Emily Baldwin, R. Grace Walton, Alex Van Engelenburg, Jessica Durrant, Laura Healy, Koh Meng Aw Yong, Michael J. Schlosser, Fallon K. Noto. Utility of the highly immunodeficient SRG rat for combined drug efficacy, pharmacokinetics, and toxicology studies in tumor-bearing animals [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 4183.

An algorithm for abnormal behavior recognition based on sharing human target tracking features

An algorithm for abnormal behavior recognition based on sharing human target tracking features

An update on Glycerophosphodiester Phosphodiesterases; From Bacteria to Human.

The hydrolysis of deacylated glycerophospholipids into sn-glycerol 3-phosphate and alcohol is facilitated by evolutionarily conserved proteins known as glycerophosphodiester phosphodiesterases (GDPDs). These proteins are crucial for the pathogenicity of bacteria and for bioremediation processes aimed at degrading organophosphorus esters that pose a hazard to both humans and the environment. Additionally, GDPDs are enzymes that respond to multiple nutrients and could potentially serve as candidate genes for addressing deficiencies in zinc, iron, potassium, and especially phosphate in important plants like rice. In mammals, glycerophosphodiesterases (GDEs) play a role in regulating osmolytes, facilitating the biosynthesis of anandamine, contributing to the development of skeletal muscle, promoting the differentiation of neurons and osteoblasts, and influencing pathological states. Due to their capacity to enhance a plant's ability to tolerate various nutrient deficiencies and their potential as pharmaceutical targets in humans, GDPDs have received increased attention in recent times. This review provides an overview of the functions of GDPD families as vital and resilient enzymes that regulate various pathways in bacteria, plants, and humans.

Identification of potential human targets of glyphosate using in silico target fishing

Glyphosate is a widely used herbicide known for its effectiveness in weed control; and it is an inhibitor of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase. Currently, it is one of the most extensively used non-specific herbicides in agroindustry. However, toxic effects of glyphosate have recently been reported, including endocrine disruption, metabolic alterations, teratogenic, tumorigenic, and hepatorenal effects. Additionally, there are environmental concerns related to possible interactions with proteins from microorganisms, aquatic organisms, and mammals.Research on the description of these interactions has gained interest, primarily with the aim of generating recommendations in terms of its use and possible regulations. On the other hand, computational methods have emerged to identify potential targets or unintended targets among numerous possible receptors. Several programs, online services, and databases are available for use in these methods.In this study, we employed a set of online tools for computational target fishing to identify receptors of glyphosate. A set of thirteen targets were selected using six fishing tools. Furthermore, docking procedures were performed to investigate the expected interactions and binding energies. Certain associations with diseases are also reported.

Shared and distinct interactions of type 1 and type 2 Epstein-Barr Nuclear Antigen 2 with the human genome

BackgroundThere are two major genetic types of Epstein-Barr Virus (EBV): type 1 (EBV-1) and type 2 (EBV-2). EBV functions by manipulating gene expression in host B cells, using virus-encoded gene regulatory proteins including Epstein-Barr Nuclear Antigen 2 (EBNA2). While type 1 EBNA2 is known to interact with human transcription factors (hTFs) such as RBPJ, EBF1, and SPI1 (PU.1), type 2 EBNA2 shares only ~ 50% amino acid identity with type 1 and thus may have distinct binding partners, human genome binding locations, and functions.ResultsIn this study, we examined genome-wide EBNA2 binding in EBV-1 and EBV-2 transformed human B cells to identify shared and unique EBNA2 interactions with the human genome, revealing thousands of type-specific EBNA2 ChIP-seq peaks. Computational predictions based on hTF motifs and subsequent ChIP-seq experiments revealed that both type 1 and 2 EBNA2 co-occupy the genome with SPI1 and AP-1 (BATF and JUNB) hTFs. However, type 1 EBNA2 showed preferential co-occupancy with EBF1, and type 2 EBNA2 preferred RBPJ. These differences in hTF co-occupancy revealed possible mechanisms underlying type-specific gene expression of known EBNA2 human target genes: MYC (shared), CXCR7 (type 1 specific), and CD21 (type 2 specific). Both type 1 and 2 EBNA2 binding events were enriched at systemic lupus erythematosus (SLE) and multiple sclerosis (MS) risk loci, while primary biliary cholangitis (PBC) risk loci were specifically enriched for type 2 peaks.ConclusionsThis study reveals extensive type-specific EBNA2 interactions with the human genome, possible differences in EBNA2 interaction partners, and a possible new role for type 2 EBNA2 in autoimmune disorders. Our results highlight the importance of considering EBV type in the control of human gene expression and disease-related investigations.

Immunomodulatory and anticytokine therapeutic potential of three Indian spices constituents and its hyaluronic acid conjugates for prevention and post COVID-19 complications: a computational modeling approach.

Targeted drug delivery to SARS-CoV-2 host target proteins for preventing or blocking COVID-19 infection is making serious concern during COVID-19 pandemic and its consequent waves around the globe. People seek reliable, effective folkloric preventive medication for immediate and precautionary relief from COVID-19. These folkloric medicines were effective and saved many patients during the past COVID-19 pandemic. The current research study aims to deliver antiviral Indian spices phytocompounds and their hyaluronic acid conjugates to human host target proteins (ACE-2, TNF-α, IL-6, IL-1β, PAR-1) of SARS-CoV-2 to inhibit virus propagation and also to regulate early clinical complications of COVID-19. Targeted drug delivery of hyaluronic acid conjugated traditional natural bioactive agent produces more effective and fewer side effects in delivering novel drugs to human host proteins of COVID-19. In silico molecular docking study of six phytocompounds from three Indian spices and standard drug atazanavir and its hyaluronic acid conjugates reveals that phytocompounds and its hyaluronic acid conjugates possess high affinity to binding pockets of SARS-CoV-2 human host targets with more binding affinity scores. Most notably HA cyclocurcumin exhibit a docking score -9.9 kcal/mol against ACE-2 (PDB ID 1R42) target protein similarly HA-Hydrazinocurcumin exhibit a docking score -9.8 kcal/mol against PAR-1 (PDB ID 3VW7). ADMET validation of phytocompounds and their hyaluronic acid conjugates reveals its best pharmacokinetic profile over standard antiviral drug especially HA cyclocurcumin conjugate possesses high HIA (86%) and good pharmacokinetic profiles. DFT analysis affirms the reason behind the higher binding affinity of hyaluronic acid conjugates of spices phytocompounds towards all screened target proteins especially HA-hydrazinocurcumin conjugate possess high softness (19.1570 eV) and low hardness (0.0522 eV) values. Finally, MD simulation of best-docked compounds against ACE-2 and PAR-1 target protein revealed that hyaluronic acid conjugates of Indian spices compounds exhibit stable RMSD values and more protein-ligand interactions during simulation than hyaluronic acid conjugates of drug atazanavir.Communicated by Ramaswamy H. Sarma.

Rapid depletion of target proteins in plants by an inducible protein degradation system.

Inducible protein knockdowns are excellent tools to test the function of essential proteins in short time scales and to capture the role of proteins in dynamic events. Current approaches destroy or sequester proteins by exploiting plant biological mechanisms such as the activity of photoreceptors for optogenetics or auxin-mediated ubiquitination in auxin degrons. It follows that these are not applicable for plants as light and auxin are strong signals for plant cells. We describe here an inducible protein degradation system in plants named E3-DART for E3-targeted Degradation of Plant Proteins. The E3-DART system is based on the specific and well-characterized interaction between the Salmonella-secreted protein H1 (SspH1) and its human target protein kinase N1 (PKN1). This system harnesses the E3 catalytic activity of SspH1 and the SspH1-binding activity of the homology region 1b (HR1b) domain from PKN1. Using Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana), we show that a chimeric protein containing the leucine-rich repeat and novel E3 ligase domains of SspH1 efficiently targets protein fusions of varying sizes containing HR1b for degradation. Target protein degradation was induced by transcriptional control of the chimeric E3 ligase using a glucocorticoid transactivation system, and target protein depletion was detected as early as 3 h after induction. This system could be used to study the loss of any plant protein with high-temporal resolution and may become an important tool in plant cell biology.

A Fusion of Taq DNA Polymerase with the CL7 Protein from Escherichia coli Remarkably Improves DNA Amplification.

DNA polymerases are important enzymes that synthesize DNA molecules and therefore are critical to various scientific fields as essential components of in vitro DNA synthesis reactions, including PCR. Modern diagnostics, molecular biology, and genetic engineering require DNA polymerases with improved performance. This study aimed to obtain and characterize a new CL7-Taq fusion DNA polymerase, in which the DNA coding sequence of Taq DNA polymerase was fused with that of CL7, a variant of CE7 (Colicin E7 DNase) from Escherichia coli. The resulting novel recombinant open reading frame was cloned and expressed in E. coli. The recombinant CL7-Taq protein exhibited excellent thermostability, extension rate, sensitivity, and resistance to PCR inhibitors. Our results showed that the sensitivity of CL7-Taq DNA polymerase was 100-fold higher than that of wild-type Taq, which required a template concentration of at least 1.8 × 105 nM. Moreover, the extension rate of CL7-Taq was 4 kb/min, which remarkably exceeded the rate of Taq DNA polymerase (2 kb/min). Furthermore, the CL7 fusion protein showed increased resistance to inhibitors of DNA amplification, including lactoferrin, heparin, and blood. Single-cope human genomic targets were readily available from whole blood, and pretreatment to purify the template DNA was not required. Thus, this is a novel enzyme that improved the properties of Taq DNA polymerase, and thus may have wide application in molecular biology and diagnostics.

Regulation of developmental gatekeeping and cell fate transition by the calpain protease DEK1 in Physcomitrium patens

Calpains are cysteine proteases that control cell fate transitions whose loss of function causes severe, pleiotropic phenotypes in eukaryotes. Although mainly considered as modulatory proteases, human calpain targets are directed to the N-end rule degradation pathway. Several such targets are transcription factors, hinting at a gene-regulatory role. Here, we analyze the gene-regulatory networks of the moss Physcomitrium patens and characterize the regulons that are misregulated in mutants of the calpain DEFECTIVE KERNEL1 (DEK1). Predicted cleavage patterns of the regulatory hierarchies in five DEK1-controlled subnetworks are consistent with a pleiotropic and regulatory role during cell fate transitions targeting multiple functions. Network structure suggests DEK1-gated sequential transitions between cell fates in 2D-to-3D development. Our method combines comprehensive phenotyping, transcriptomics and data science to dissect phenotypic traits, and our model explains the protease function as a switch gatekeeping cell fate transitions potentially also beyond plant development.

Two-Step Accuracy Improvement for Multitarget Detection in Complex Environment Using UWB Radar

Detecting multiple human targets in indoor scenarios using ultra-wideband (UWB) radar usually involves false detection results caused by the secondary reflections, which might reduce the target detection accuracy and cause a more severe deterioration when the number of targets increases. This article proposed a two-step accuracy improvement method for multitarget detection in environments with multiple human targets of more than three and strong secondary reflections by the surroundings, especially the walls. Based on the rough detection results acquired by the modified CA-CFAR (MCA-CFAR) processing, the first step achieves the primary false alarm suppression using a short-window accumulation in the time domain. Then, the second step applies the decision confidence on the detection results from the first step to assess the reliability of results for improved accuracy. The two-step accuracy improvement could thus have a higher accuracy through cascading false alarm suppression. The effectiveness and accuracy of the proposed algorithm are verified based on the experimental results.

Epidrugs: alternative chemotherapy targeting Theileria annulata schizont stage parasites.

The growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata-infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC50) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata-infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCETheileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata, contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper's significance in advancing veterinary science and improving livestock health globally.