Galectin-3 Inhibitors Research Articles

P10.31.A OSIMERTINIB AND OTX008 IMPAIR SURVIVAL OF GLIOBLASTOMA CELLS BY INHIBITING RAS

Abstract BACKGROUND Tyrosine kinase receptors play a critical role in the progression of almost all malignancies. In 83% of glioblastoma the endothelial growth factor receptor (EGFR) is affected by genetic alterations leading to cell proliferation and therapy resistance. This leads to a constitutive activation of EGFR, following the activation of the PI3K/AKT and RAS/MAPK signalling pathways. The EGFR-inhibitor Osimertinib showed anti-tumor effect in lung cancer patients with cerebral metastasis. OTX008 is a selective inhibitor of Galectin-1, a small protein, which is involved in stabilising the active form of H-Ras and thereby enables the downstream PI3K/AKT and RAS/MAPK pathways. This study was performed to investigate whether OTX008, Osimertinib and a combination of both have an antiproliferative effect and inhibit the Ras downstream signalling pathways in glioblastoma cells. MATERIAL AND METHODS U87 and A172 glioblastoma cells were grown to 80% confluence and treated with Osimertinib in concentrations ranging from 3 μM to 7 μM. After 72 hours cell viability was quantified via XTT-assay. Dose-response curves were calculated by probit analysis. For investigation of the inhibitory effect of OTX008 and Osimertinib on the constitutive activation of Ras U87 cells were treated with 15 μM OTX008, 3 μM Osimertinib, a combination of 15 μM OTX008 and 3 μM Osimertinib and a combination of DMSO and hydrochloricacid. After 72 hours the cells were collected and lysed using Active Ras Detection Kit from Cell Signaling Technology for western blot analysis. RESULTS Osimertinib reduced cell survival in all tested glioblastoma cell lines with a mean ED50 of 6.81 (95%CI: 6.75-6.86) μM. Using western blot analysis to evaluate the effect on the effector protein Ras at the beginning of the PI3K/AKT and RAS/MAPK pathways the effect of OTX008 and Osimertinib on the proliferation of glioblastoma cell line U87 was shown with the inhibition of Ras. CONCLUSION The constitutive activation of the PI3K/Akt and RAS/MAPK pathways is associated with uncontrolled cell growth and therapeutic resistance. The EGFR-inhibitor Osimertinib and the selective Galectin-1 inhibitor OTX008 showed antiproliferative effect in immortalized glioblastoma cell line U87 and were also able to inhibit activation of Ras. Future experiments should focus on the downstream signalling of the pathways, especially in EGFR mutated glioblastoma stem cells.

Strong Binding of C-Glycosylic1,2-Thiodisaccharides to Galectin-3─Enthalpy-Driven Affinity Enhancement by Water-Mediated Hydrogen Bonds.

Galectin-3 is involved in multiple pathways of many diseases, including cancer, fibrosis, and diabetes, and it is a validated pharmaceutical target for the development of novel therapeutic agents to address unmet medical needs. Novel 1,2-thiodisaccharides with a C-glycosylic functionality were synthesized by the photoinitiated thiol-ene click reaction of O-peracylated 1-C-substituted glycals and 1-thio-glycopyranoses. Subsequent global deprotection yielded test compounds, which were studied for their binding to human galectin-3 by fluorescence polarization and isothermal titration calorimetry to show low micromolar Kd values. The best inhibitor displayed a Kd value of 8.0 μM. An analysis of the thermodynamic binding parameters revealed that the binding Gibbs free energy (ΔG) of the new inhibitors was dominated by enthalpy (ΔH). The binding mode of the four most efficient 1,2-thiodisaccharides was also studied by X-ray crystallography that uncovered the unique role of water-mediated hydrogen bonds in conferring enthalpy-driven affinity enhancement for the new inhibitors. This 1,2-thiodisaccharide-type scaffold represents a new lead for galectin-3 inhibitor discovery and offers several possibilities for further development.

Inhibition of galectin-3 post-infarction impedes progressive fibrosis by regulating inflammatory profibrotic cascades.

Acute myocardial infarction (MI) causes inflammation, collagen deposition, and reparative fibrosis in response to myocyte death and, subsequently, a pathological myocardial remodelling process characterized by excessive interstitial fibrosis, driving heart failure (HF). Nonetheless, how or when to limit excessive fibrosis for therapeutic purposes remains uncertain. Galectin-3, a major mediator of organ fibrosis, promotes cardiac fibrosis and remodelling. We performed a preclinical assessment of a protein inhibitor of galectin-3 (its C-terminal domain, Gal-3C) to limit excessive fibrosis resulting from MI and prevent ventricular enlargement and HF. Gal-3C was produced by enzymatic cleavage of full-length galectin-3 or by direct expression of the truncated form in Escherichia coli. Gal-3C was intravenously administered for 7 days in acute MI models of young and aged rats, starting either pre-MI or 4 days post-MI. Echocardiography, haemodynamics, histology, and molecular and cellular analyses were performed to assess post-MI cardiac functionality and pathological fibrotic progression. Gal-3C profoundly benefitted left ventricular ejection fraction, end-systolic and end-diastolic volumes, haemodynamic parameters, infarct scar size, and interstitial fibrosis, with better therapeutic efficacy than losartan and spironolactone monotherapies over the 56-day study. Gal-3C therapy in post-MI aged rats substantially improved pump function and attenuated ventricular dilation, preventing progressive HF. Gal-3C in vitro treatment of M2-polarized macrophage-like cells reduced their M2-phenotypic expression of arginase-1 and interleukin-10. Gal-3C inhibited M2 polarization of cardiac macrophages during reparative response post-MI. Gal-3C impeded progressive fibrosis post-MI by down-regulating galectin-3-mediated profibrotic signalling cascades including a reduction in endogenous arginase-1 and inducible nitric oxide synthase (iNOS). Gal-3C treatment improved long-term cardiac function post-MI by reduction in the wound-healing response, and inhibition of inflammatory fibrogenic signalling to avert an augmentation of fibrosis in the periinfarct region. Thus, Gal-3C treatment prevented the infarcted heart from extensive fibrosis that accelerates the development of HF, providing a potential targeted therapy.

Discovery of N-Arylsulfonyl-Indole-2-Carboxamide Derivatives as Galectin-3 and Galectin-8 C-Terminal Domain Inhibitors.

Both galectin-3 and galectin-8 are involved in cell adhesion, migration, apoptosis, angiogenesis, and inflammatory processes by recognizing galactose-containing glycoproteins. Inhibiting galectin-3/8 activities is a potential treatment for cancer and tissue fibrosis. Herein, a series of novel N-arylsulfonyl-5-aryloxy-indole-2-carboxamide derivatives was disclosed as dual inhibitors toward galectin-3 and galectin-8 C-terminal domain with Kd values of low micromolar level (Cpd53, gal-3: Kd= 4.12 μM, gal-8C: Kd= 6.04 μM; Cpd57, gal-3: Kd= 12.8 μM, gal-8C: Kd= 2.06 μM), which are the most potent and selective noncarbohydrate-based inhibitors toward gal-3/8 isoforms to date. The molecular docking investigations suggested that the unique amino acids Arg144 in galectin-3 and Ser213 in galectin-8C could contribute to their potency and selectivity. The scratch wound assay demonstrated that Cpd53 and Cpd57 were able to inhibit the MRC-5 lung fibroblast cells migration as well. This class of inhibitors could serve as a new starting point for further discovering structurally distinct gal-3 and gal-8C inhibitors to be used in cancer and tissue fibrosis treatment.

Epithelial Galectin-3 Induced the Mitochondrial Complex Inhibition and Cell Cycle Arrest of CD8+ T Cells in Severe/Critical COVID-19.

Previous research suggested that the dramatical decrease in CD8+ T cells is a contributing factor in the poor prognosis and disease progression of COVID-19 patients. However, the underlying mechanisms are not fully understood. In this study, we conducted Single-cell RNA sequencing (scRNA-seq) and single-cell T cell receptor sequencing (scTCR-seq) analysis, which revealed a proliferative-exhausted MCM+FASLGlow CD8+ T cell phenotype in severe/critical COVID-19 patients. These CD8+ T cells were characterized by G2/M cell cycle arrest, downregulation of respiratory chain complex genes, and inhibition of mitochondrial biogenesis. CellChat analysis of infected lung epithelial cells and CD8+ T cells found that the galectin signaling pathway played a crucial role in CD8+ T cell reduction and dysfunction. To further elucidate the mechanisms, we established SARS-CoV-2 ORF3a-transfected A549 cells, and co-cultured them with CD8+ T cells for ex vivo experiments. Our results showed that epithelial galectin-3 inhibited the transcription of the mitochondrial respiratory chain complex III/IV genes of CD8+ T cells by suppressing the nuclear translocation of nuclear respiratory factor 1 (NRF1). Further findings showed that the suppression of NRF1 translocation was associated with ERK-related and Akt-related signaling pathways. Importantly, the galectin-3 inhibitor, TD-139, promoted nuclear translocation of NRF1, thus enhancing the expression of the mitochondrial respiratory chain complex III/IV genes and the mitochondrial biogenesis of CD8+ T cells. Our study provided new insights into the immunopathogenesis of COVID-19 and identified potential therapeutic targets for the prevention and treatment of severe/critical COVID-19 patients.

Pattern Analysis of Serum Galectins-1, -3, and -9 in Breast Cancer.

Galectins have been shown to have roles in cancer progression via their contributions to angiogenesis, metastasis, cell division, and the evasion of immune destruction. This study analyzes galectin-1, -3, and -9 serum concentrations in breast cancer patients through enzyme-linked immunosorbent assay (ELISA) against the characteristics of the patient and the tumor such as stage, molecular subtype, and receptor expression. Galectin-9 was found to be statistically significantly increased in HER2-enriched tumors and reduced in patients with hormone-receptor-positive tumors. Galectin-1 was found to be statistically significantly increased in the serum of patients who had undergone hormonal, immunotherapy, or chemotherapy. These findings provide insight into the changes in galectin levels during the progress of cancer, the response to treatment, and the molecular phenotype. These findings are valuable in the further understanding of the relationships between galectin and tumor biology and can inform future research on therapeutic targets for galectin inhibitors and the utility of galectin biomarkers.

Catalpol Prevents Glomerular Angiogenesis Induced by Advanced Glycation End Products via Inhibiting Galectin-3.

The main characteristics of diabetic nephropathy (DN) at the early stage are abnormal angiogenesis of glomerular endothelial cells (GECs) and macrophage infiltration. Galectin-3 plays a pivotal role in the pathogenesis of DN via binding with its ligand, advanced glycation end products (AGEs). Catalpol, an iridoid glucoside extracted from Rehmannia glutinosa, has been found to ameliorate vascular inflammation, reduce endothelial permeability, and protect against endothelial damage in diabetic milieu. However, little is known about whether catalpol could exert an anti-angiogenesis and anti-inflammation effect induced by AGEs. Mouse GECs (mGECs) and RAW 264.7 macrophages were treated with different concentrations of AGEs (0, 50, 100, 200 and 400 µg/mL) for different time (0, 6, 12, 24 and 48 h) to determine the optimal concentration of AGEs and treatment time. Cells were treated with catalpol (10 µmol/L), GB1107 (1 µmol/L, galectin-3 inhibitor), PX-478 (50 µmol/L, HIF-1α inhibitor), adenovirus-green fluorescent protein (Ad-GFP) [3×107 plaque-forming unit (PFU)/mL] or Ad-galectin-3-GFP (2×108 PFU/mL), which was followed by incubation with 50 µg/mL AGEs. The levels of galectin-3, vascular endothelial growth factor A (VEGFA) and pro-angiogenic factors angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), tunica interna endothelial cell kinase-2 (Tie-2) were detected by enzymelinked immunosorbent assay (ELISA). Cell counting kit-8 (CCK-8) assay was used to evaluate the proliferation of these cells. The expression levels of galectin-3, vascular endothelial growth factor receptor 1 (VEGFR1), VEGFR2, and hypoxia-inducible factor-1α (HIF-1α) in mGECs and those of galectin-3 and HIF-1α in RAW 264.7 macrophages were detected by Western blotting and immunofluorescence (IF) staining. The rat DN model was established. Catalpol (100 mg/kg) or GB1107 (10 mg/kg) was administered intragastrically once a day for 12 weeks. Ad-galectin-3-GFP (6×107 PFU/mL, 0.5 mL) or Ad-GFP (6×106 PFU/mL, 0.5 mL) was injected into the tail vein of rats 48 h before the sacrifice of the animals. The expression of galectin-3, VEGFR1, VEGFR2, and HIF-1α in renal cortices was analyzed by Western blotting. The expression of galectin-3, F4/80 (a macrophage biomarker), and CD34 (an endothelium biomarker) in renal cortices was detected by IF staining, and collagen accumulation by Masson staining. The expression levels of galectin-3 and VEGFA were significantly higher in mGECs and RAW 264.7 macrophages treated with 50 µg/mL AGEs for 48 h than those in untreated cells. Catalpol and GB1107 could block the AGEs-induced proliferation of mGECs and RAW 264.7 macrophages. Over-expression of galectin-3 was found to reduce the inhibitory effect of catalpol on the proliferation of cells. Catalpol could significantly decrease the levels of Ang-1, Ang-2 and Tie-2 released by AGEs-treated mGECs, which could be reversed by over-expression of galectin-3. Catalpol could significantly inhibit AGEs-induced expression of galectin-3, HIF-1α, VEGFR1, and VEGFR2 in mGECs. The inhibitory effect of catalpol on galectin-3 in AGEs-treated mGECs was impaired by PX-478. Moreover, catalpol attenuated the AGEs-activated HIF-1α/galectin-3 pathway in RAW 264.7 macrophages, which was weakened by PX-478. Additionally, catalpol significantly inhibited the expression of galectin-3, macrophage infiltration, collagen accumulation, and angiogenesis in the kidney of diabetic rats. Over-expression of galectin-3 could antagonize these inhibitory effects of catalpol. Catalpol prevented the angiogenesis of mGECs and macrophage proliferation via inhibiting galectin-3. It could prevent the progression of diabetes-induced renal damage.

Role of Galectin in Cardiovascular Conditions including Cirrhotic Cardiomyopathy.

Abnormal cardiac function in the setting of cirrhosis and in the absence of a primary cardiac disease is known as cirrhotic cardiomyopathy. The pathogenesis of cirrhotic cardiomyopathy is multifactorial but broadly is comprised of two pathways. The first is due to cirrhosis and synthetic liver failure with abnormal structure and function of many substances, including proteins, lipids, hormones, and carbohydrates such as lectins. The second is due to portal hypertension which invariably accompanies cirrhosis. Portal hypertension leads to a leaky, congested gut with resultant endotoxemia and systemic inflammation. This inflammatory phenotype comprises oxidative stress, cellular apoptosis, and inflammatory cell infiltration. Galectins exert all these pro-inflammatory mechanisms across many different tissues and organs, including the heart. Effective therapies for improving cardiac function in patients with cirrhosis are not available. Conventional strategies for other noncirrhotic heart diseases, including vasodilators, are not feasible because of the significant baseline vasodilation in cirrhotic patients. Therefore, exploring new treatment modalities for cirrhotic cardiomyopathy is of great importance. Galectin-3 inhibitors such as modified citrus pectin, N-acetyllactosamine, TD139 and GB0139 exert anti-apoptotic, anti-oxidative and anti-inflammatory effects and thus have potential therapeutic interest. This review briefly summarizes the physiological and pathophysiological role of galectin and specifically examines its role in cardiac disease processes. We present a more detailed discussion of galectin in cardiovascular complications of cirrhosis, particularly cirrhotic cardiomyopathy. Finally, therapeutic studies of galectin-3 inhibitors in cirrhotic cardiomyopathy are reviewed.

ST6GALNAC4 promotes hepatocellular carcinogenesis by inducing abnormal glycosylation

Hepatocellular carcinoma (HCC) is one of the most lethal tumor types worldwide. Glycosylation has shown promise in the study of tumor mechanisms and treatment. The glycosylation status of HCC and the underlying molecular mechanisms are still not fully elucidated. Using bioinformatic analysis we obtained a more comprehensive characterization of glycosylation of HCC. Our analysis presented that high glycosylation levels might correlate with tumor progression and poor prognosis. Subsequent Experiments identified key molecular mechanisms for ST6GALNAC4 promoting malignant progression by inducing abnormal glycosylation. We confirmed the contribution of ST6GALNAC4 to proliferation, migration, and invasion in vitro and in vivo. Mechanistic studies revealed that ST6GALNAC4 may be induced abnormal TGFBR2 glycosylation, resulting in the higher protein levels of TGFBR2 and TGFbeta pathway increased activation. Our study also provided a further understand of immunosuppressive function of ST6GALNAC4 through T antigen-galectin3+ TAMs axis. This study has provided one such possibility that galectin3 inhibitors might be an acceptable treatment choice for HCC patients with high T antigen expression.

Galectin-3 Plays an Important Role in BMP7-Induced Cementoblastic Differentiation of Human Periodontal Ligament Cells by Interacting with Extracellular Components.

Human periodontal ligament stem cells (hPDLSCs) contain multipotent postnatal stem cells that differentiate into PDL progenitors, osteoblasts, and cementoblasts. Previously, we obtained cementoblast-like cells from hPDLSCs using bone morphogenetic protein 7 (BMP7) treatment. Differentiation into appropriate progenitor cells requires interactions and changes between stem or progenitor cells and their so-called environment niches, and cell surface markers play an important role. However, cementoblast-specific cell surface markers have not yet been fully studied. Through decoy immunization with intact cementoblasts, we developed a series of monoclonal antibodies against cementoblast-specific membrane/extracellular matrix (ECM) molecules. One of these antibodies, the anti-CM3 antibody, recognized an approximate 30 kDa protein in a mouse cementoblast cell line, and the CM3 antigenic molecule accumulated in the cementum region of human tooth roots. Using mass spectrometric analysis, we found that the antigenic molecules recognized by the anti-CM3 antibody were galectin-3. As cementoblastic differentiation progressed, the expression of galectin-3 increased, and it localized at the cell surface. Inhibition of galectin-3 via siRNA and a specific inhibitor showed the complete blockage of cementoblastic differentiation and mineralization. In contrast, ectopic expression of galectin-3 induced cementoblastic differentiation. Galectin-3 interacted with laminin α2 and BMP7, and these interactions were diminished by galectin-3 inhibitors. These results suggested that galectin-3 participates in binding to the ECM component and trapping BMP7 to induce, in a sustained fashion, the upregulation of cementoblastic differentiation. Finally, galectin-3 could be a potential cementoblast-specific cell surface marker, with functional importance in cell-to-ECM interactions.

Reduction of Tendon Fibrosis Using Galectin-3 Inhibitors.

Fibrosis is a complication of both tendon injuries and repairs. The authors aimed to develop a mouse model to assess tendon fibrosis and to identify an antifibrotic agent capable of overcoming it. The Achilles tendon of adult C57Bl/6 mice was exposed via skin incision, followed by 50% tendon injury and abrasion with sandpaper. Sham operations were conducted on contralateral hindlimbs. Histologic analyses and immunofluorescent staining for fibrotic markers (collagen type 1 [ Col1 ], α-smooth muscle actin [ α-SMA ]) were used to confirm that the model induced tendon fibrosis. A second experiment further examined the role of α-SMA in adhesion formation using α-SMA.mTmG mice (6 to 8 weeks old; n = 3) with the same injury model. Lastly, α-SMA.mTmG mice were randomized to either condition 1 (tendon injury [control group]) or condition 2 (tendon injury with galectin-3 inhibitor [Gal3i] treatment at time of injury [treatment group]). Histologic analyses confirmed tendon thickening and collagen deposition after tendon injury and abrasion compared with control. Immunofluorescence showed higher levels of Col1 and α-SMA protein expression after injury compared with sham ( P < 0.05). Real-time quantitative polymerase chain reaction also demonstrated increased gene expression of Col1 and α-SMA after injury compared with sham ( P < 0.05). Gal3 protein expression also increased after injury and colocalized with α-SMA+ fibroblasts surrounding the fibrotic tendon. Gal3i treatment decreased collagen deposition and scarring observed in the treatment group ( P < 0.05). The authors' study provides a reproducible and reliable model to investigate tendon fibrosis. Findings suggest the potential of Gal3i to overcome fibrosis resulting from tendon injuries. Tendon injuries are common presentations to hand surgeons. Complications include adhesion formation, which results in reduced strength and frequent reinjury. Advancements in management require a better understanding of the mechanisms behind tendon fibrosis in order to identify ways to overcome it.

1602-P: Galectin-3 Inhibition Protects ApoE Knockout Mice against Western Diet–Induced Nonalcoholic Steatohepatitis and Glucose Intolerance

Nonalcoholic Fatty Liver Disease represents a broad spectrum of histological abnormalities ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which may progress to cirrhosis or hepatocellular carcinoma. NASH is characterized by steatosis, hepatic inflammation, hepatocellular ballooning, and fibrosis. Therefore, the aim of this work was to evaluate the effect of modified citrus pectin (MCP), inhibitor of galectin-3, in ApoE KO mice fed a western diet (WD). ApoE KO mice were divided into 3 groups: standard diet-fed (SD) group, WD-fed group, and WD-fed + MCP-treated group (WD+MCP). WD-fed mice displayed increased hepatic triacylglycerides (TAG-290%), and plasma ALT (115%) and AST (130%) compared with SD group. MCP treatment decreased hepatic TAG (30%) in WD-fed mice, plasma ALT and AST were reverted. WD-fed mice also displayed increased hepatic collagen deposition (18%), and MCP treatment inhibited this increase. These results were similar with hydroxyproline concentration in liver. WD-fed mice showed an impaired glucose tolerance compared with SD-fed mice, while MCP treatment improved glucose tolerance in WD-fed mice. Also, WD-fed mice presented increased fasting insulin (140%) compared with SD-fed mice, and MCP treatment reverted this increase by 100%. Gene expression of F4/80 was increased by 76% in the WD group compared with SD group, however had not significant difference between WD+MCP and SD group. Gene expression of matrix metalloproteinase 2 was increased by 5-fold in WD group compared with SD group, while this increase was inhibited with MCP treatment. Also, it was observed an increase in JNK phosphorylation by 3-fold in WD group, and MCP treatment inhibited this increase. Taken together, we confirm WD-fed ApoE KO mice as a suitable experimental model of NASH, and the inhibition of galectin-3 by the treatment with MCP protects ApoE KO mice against the development of NASH and glucose intolerance. Disclosure L. Araujo: None. F. Sucupira: None. C.C.B. Dias: None. A. Amaral: None. J. Camporez: None. Funding São Paulo Research Foundation (FAPESP)

Discovery and exploration of monosaccharide linked dimers of galectin-3 inhibitors to target fibrosis

Discovery and exploration of monosaccharide linked dimers of galectin-3 inhibitors to target fibrosis

Chimera and Tandem-Repeat Type Galectins: The New Targets for Cancer Immunotherapy.

In humans, a total of 12 galectins have been identified. Their intracellular and extracellular biological functions are explored and discussed in this review. These galectins play important roles in controlling immune responses within the tumour microenvironment (TME) and the infiltration of immune cells, including different subsets of T cells, macrophages, and neutrophils, to fight against cancer cells. However, these infiltrating cells also have repair roles and are hijacked by cancer cells for pro-tumorigenic activities. Upon a better understanding of the immunomodulating functions of galectin-3 and -9, their inhibitors, namely, GB1211 and LYT-200, have been selected as candidates for clinical trials. The use of these galectin inhibitors as combined treatments with current immune checkpoint inhibitors (ICIs) is also undergoing clinical trial investigations. Through their network of binding partners, inhibition of galectin have broad downstream effects acting on CD8+ cytotoxic T cells, regulatory T cells (Tregs), Natural Killer (NK) cells, and macrophages as well as playing pro-inflammatory roles, inhibiting T-cell exhaustion to support the fight against cancer cells. Other galectin members are also included in this review to provide insight into potential candidates for future treatment(s). The pitfalls and limitations of using galectins and their inhibitors are also discussed to cognise their clinical application.

Role of galectin-3 in cardiac dysfunction induced by subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) is a severe acute cerebrovascular event that not only impairs the central nervous system but also negatively affects various other organs, including the heart. The underlying mechanisms, however, remain unclear. In this study, we discovered that mice with SAH exhibited significant cardiac injuries, such as extended QT and QTc intervals, cardiac fibrosis, and reduced cardiac ejection fractions. This phenomenon was accompanied by increased galectin-3 expression in the cardiac ventricle and can be reversed by galectin-3 inhibitor TD139. Interestingly, we also observed increased co-expression of galectin-3 in macrophage within the heart tissue of SAH mice. Additionally, when macrophage activation was suppressed using the beta-blocker propranolol, cardiac function improved, and galectin-3 expression in the cardiac tissue decreased. Collectively, our findings offer new insights into the role of galectin-3 in SAH-related cardiac dysfunction and suggest a macrophage-galectin-3 axis as a potential therapeutic strategy.

Abstract 5493: Novel strategy to make KRAS targeted therapies more effective for PDAC treatment

Abstract Introduction: Pancreatic Ductal Adenocarcinoma (PDAC) patients exhibit extremely poor prognosis. KRAS mutation on codon-12 is present in 70-95% of PDAC cases and it drives PDAC growth and progression. Galectin-1 (Gal-1) is present in both PDAC and stromal cells, being involved in tumor microenvironment, immune cell activation and metastasis. Therefore, this study discusses the efficiency of combined inhibition of mutated KRASG12D and Gal-1 inhibition to effectively suppress PDAC growth and progression. For this we have delivered KRASG12D inhibiting siRNA (siKRASG12D) using a superparamagnetic iron oxide nanoparticle (SPION) and a galectin inhibitor. Methods: SPION nano-formulation was used to deliver siKRASG12D and investigate in conjunction with Gal-1 inhibitor for its anticancer efficacy. Particles were investigated for size, physico-chemical characterization (Dynamic light scattering), hemocompatibility (hemolysis assay) and the complexation of siKRAS (gel retardation assay). Cellular internalization and uptake of the particles were investigated. Anti-cancer efficacy was determined using in vitro functional assays for cell viability (MTT), migration (Boyden chambers), invasion (Matrigel), clonogenicity, tumor spheroid formation, and in a KrasG12D;LSL-Trp53R172H syngeneic mouse model. Results: Our results demonstrate that SP-siKRAS efficiently internalized in PDAC cells and suppressed KRASG12D as well as its downstream targets, YAP and PDL-1. Combined targeting of siKRAS and Gal-1 inhibited cell proliferation, clonogenicity, migration, and invasion of PDAC cells and tumor spheroid growth in 3D cell models, which recapitulate the heterogeneity and pathophysiology of PDAC. We have used -KrasG12D;LSL-Trp53R172H syngeneic mouse model of PDAC for investigating efficacy of combined SP-siKRAS formulation and galectin-1 inhibitor. Our results showed that the combination treatment inhibited the fibrotic tumor growth and increased survival rate. The combined treatment increased infiltration of total T cell population and CD8+T cells, reduced the population of myeloid-derived suppressor cells (MDSCs) by 50% (CD45+, CD3-, CD11b+, Ly6C high, Ly6G-) and T-Regulatory cells (Treg) by 57% (FoxP3+CD25+CD45+CD3+) and increased memory T cells by 34% in mice. Conclusion: This gene therapy targeting KRAS G12D mutation with a Gal-1 inhibition has a potential to modulate the oncogenic network and tumor microenvironment resulting in the repression of growth, metastasis, chemoresistance, and improvement in patient survival. This study will develop a novel sustainable therapeutic approach to target PDAC growth and improve patient survivability. Citation Format: Ana I. Bulnes, Orlando Garcia, Poornima Shaji, Swathi Holla, Nirnoy Dan, Anupam Dhasmana, Shabnam Malik, Murali Yallapu, Stephen Behrman, Subhash Chauhan, Sheema S. Khan. Novel strategy to make KRAS targeted therapies more effective for PDAC treatment. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5493.

An Oral Galectin Inhibitor in COVID-19-A Phase II Randomized Controlled Trial.

SARS-CoV-2 vaccines play an important role in reducing disease severity, hospitalization, and death, although they failed to prevent the transmission of SARS-CoV-2 variants. Therefore, an effective inhibitor of galectin-3 (Gal-3) could be used to treat and prevent the transmission of COVID-19. ProLectin-M (PL-M), a Gal-3 antagonist, was shown to interact with Gal-3 and thereby prevent cellular entry of SARS-CoV-2 in previous studies. The present study aimed to further evaluate the therapeutic effect of PL-M tablets in 34 subjects with COVID-19. The efficacy of PL-M was evaluated in a randomized, double-blind, placebo-controlled clinical study in patients with mild to moderately severe COVID-19. Primary endpoints included changes in the absolute RT-PCR Ct values of the nucleocapsid and open reading frame (ORF) genes from baseline to days 3 and 7. The incidence of adverse events, changes in blood biochemistry, inflammatory biomarkers, and levels of antibodies against COVID-19 were also evaluated as part of the safety evaluation. PL-M treatment significantly (p = 0.001) increased RT-PCR cycle counts for N and ORF genes on days 3 (Ct values 32.09 ± 2.39 and 30.69 ± 3.38, respectively) and 7 (Ct values 34.91 ± 0.39 and 34.85 ± 0.61, respectively) compared to a placebo treatment. On day 3, 14 subjects in the PL-M group had cycle counts for the N gene above the cut-off value of 29 (target cycle count 29), whereas on day 7, all subjects had cycle counts above the cut-off value. Ct values in placebo subjects were consistently less than 29, and no placebo subjects were RT-PCR-negative until day 7. Most of the symptoms disappeared completely after receiving PL-M treatment for 7 days in more patients compared to the placebo group. PL-M is safe and effective for clinical use in reducing viral loads and promoting rapid viral clearance in COVID-19 patients by inhibiting SARS-CoV-2 entry into cells through the inhibition of Gal-3.

Safety and pharmacokinetics of GB1211, an oral galectin-3 inhibitor: a single- and multiple-dose first-in-human study in healthy participants

PurposeGalectin-3, a β-galactoside-binding lectin, plays a key role in several cellular pathways involved in chronic inflammation, heart disease and cancer. GB1211 is an orally bioavailable galectin-3 inhibitor, developed to be systemically active. We report safety and pharmacokinetics (PK) of GB1211 in healthy participants.MethodsThis phase 1, double-blind, placebo-controlled, first-in-human study (NCT03809052) included a single ascending-dose phase (with a food-effect cohort) where participants across seven sequential cohorts were randomized 3:1 to receive oral GB1211 (5, 20, 50, 100, 200 or 400 mg) or placebo. In the multiple ascending-dose phase, participants received 50 or 100 mg GB1211 or placebo twice daily for 10 days. All doses were administered in the fasted state except in the food-effect cohort where doses were given 30 min after a high-fat meal.ResultsAll 78 participants received at least one GB1211 dose (n = 58) or placebo (n = 20) and completed the study. No safety concerns were identified. Following single and multiple oral doses under fasted conditions, maximum GB1211 plasma concentrations were reached at 1.75–4 h (median) post-dose; mean half-life was 11–16 h. There was a ~ twofold GB1211 accumulation in plasma with multiple dosing, with steady-state reached within 3 days; 30% of the administered dose was excreted in urine as unchanged drug. Absorption in the fed state was delayed by 2 h but systemic exposure was unaffected.ConclusionGB1211 was well tolerated, rapidly absorbed, and displayed favorable PK, indicating a potential to treat multiple disease types. These findings support further clinical development of GB1211.Clinical trial registrationThe study was registered with ClinicalTrials.gov (identifier: NCT03809052).

O-glycan structures in apo(a) subunit of human lipoprotein(a) suppresses the pro-angiogenic activity of galectin-1 on human umbilical vein endothelial cells.

Apolipoprotein(a) [apo(a)] is a highly polymorphic O-glycoprotein circulating in human plasma as lipoprotein(a) [Lp(a)]. The O-glycan structures of apo(a) subunit of Lp(a) serve as strong ligands of galectin-1, an O-glycan binding pro-angiogenic lectin abundantly expressed in placental vascular tissues. But the pathophysiological significance of apo(a)-galectin-1 binding is not yet been revealed. Carbohydrate-dependent binding of galectin-1 to another O-glycoprotein, neuropilin-1 (NRP-1) on endothelial cells activates vascular endothelial growth factor receptor 2 (VEGFR2) and mitogen-activated protein kinase (MAPK) signaling. Using apo(a), isolated from human plasma, we demonstrated the potential of the O-glycan structures of apo(a) in Lp(a) to inhibit angiogenic properties such as proliferation, migration, and tube-formation in human umbilical vein endothelial cells (HUVECs) as well as neovascularization in chick chorioallantoic membrane. Further, in vitro protein-protein interaction studies have confirmed apo(a) as a superior ligand to NRP-1 for galectin-1 binding. We also demonstrated that the protein levels of galectin-1, NRP-1, VEGFR2, and downstream proteins in MAPK signaling were reduced in HUVECs in the presence of apo(a) with intact O-glycan structures compared to that of de-O-glycosylated apo(a). In conclusion, our study shows that apo(a)-linked O-glycans prevent the binding of galectin-1 to NRP-1 leading to the inhibition of galectin-1/neuropilin-1/VEGFR2/MAPK-mediated angiogenic signaling pathway in endothelial cells. As higher plasma Lp(a) level in women is an independent risk factor for pre-eclamsia, a pregnancy-associated vascular complication, we propose that apo(a) O-glycans-mediated inhibition of the pro-angiogenic activity of galectin-1 may be one of the underlying molecular mechanism of pathogenesis of Lp(a) in pre-eclampsia.

Cigarette smoke induces epithelial-to-mesenchymal transition, stemness, and metastasis in lung adenocarcinoma cells via upregulated RUNX-2/galectin-3 pathway

AimsAn elevated level of galectin-3, a carbohydrate-binding lectin implicated in tumorigenesis, metastasis, and epithelial-mesenchymal transition (EMT), has been found in cigarette smokers. However, the regulation of its expression and role in the pathogenesis of CS-induced EMT and lung cancer metastasis is unclear. Here, we have investigated the mechanism of CS-induced and galectin-3-mediated EMT in airway epithelial cells (AECs). Main methodsA549 adenocarcinoma cells and primary small airway epithelial cells cultured on an air-liquid interface (ALI) were exposed to cigarette smoke extract (CSE), and MTT, trypan blue, migration, invasion, tumor spheroid and colony formation assays were performed to assess EMT phenotype. Immunoblotting was performed to assess EMT and stemness markers and other regulatory proteins. Key findingsCSE exposure affected cell survival and morphology, migration, invasion, and clonogenicity of AECs, which were concomitant with an increase in the expression of EMT markers, galectin-3, and runt-related transcription factor-2 (RUNX-2), an osteogenic transcription factor and upstream regulator of galectin-3. Chemical inhibition or silencing of RUNX-2 downregulated galectin-3 and modulated EMT marker expression, migration, invasion, and clonogenicity in CSE-exposed AECs. Recombinant human galectin-3 also induced EMT and stemness-associated changes in the AECs, and GB1107, a galectin-3 inhibitor, ameliorated these changes. Further, CSE-induced intracellular ROS enabled an increase in RUNX-2 and galectin-3 expression, which were reversed by n-acetyl-cysteine. SignificanceThese results provide a novel mechanistic insight into CSE-induced EMT via RUNX-2/galectin-3 axis mediated through ROS, which promoted EMT-associated changes, including invasion, migration, and stemness in AECs, which could be implicated in CS-induced lung cancer progression.