Receptor Targeting Research Articles

Systemically administered low-affinity HER2 CAR T cells mediate antitumor efficacy without toxicity

BackgroundThe paucity of tumor-specific targets for chimeric antigen receptor (CAR) T-cell therapy of solid tumors necessitates careful preclinical evaluation of the therapeutic window for candidate antigens. Human epidermal growth factor...

Abstract B030: ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model

Abstract Background: ABC101-1 is an antibody-drug conjugate (ADC) targeting receptor tyrosine-protein kinase erbB-2, also known as HER2. ABC101-1 consists of a humanized IgG1 monoclonal antibody called trastuzumab conjugated to a microtubulin inhibitor, DM1, through interchain cysteines with a drug-to-antibody ratio (DAR) of 8. Our novel non-cleavable linker platform using maleimide as a connector enabled the construction of homogeneous ABC101-1 with DAR of 8, via a straightforward process resulting in high conjugation yield. Employing non-cleavable linkers may ensure that the cytotoxic drug is delivered stably to tumor cells, enhancing the therapeutic efficacy of ADC. Herein, we describe anti-breast cancer activity through in vitro cytotoxicity and in vivo efficacy, pharmacokinetics, and binding affinity of ABC101-1. Methods: In vitro cytotoxicity of ADC and linker-payload was evaluated by measuring cell viability using Cell Count Kit-8 (CCK8) analysis. In vivo anti-tumor activity was investigated using SKBR3 cell line-derived xenograft (CDX) model. A pharmacokinetic (PK) profile was assessed by performing indirect ELISA to determine concentrations of total mAb in the serum. The binding affinity of ABC101-1 to HER2 protein was assessed using the indirect ELISA measuring the concentrations of total mAb. Results: In this study, ABC101-1 exhibited potent in vitro cytotoxicity with picomolar activity on HER2-positive breast cancer cell line. In the CDX breast cancer model, ABC101-1 led to stronger tumor growth inhibition in both early and late phases than ado-trastuzumab emtansine (DAR 3.5) at the same dose. Also, the favorable pharmacokinetic profile of ABC101-1 was observed, along with the preserved antigen-binding ability upon the conjugation to linker-payload. Conclusions: ABC101-1 displayed preclinical efficacy in in vitro and in vivo studies as well as a favorable PK, thereby suggesting its therapeutic potential as an anti-tumor drug in breast cancer therapy. This study would reveal that our novel linker platform is a promising and efficient strategy to develop effective and homogeneous ADCs with DAR 8. Our finding may support the further pre-clinical evaluation of ABC101-1. Citation Format: Seo Woo Kim, Hana Yu, Geon Ho Lee, Tae Gyu Lim, Minji Jeong, Hyeonjoo Hwang, Jee Eun Moon, Yong Beom Lee, Sung-Ha Jin, Jin Hyun Jeong. ABC101-1, a novel microtubule inhibitor antibody-drug conjugate targeting HER2, has potent anti-tumor activity in a breast cancer model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B030.

Measuring Hydroxyl Exchange Rates in Glycans Using a Synergistic Combination of Saturation Transfer and Relaxation Dispersion NMR.

Molecular recognition events mediated by glycans play pivotal roles in controlling the fate of diverse biological processes such as cellular communication and the immune response. The affinity of glycans for their target receptors is governed primarily by the hydrogen bonds formed by hydroxyl groups decorating the glycan surface. Hydroxyl exchange rate constants are therefore vital parameters that report on glycan structure and dynamics. Here we present a strategy for characterizing hydroxyl hydrogen/deuterium (H/D) exchange in glycans that employs a synergistic combination of 13C chemical exchange saturation transfer (CEST) and Carr-Purcell-Meiboom-Gill relaxation dispersion (CPMG) NMR methods. We show that the combination of CEST and CPMG experiments facilitates the sensitive detection of the small (∼0.1 ppm) two-bond deuterium isotope shift on a 13C nucleus when the attached hydroxyl group fluctuates between protonated and deuterated states. This shift is leveraged for measuring site-specific kinetic H/D exchange rate constants as well as thermodynamic free energies of isotope fractionation. The CEST and CPMG modules are integrated with a selective J-cross-polarization scheme that provides the flexibility for rapid characterization of H/D exchange at a specific hydroxyl site. Moreover, our approach enables the precise isothermal measurement of hydroxyl exchange rate constants without the need for cumbersome isotope labeling. The H/D exchange rate constants of three different glycans assessed using this method highlight its potential for detecting transient intra- and intermolecular hydrogen bonds. In addition, the trends in H/D exchange rate constants establish site-specific steric accessibility as a key determinant of solvent exchange dynamics in glycans.

Bioorthogonal chemical labeling of endogenous neurotransmitter receptors in living mouse brains

Neurotransmitter receptors are essential components of synapses for communication between neurons in the brain. Because the spatiotemporal expression profiles and dynamics of neurotransmitter receptors involved in many functions are delicately governed in the brain, in vivo research tools with high spatiotemporal resolution for receptors in intact brains are highly desirable. Covalent labeling by chemical reaction (chemical labeling) of proteins without genetic manipulation is now a powerful method for analyzing receptors in vitro. However, selective target receptor labeling in the brain has not yet been achieved. This study shows that ligand-directed alkoxyacylimidazole (LDAI) chemistry can be used to selectively tether synthetic probes to target endogenous receptors in living mouse brains. The reactive LDAI reagents with negative charges were found to diffuse well over the whole brain and could selectively label target endogenous receptors, including AMPAR, NMDAR, mGlu1, and GABAAR. This simple and robust labeling protocol was then used for various applications: three-dimensional spatial mapping of endogenous receptors in the brains of healthy and disease-model mice; multi-color receptor imaging; and pulse-chase analysis of the receptor dynamics in postnatal mouse brains. Here, results demonstrated that bioorthogonal receptor modification in living animal brains may provide innovative molecular tools that contribute to the in-depth understanding of complicated brain functions.

Interaction of human dendritic cell receptor DEC205/CD205 with keratins

DEC205 (CD205) is one of the major endocytic receptors on dendritic cells and has been widely used as a receptor target in immune therapies. It has been shown that DEC205 can recognize dead cells through keratins in a pH-dependent manner. But the mechanism underlying the interaction between DEC205 and keratins remains unclear. Here we determine the crystal structures of an N-terminal fragment of human DEC205 (CysR∼CTLD3). The structural data show that DEC205 shares similar overall features with the other mannose receptor family members such as the mannose receptor and Endo180, but the individual domains of DEC205 in the crystal structure exhibit distinct structural features that may lead to specific ligand binding properties of the molecule. Among them, CTLD3 of DEC205 adopts a unique fold of CTLD, which may correlate with the binding of keratins. Furthermore, we examine the interaction of DEC205 with keratins by mutagenesis and biochemical assays based on the structural information and identify an XGGGX motif on keratins that can be recognized by DEC205, thereby providing insights for the interaction between DEC205 and keratins. Overall, these findings not only improve the understanding of the diverse ligand specificities of the mannose receptor family members at molecular level, but may also give clues for the interactions of keratins with their binding partners in the corresponding pathways.

Identification of Low-Density Lipoprotein Receptor-Related Protein 1 as a CXCL14 Receptor Using Chemically Synthesized Tetrafunctional Probes.

CXCL14 is a primordial CXC-type chemokine that transports CpG oligodeoxynucleotides (ODN) into endosomes and lysosomes in dendritic cells, thereby leading to the activation of the Toll-like receptor 9 (TLR9)-mediated innate immune system. However, the underlying molecular mechanism by which the CXCL14-CpG ODN complex enters cells remains elusive. Herein, we describe the chemical synthesis of CXCL14-derived photoaffinity probes and their application to the identification of target receptors for CXCL14 using quantitative proteomics. By utilizing native chemical ligation and maleimide-thiol coupling chemistry, we synthesized site-specifically modified CXCL14-based photoaffinity probes that contain photoreactive 2-aryl-5-carboxytetrazole (ACT) and a hydrazine-labile cleavable linker. CXCL14-based probes were found to be capable of binding CpG ODN to immune cells, whose bioactivities were comparable to native CXCL14. Application of CXCL14-derived probes to quantitative proteomic experiments enabled the identification of dozens of target receptor candidates for CXCL14 in mouse macrophage-derived RAW264.7 cells, and we discovered that low-density lipoprotein receptor-related protein 1 (LRP1) is a novel receptor for CXCL14 by competitive proteome profiling. We further showed that disruption of LRP1 affected the incorporation of the CXCL14-CpG ODN complex in the cells. Overall, this report highlights the power of synthetic CXCL14-derived photoaffinity probes combined with chemical proteomics to discover previously unidentified receptors for CXCL14, which could promote an understanding of the molecular functions of CXCL14 and the elaborate machinery of innate immune systems.

A novel aminopeptidase N/CD13 inhibitor selectively targets an endothelial form of CD13 after coupling to proteins

Aminopeptidase N/CD13, a membrane-bound enzyme upregulated in tumor vasculature and involved in angiogenesis, can be used as a receptor for the targeted delivery of drugs to tumors through ligand-directed targeting approaches. We describe a novel peptide ligand (VGCARRYCS, called “G4”) that recognizes CD13 with high affinity and selectivity. Enzymological and computational studies showed that G4 is a competitive inhibitor that binds to the catalytic pocket of CD13 through its N-terminal region. Fusing the peptide C-terminus to tumor necrosis factor-alpha (TNF) or coupling it to a biotin/avidin complex causes loss of binding and inhibitory activity against different forms of CD13, including natural or recombinant ectoenzyme and a membrane form expressed by HL60 promyelocytic leukemia cells (likely due to steric hindrance), but not binding to a membrane form of CD13 expressed by endothelial cells (ECs). Furthermore, G4-TNF systemically administered to tumor-bearing mice exerted anticancer effects through a CD13-targeting mechanism, indicating the presence of a CD13 form in tumor vessels with an accessible binding site. Biochemical studies showed that most CD13 molecules expressed on the surface of ECs are catalytically inactive. Other functional assays showed that these molecules can promote endothelial cell adhesion to plates coated with G4-avidin complexes, suggesting that the endothelial form of CD13 can exert catalytically independent biological functions. In conclusion, ECs express a catalytically inactive form of CD13 characterized by an accessible conformation that can be selectively targeted by G4-protein conjugates. This form of CD13 may represent a specific target receptor for ligand-directed targeted delivery of therapeutics to tumors.

Antibody-Conjugated Polymersomes with Encapsulated Indocyanine Green J-Aggregates and High Near-Infrared Absorption for Molecular Photoacoustic Cancer Imaging.

Imaging plays a critical role in all stages of cancer care from early detection to diagnosis, prognosis, and therapy monitoring. Recently, photoacoustic imaging (PAI) has started to emerge into the clinical realm due to its high sensitivity and ability to penetrate tissues up to several centimeters deep. Herein, we encapsulated indocyanine green J (ICGJ) aggregate, one of the only FDA-approved organic exogenous contrast agents that absorbs in the near-infrared range, at high loadings up to ∼40% w/w within biodegradable polymersomes (ICGJ-Ps) composed of poly(lactide-co-glycolide-b-polyethylene glycol) (PLGA-b-PEG). The small Ps hydrodynamic diameter of 80 nm is advantageous for in vivo applications, while directional conjugation with epidermal growth factor receptor (EGFR) targeting cetuximab antibodies renders molecular specificity. Even when exposed to serum, the ∼11 nm-thick membrane of the Ps prevents dissociation of the encapsulated ICGJ for at least 48 h with a high ratio of ICGJ to monomeric ICG absorbances (i.e., I895/I780 ratio) of approximately 5.0 that enables generation of a strong NIR photoacoustic (PA) signal. The PA signal of polymersome-labeled breast cancer cells is proportional to the level of cellular EGFR expression, indicating the feasibility of molecular PAI with antibody-conjugated ICGJ-Ps. Furthermore, the labeled cells were successfully detected with PAI in highly turbid tissue-mimicking phantoms up to a depth of 5 mm with the PA signal proportional to the amount of cells. These data show the potential of molecular PAI with ICGJ-Ps for clinical applications such as tumor margin detection, evaluation of lymph nodes for the presence of micrometastasis, and laparoscopic imaging procedures.

OP24 SPP1 in colitis-associated colon cancer

Abstract Background Patients with ulcerative colitis (UC) and Crohn’s disease (CD) face a lifelong risk of developing colitis-associated carcinoma (CAC). Current insights into CAC development originate from murine CAC models, while human CAC studies primarily focus on mutational analyses. Although the mutational landscape reveals distinct patterns and frequencies compared to colorectal cancer, it falls short in elucidating the inflammatory mechanisms of CAC development. Consequently, we adopted a multi-omics approach to unravel CAC carcinogenesis, utilizing human samples from patients with CD, CD-associated CAC (CD-CAC), UC, and UC-associated CAC (UC-CAC). Findings were further validated in vitro using human colon organoids. Methods A Nanostring Immunology v2 panel was employed on RNA (archival FFPE sections) from 40 patients suffering from either IBD or CAC, alongside inflammation-free healthy controls (n=10 per group). Our data revealed a robust upregulation of the SPP1 (Secreted Phosphoprotein 1) gene encoding osteopontin (OPN) in CAC patients. Prospectively, we extended our methodology to an unbiased imaging-based spatial RNA in situ sequencing (ISS) approach (477 genes, Xenium, 10X Genomics) using FFPE sections from CAC and UC patients (n=3 per group). This provided insights into the spatial expression of SPP1 and its target receptors at a subcellular resolution. Findings were validated using whole FFPE section confocal microscopy. In addition, in vitro models, including human UC organoids, were analyzed by bulk RNA-Seq, RT-qPCR, and downstream functional assays. Results Nanostring Immunology v2 gene expression data revealed upregulation of the SPP1 gene encoding OPN (Fig 1A) and EMT transcription factors FN1 and ZEB1 in CAC patients. Xenium RNA in situ sequencing (ISS) unveiled the spatial heterogeneity of SPP1 expression in CAC (Fig 1B). Unsupervised graph-based clustering identified an SPP1+CD68+ macrophage cluster exclusively in CAC (Fig 1C), validated through OPN/CD68 immunostainings (Fig 1D). In vitro OPN stimulation across multiple human and mice colon organoid models excluded OPN as the trigger of EMT. Interestingly, Xenium ISS and immunostainings revealed a mutually exclusive spatial location of SPP1/OPN+ macrophages and CD8+ T cells (Fig 1E, F and G). Conclusion SPP1 is significantly upregulated in CAC (RNA and protein), expressed by CD68+ macrophages. In vitro OPN stimulation demonstrated no direct effect on intestinal epithelial organoids. The mutually exclusive spatial locations of SPP1/OPN+ macrophages and CD8+ T cells suggests a crucial role of SPP1/OPN in modulating the immunosurveillance of CAC cells by CD8+ tumour lymphocytes.

Targeting the Endocannabinoid System Present in the Glioblastoma Tumour Microenvironment as a Potential Anti-Cancer Strategy.

The highly aggressive and invasive glioblastoma (GBM) tumour is the most malignant lesion among adult-type diffuse gliomas, representing the most common primary brain tumour in the neuro-oncology practice of adults. With a poor overall prognosis and strong resistance to treatment, this nervous system tumour requires new innovative treatment. GBM is a polymorphic tumour consisting of an array of stromal cells and various malignant cells contributing to tumour initiation, progression, and treatment response. Cannabinoids possess anti-cancer potencies against glioma cell lines and in animal models. To improve existing treatment, cannabinoids as functionalised ligands on nanocarriers were investigated as potential anti-cancer agents. The GBM tumour microenvironment is a multifaceted system consisting of resident or recruited immune cells, extracellular matrix components, tissue-resident cells, and soluble factors. The immune microenvironment accounts for a substantial volume of GBM tumours. The barriers to the treatment of glioblastoma with cannabinoids, such as crossing the blood-brain barrier and psychoactive and off-target side effects, can be alleviated with the use of nanocarrier drug delivery systems and functionalised ligands for improved specificity and targeting of pharmacological receptors and anti-cancer signalling pathways. This review has shown the presence of endocannabinoid receptors in the tumour microenvironment, which can be used as a potential unique target for specific drug delivery. Existing cannabinoid agents, studied previously, show anti-cancer potencies via signalling pathways associated with the hallmarks of cancer. The results of the review can be used to provide guidance in the design of future drug therapy for glioblastoma tumours.

Mannose Ligands for Mannose Receptor Targeting.

The mannose receptor (MR, CD 206) is an endocytic receptor primarily expressed by macrophages and dendritic cells, which plays a critical role in both endocytosis and antigen processing and presentation. MR carbohydrate recognition domains (CRDs) exhibit a high binding affinity for branched and linear oligosaccharides. Furthermore, multivalent mannose presentation on the various templates like peptides, proteins, polymers, micelles, and dendrimers was proven to be a valuable approach for the selective and efficient delivery of various therapeutically active agents to MR. This review provides a detailed account of the most relevant and recent aspects of the synthesis and application of mannosylated bioactive formulations for MR-mediated delivery in treatments of cancer and other infectious diseases. It further highlights recent findings related to the necessary structural features of the mannose-containing ligands for successful binding to the MR.

Garlic as an effective antifungal inhibitor: A combination of reverse docking, molecular dynamics simulation, ADMET screening, DFT, and retrosynthesis studies

Fungal infections profoundly affect human health, causing a substantial number of infections and millions of fatalities annually on a global scale. The identification of new drugs targeting this infection is a challenge that is not yet complete. Natural products, including medicinal and aromatic plants, substances that act as sources of beneficial chemical compounds for the development of efficient therapies, are among the medicines that can be used to combat this type of infection. In this study, seven bioactive molecules derived from garlic plant as potential antifungal inhibitors were investigated using computational methods. Alliin and S-allyl-cysteine, bioactive molecules generated from garlic, showed good stability at the active site of the studied receptor (PDB code: 5TZ1). They provided binding energies of −4.80 and −4.90 Kcal/mol, and inhibition constant (Ki) values of 303.78 and 253.68 µM, respectively. Similarly, alliin and S-allyl-cysteine were stabilized in the active site of the target receptor by conventional hydrogen bonds with residues Ser507 (2.47 Å), Ser378 (3.01 Å), Met508 (2.62 Å, 3.46 Å), and His377 (3.00 Å), Ser378 (3.09 Å), Met508 (2.01 Å), Ser507 (2.26 Å), respectively. These results were confirmed by molecular dynamic simulation. The selected molecules comply with the most important drug rules such as Lipinski, Veber and Egan, have good ADME properties and are not toxic; therefore, these bioactive molecules have good pharmacokinetic properties and bioavailability. The retrosynthesis method has created a pathway for the synthesis of these candidate inhibitors. As a result, the outcomes of this study strongly suggest that Alliin, S-allyl-cysteine, are potential antifungal inhibitors in the future.

Integrins in Health and Disease-Suitable Targets for Treatment?

Integrin receptors are heterodimeric surface receptors that play multiple roles regarding cell-cell communication, signaling, and migration. The four members of the β2 integrin subfamily are composed of an alternative α (CD11a-d) subunit, which determines the specific receptor properties, and a constant β (CD18) subunit. This review aims to present insight into the multiple immunological roles of integrin receptors, with a focus on β2 integrins that are specifically expressed by leukocytes. The pathophysiological role of β2 integrins is confirmed by the drastic phenotype of patients suffering from leukocyte adhesion deficiencies, most often resulting in severe recurrent infections and, at the same time, a predisposition for autoimmune diseases. So far, studies on the role of β2 integrins in vivo employed mice with a constitutive knockout of all β2 integrins or either family member, respectively, which complicated the differentiation between the direct and indirect effects of β2 integrin deficiency for distinct cell types. The recent generation and characterization of transgenic mice with a cell-type-specific knockdown of β2 integrins by our group has enabled the dissection of cell-specific roles of β2 integrins. Further, integrin receptors have been recognized as target receptors for the treatment of inflammatory diseases as well as tumor therapy. However, whereas both agonistic and antagonistic agents yielded beneficial effects in animal models, the success of clinical trials was limited in most cases and was associated with unwanted side effects. This unfavorable outcome is most probably related to the systemic effects of the used compounds on all leukocytes, thereby emphasizing the need to develop formulations that target distinct types of leukocytes to modulate β2 integrin activity for therapeutic applications.

A transcriptomic approach to explore the immune landscape of patient with pancreatic ductal adenocarcinoma with prognostic impact.

681 Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by its immunologically cold tumor microenvironment (TME) with scarce T cell infiltration and few molecular signatures of immune activation. To date, the immunotherapies including the immune checkpoint blockade of PD-1, and CTLA-4 either single or in combination have shown modest results in PDAC tumors. Recently, the Lymphocyte Activation Gene (LAG3) has emerged as a promising checkpoint target as indicated by preclinical and clinical data. The aim of this study is to explore the immune profile of a series of PDAC and to evaluate its prognostic impact. Methods: A 5-mm thick section of Formalin-Fixed Paraffin-Embedded (FFPE) tissue from a retrospective cohort of 28 PDAC cases were analyzed using the Precision Immunooncology panel (PIP, HTG Molecular Diagnostics) on a NextSeq 550 sequencer (Illumina). The RNA expression of 1392 immune-related genes were analyzed. Principal Components (PC) and Manhattan distance were studied for data visualization and clustering. Maxstat algorithm (maxstat v0.7-25) was used to establish optimal cut-offs for variable categorization. Log-rank and Cox regression were used for both univariate and multivariate for survival. All tests were two-tailed. The statistical analysis was performed using R studio (R version 4.0.3). Lastly, immunohistochemistry (IHC) for CD4, CD8, CD20, PD1, PD-L1, and LAG-3 proteins was performed on tissue microarrays (TMAs) to assess their correlation with RNA expression. Results: Unsupervised analysis of the gene expression identified two clusters of patients with differentially prognostic information. These two groups were defined with a logistic regression model of 14 genes ( BTLA, CXCR5, DLX6, KLHDC9, KRT13, LAG3, LGSN, MICB, SIGLEC5, SPINK5, SPN, TBX21, UPK2, ZIC5) showing for cluster1 and cluster 2 a median survival of 13 (0-97.583) and 57 (0.67-256.08) months, respectively (p-value = 0.0088). This predictive model was independently validated in the PDAC dataset of the Cancer Genome Atlas (TCGA) (p<0.0001). Interestingly, cluster 1, was characterized by the significant overexpression of LAG3 which has recently been proposed as the next immune checkpoint (IC) receptor target. Moreover, Cluster 1, exhibited the overexpression of other ICs such as PD1, PD-L1, IDO1, LIF and CTLA4, defining cluster 1 as an immunologically hot tumor. IHC for LAG3 and other ICs showed a moderate correlation with HTG results. Conclusions: In this study, a prognostic transcriptomic-based signature of 14 genes has been defined and validated for PDAC. This signature clearly identifies two prognostic groups that could constitute the basis for tailored immunotherapy with specific IC inhibitors. LAG-3 is a promising target for immunotherapy in PDAC patients.

I-shaped antibody engineering enables conformational tuning of biotherapeutic receptor agonists

The ability to leverage antibodies to agonize disease relevant biological pathways has tremendous potential for clinical investigation. Yet while antibodies have been successful as antagonists, immune mediators, and targeting agents, they are not readily effective at recapitulating the biology of natural ligands. Among the important determinants of antibody agonist activity is the geometry of target receptor engagement. Here, we describe an engineering approach inspired by a naturally occurring Fab-Fab homotypic interaction that constrains IgG in a unique i-shaped conformation. i-shaped antibody (iAb) engineering enables potent intrinsic agonism of five tumor necrosis factor receptor superfamily (TNFRSF) targets. When applied to bispecific antibodies against the heterodimeric IL-2 receptor pair, constrained bispecific IgG formats recapitulate IL-2 agonist activity. iAb engineering provides a tool to tune agonist antibody function and this work provides a framework for the development of intrinsic antibody agonists with the potential for generalization across broad receptor classes.

Design of novel isoxazole derivatives as tubulin inhibitors using computer-aided techniques: QSAR modeling, in silico ADMETox, molecular docking, molecular dynamics, biological efficacy, and retrosynthesis

In the current work, computational methods were used to investigate new isoxazole derivatives that could be used as tubulin inhibitors. The study aims to develop a reliable quantitative structure-activity relationship (QSAR) model, following the criteria set by Golbraikh, Tropsha, and Roy. As a result, seven candidate compounds were developed, all having higher activity than the well-established anticancer agent Cisplatin (Cisp). According to the ADMETox in silico test, the candidates Pr4, Pr5, and P6 can be toxic. As a result, we have chosen to focus our study on compounds Pr1, Pr2, and Pr3. Molecular docking analysis revealed that drug candidate Pr2 exhibits the highest stability within the oxidized quinone reductase 2 (PDB ID: 4zvm), target receptor (ΔG(Pr2) = ΔG(Pr3) = −10.4 < ΔG(Pr1) = −10.0 < ΔG(Cisp) = −7.3 kcal/mol). This finding aligns with the activity predictions made by the QSAR model. Furthermore, molecular dynamics simulations of the Pr2–4zvm complex over 100 ns confirm the ligand’s robust stability within the receptor’s active site, supporting the results obtained from molecular docking and the QSAR model predictions. The CaverDock software was utilized to identify the tunnels likely to be followed by ligands moving from the active site to the receptor surface. This analysis also helped in determining the biological efficacy of the target compounds. The results indicated that the Pr2 compound is more effective than the others. Finally, the computer-assisted retrosynthesis process of two high confidence sequences was used to synthesize drug candidates. Communicated by Ramaswamy H. Sarma

Restricted effects of androgens on glucocorticoid signaling in the mouse prefrontal cortex and midbrain.

Glucocorticoids are key executors of the physiological response to stress. Previous studies in mice showed that the androgen receptor (AR) influenced the transcriptional outcome of glucocorticoid treatment in white and brown adipocytes and in the liver. In the brain, we observed that chronic hypercorticism induced changes in gene expression that tended to be more pronounced in male mice. In the present study, we investigated if glucocorticoid signaling in the brain could be modulated by androgen. After chronic treatment with corticosterone, dihydrotestosterone, a combination of both, and corticosterone in combination with the AR antagonist enzalutamide, we compared the expression of glucocorticoid receptor (NR3C1, also abbreviated GR) target genes in brain regions where AR and GR are co-expressed, namely: prefrontal cortex, hypothalamus, hippocampus, ventral tegmental area and substantia nigra. We observed that androgen affected glucocorticoid signaling only in the prefrontal cortex and the substantia nigra. Dihydrotestosterone and corticosterone independently and inversely regulated expression of Sgk1 and Tsc22d3 in prefrontal cortex. AR antagonism with enzalutamide attenuated corticosterone-induced expression of Fkbp5 in the prefrontal cortex and of Fkbp5 and Sgk1 in the substantia nigra. Additionally, in the substantia nigra, AR antagonism increased expression of Th and Slc18a1, two genes coding for key components of the dopaminergic system. Our data indicate that androgen influence over glucocorticoid stimulation in the brain is not a dominant phenomenon in the context of high corticosterone levels, but can occur in the prefrontal cortex and substantia nigra.

From MASH to HCC: the role of Gas6/TAM receptors.

Metabolic dysfunction-associated steatohepatitis (MASH) is the replacement term for what used to be called nonalcoholic steatohepatitis (NASH). It is characterized by inflammation and injury of the liver in the presence of cardiometabolic risk factors and may eventually result in the development of hepatocellular carcinoma (HCC), the most common form of primary liver cancer. Several pathogenic mechanisms are involved in the transition from MASH to HCC, encompassing metabolic injury, inflammation, immune dysregulation and fibrosis. In this context, Gas6 (Growth Arrest-Specific 6) and TAM (Tyro3, Axl, and MerTK) receptors may play important roles. The Gas6/TAM family is involved in the modulation of inflammation, lipid metabolism, fibrosis, tumor progression and metastasis, processes which play an important role in the pathophysiology of acute and chronic liver diseases. In this review, we discuss MASH-associated HCC and the potential involvement of the Gas6/TAM system in disease development and progression. In addition, since therapeutic strategies for MASH and HCC are limited, we also speculate regarding possible future treatments involving the targeting of Gas6 or TAM receptors.

FGF21 Induces Skeletal Muscle Atrophy and Increases Amino Acids in Female Mice: A Potential Role for Glucocorticoids.

Fibroblast growth factor-21 (FGF21) is an intercellular signaling molecule secreted by metabolic organs, including skeletal muscle, in response to intracellular stress. FGF21 crosses the blood-brain barrier and acts via the nervous system to coordinate aspects of the adaptive starvation response, including increased lipolysis, gluconeogenesis, fatty acid oxidation, and activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. Given its beneficial effects for hepatic lipid metabolism, pharmaceutical FGF21 analogues are used in clinical trials treatment of fatty liver disease. We predicted pharmacologic treatment with FGF21 increases HPA axis activity and skeletal muscle glucocorticoid signaling and induces skeletal muscle atrophy in mice. Here we found a short course of systemic FGF21 treatment decreased muscle protein synthesis and reduced tibialis anterior weight; this was driven primarily by its effect in female mice. Similarly, intracerebroventricular FGF21 reduced tibialis anterior muscle fiber cross-sectional area; this was more apparent among female mice than male littermates. In agreement with the reduced muscle mass, the topmost enriched metabolic pathways in plasma collected from FGF21-treated females were related to amino acid metabolism, and the relative abundance of plasma proteinogenic amino acids was increased up to 3-fold. FGF21 treatment increased hypothalamic Crh mRNA, plasma corticosterone, and adrenal weight, and increased expression of glucocorticoid receptor target genes known to reduce muscle protein synthesis and/or promote degradation. Given the proposed use of FGF21 analogues for the treatment of metabolic disease, the study is both physiologically relevant and may have important clinical implications.

Inhibition of CSF1R and KIT With Pexidartinib Reduces Inflammatory Signaling and Cell Viability in Endometriosis.

Endometriosis is a common and debilitating disease, affecting ∼170 million women worldwide. Affected patients have limited therapeutic options such as hormonal suppression or surgical excision of the lesions, though therapies are often not completely curative. Targeting receptor tyrosine kinases (RTKs) could provide a nonhormonal treatment option for endometriosis. We determined that 2 RTKs, macrophage-colony stimulating factor 1 receptor (CSF1R) and mast/stem cell growth factor receptor KIT (KIT), are overexpressed in endometriotic lesions and could be novel nonhormonal therapeutic targets for endometriosis. The kinase activity of CSF1R and KIT is suppressed by pexidartinib, a small molecule inhibitor that was recently approved by the US Food and Drug Administration. Using immunohistochemistry, we detected CSF1R and KIT in endometriotic tissues obtained from peritoneal lesions, colorectal lesions, and endometriomas. Specifically, we show that KIT is localized to the epithelium of the lesions, while CSF1R is expressed in the stroma and macrophages of the endometriotic lesions. Given the high epithelial expression of CSF1R and KIT, 12Z endometriotic epithelial cells were used to evaluate the efficacy of dual CSF1R and KIT inhibition with pexidartinib. We found that pexidartinib suppressed activation in 12Z cells of JNK, STAT3, and AKT signaling pathways, which control key proinflammatory and survival networks within the cell. Using quantitative real-time polymerase chain reaction, we determined that pexidartinib suppressed interleukin 8 (IL8) and cyclin D1 (CCND1) expression. Lastly, we demonstrated that pexidartinib decreased cell growth and viability. Overall, these results indicate that pexidartinib-mediated CSF1R and KIT inhibition reduces proinflammatory signaling and cell viability in endometriosis.