Galectin-8N-Selective 4-Halophenylphthalazinone-Galactals Double π-Stack in a Unique Pocket.

Galectin-8 contains two different carbohydrate recognition domains (CRDs). Selective inhibitors for at least one CRD are desirable for galectin-8 biology studies and potentially for pharmacological purposes. Structure-guided design led to the discovery of potent and selective glycomimetic-heterocycle hybrid ligands, with a 4-(p-bromophenyl)phthalazinone derivative displaying a 34 µM K d for galectin-8N (N-terminal CRD), no binding to galectin-8C (C-terminal CRD), -1, -3, -4N, -7, -9C, or -9N, and >40-fold selectivity over galectin-4C. Selectivity was achieved with the halogenated 4-phenylphthalazinone moiety occupying a galectin-8N-specific sub-pocket. A 1.30 Å resolution X-ray structure revealed the phthalazinone moiety stacking with Arg45 and the 4-bromophenyl moiety stacking both Arg59 and Tyr141 of galectin-8N. Physicochemical and in vitro ADME studies revealed a desirable LogD, which also translated to good passive permeability. The chemical, microsome, and plasma stability support these compounds as promising tool compounds and candidates for hit-to-lead optimization.

Myocardin related transcription factor and galectin-3 drive lipid accumulation in human blood vessels.

OBJECTIVE: Diabetes and hypertension are important risk factors for vascular disease, including atherosclerosis. A driving factor in this process is lipid accumulation in smooth muscle cells of the vascular wall. The glucose- and mechano-sensitive transcriptional coactivator, myocardin-related transcription factor A (MRTF-A/MKL1) can promote lipid accumulation in cultured human smooth muscle cells and contribute to the formation of smooth muscle-derived foam cells. The purpose of this study was to determine if intact human blood vessels ex vivo can be used to evaluate lipid accumulation in the vascular wall, and if this process is dependent on MRTF and/or galectin-3/LGALS3. Galectin-3 is an early marker of smooth muscle transdifferentiation and a potential mediator for foam cell formation and atherosclerosis. APPROACH AND RESULTS: Human mammary arteries and saphenous veins were exposed to altered cholesterol and glucose levels in an organ culture model. Accumulation of lipids, quantified by Oil Red O, was increased by cholesterol loading and elevated glucose concentrations. Pharmacological inhibition of MRTF with CCG-203971 decreased lipid accumulation, whereas adenoviral-mediated overexpression of MRTF-A had the opposite effect. Cholesterol-induced expression of galectin-3 was decreased after inhibition of MRTF. Importantly, pharmacological inhibition of galectin-3 with GB1107 reduced lipid accumulation in the vascular wall after cholesterol loading. CONCLUSION: Ex vivo organ culture of human arteries and veins can be used to evaluate lipid accumulation in the intact vascular wall, as well as adenoviral transduction and pharmacological inhibition. Although MRTF and galectin-3 may have beneficial, anti-inflammatory effects under certain circumstances, our results, which demonstrate a significant decrease in lipid accumulation, support further evaluation of MRTF- and galectin-3-inhibitors for therapeutic intervention against atherosclerotic vascular disease.

Determining the Affinity and Kinetics of Small Molecule Inhibitors of Galectin-1 Using Surface Plasmon Resonance.

The beta-galactoside-binding mammalian lectin galectin-1 can bind, via its carbohydrate recognition domain (CRD), to various cell surface glycoproteins and has been implicated in a range of cancers. As a consequence of binding to sugar residues on cell surface receptors, it has been shown to have a pleiotropic effect across many cell types and mechanisms, resulting in immune system modulation and cancer progression. As a result, it has started to become a therapeutic target for both small and large molecules. In previous studies, we used fluorescence polarization (FP) assays to determine KD values to screen and triage small molecule glycomimetics that bind to the galectin-1 CRD. In this study, surface plasmon resonance (SPR) was used to compare human and mouse galectin-1 affinity measures with FP, as SPR has not been applied for compound screening against this galectin. Binding affinities for a selection of mono- and di-saccharides covering a 1000-fold range correlated well between FP and SPR assay formats for both human and mouse galectin-1. It was shown that slower dissociation drove the increased affinity at human galectin-1, whilst faster association was responsible for the effects in mouse galectin-1. This study demonstrates that SPR is a sound alternative to FP for early drug discovery screening and determining affinity estimates. Consequently, it also allows association and dissociation constants to be measured in a high-throughput manner for small molecule galectin-1 inhibitors.

Discovery of the Selective and Orally Available Galectin-1 Inhibitor GB1908 as a Potential Treatment for Lung Cancer.

We have previously described a new series of selective and orally available galectin-1 inhibitors resulting in the thiazole-containing glycomimetic GB1490. Here, we show that the introduction of polar substituents to the thiazole ring results in galectin-1-specific compounds with low nM affinities. X-ray structural analysis of a new ligand-galectin-1 complex shows changes in the binding mode and ligand-protein hydrogen bond interactions compared to the GB1490-galectin-1 complex. These new high affinity ligands were further optimized with respect to affinity and ADME properties resulting in the galectin-1-selective GB1908 (Kd galectin-1/3 0.057/6.0 µM). In vitro GB1908 inhibited galectin-1-induced apoptosis in Jurkat cells (IC50 = 850 nM). Pharmacokinetic experiments in mice revealed that a dose of 30 mg/kg b.i.d. results in free levels of GB1908 in plasma over galectin-1 Kd for 24 h. GB1908 dosed with this regimen reduced the growth of primary lung tumor LL/2 in a syngeneic mouse model.

Interplay of halogen bonding and solvation in protein-ligand binding.

Halogen bonding is increasingly utilized in efforts to achieve high affinity and selectivity of molecules designed to bind proteins, making it paramount to understand the relationship between structure, dynamics, and thermodynamic driving forces. We present a detailed analysis addressing this problem using a series of protein-ligand complexes involving single halogen substitutions - F, Cl, Br, and I - and nearly identical structures. Isothermal titration calorimetry reveals an increasingly favorable binding enthalpy from F to I that correlates with the halogen size and σ-hole electropositive character, but is partially counteracted by unfavorable entropy, which is constant from F to Cl and Br, but worse for I. Consequently, the binding free energy is roughly equal for Cl, Br, and I. QM and solvation-free-energy calculations reflect an intricate balance between halogen bonding, hydrogen bonds, and solvation. These advances have the potential to aid future drug design initiatives involving halogenated compounds.

Galectin-8 inhibition and functions in immune response and tumor biology.

Galectins are among organisms' most abundantly expressed lectins (carbohydrate-binding proteins) that specifically bind ß-galactosides. They act not only outside the cell, where they bind to extracellular matrix glycans, but also inside the cell, where they have a significant impact on signaling pathways. Galectin-8 is a galectin family protein encoded by the LGALS8 gene. Its role is evident in both T- and B-cell immunity and in the innate immune response, where it acts directly on dendritic cells and induces some pro-inflammatory cytokines. Galectin-8 also plays an important role in the defense against bacterial and viral infections. It is known to promote antibacterial autophagy by recognizing and binding glycans present on the vacuolar membrane, thus acting as a danger receptor. The most important role of galectin-8 is the regulation of cancer growth, metastasis, tumor progression, and tumor cell survival. Importantly, the expression of galectins is typically higher in tumor tissues than in noncancerous tissues. In this review article, we focus on galectin-8 and its function in immune response, microbial infections, and cancer. Given all of these functions of galectin-8, we emphasize the importance of developing new and selective galectin-8 inhibitors and report the current status of their development.

Development of a novel AAK1 inhibitor via Kinobeads-based screening.

A chemical proteomics approach using Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor-immobilized sepharose (TIM-063-Kinobeads) identified main targets such as CaMKKα/1 and ß/2, and potential off-target kinases, including AP2-associated protein kinase 1 (AAK1), as TIM-063 interactants. Because TIM-063 interacted with the AAK1 catalytic domain and inhibited its enzymatic activity moderately (IC50 = 8.51 µM), we attempted to identify potential AAK1 inhibitors from TIM-063-derivatives and found a novel AAK1 inhibitor, TIM-098a (11-amino-2-hydroxy-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinolin-7-one) which is more potent (IC50 = 0.24 µM) than TIM-063 without any inhibitory activity against CaMKK isoforms and a relative AAK1-selectivity among the Numb-associated kinases family. TIM-098a could inhibit AAK1 activity in transfected cultured cells (IC50 = 0.87 µM), indicating cell-membrane permeability of the compound. Overexpression of AAK1 in HeLa cells significantly reduced the number of early endosomes, which was blocked by treatment with 10 µM TIM-098a. These results indicate TIM-063-Kinobeads-based chemical proteomics is efficient for identifying off-target kinases and re-evaluating the kinase inhibitor (TIM-063), leading to the successful development of a novel inhibitory compound (TIM-098a) for AAK1, which could be a molecular probe for AAK1. TIM-098a may be a promising lead compound for a more potent, selective and therapeutically useful AAK1 inhibitor.

Discovery of Selective and Orally Available Galectin-1 Inhibitors.

A new series of orally available α-d-galactopyranosides with high affinity and specificity toward galectin-1 have been discovered. High affinity and specificity were achieved by changing six-membered aryl-triazolyl substituents in a series of recently published galectin-3-selective α-d-thiogalactosides (e.g., GB1107 Kd galectin-1/3 3.7/0.037 µM) for five-membered heterocycles such as thiazoles. The in vitro pharmacokinetic properties were optimized, resulting in several galectin-1 inhibitors with favorable properties. One compound, GB1490 (Kd galectin-1/3 0.4/2.7 µM), was selected for further characterization toward a panel of galectins showing a selectivity of 6- to 320-fold dependent on galectin. The X-ray structure of GB1490 bound to galectin-1 reveals the compound bound in a single conformation in the carbohydrate binding site. GB1490 was shown to reverse galectin-1-induced apoptosis of Jurkat cells at low µM concentrations. No cell cytotoxicity was observed for GB1490 up to 90 µM in the A549 cells. In pharmacokinetic studies in mice, GB1490 showed high oral bioavailability (F% > 99%).

Ligand Sulfur Oxidation State Progressively Alters Galectin-3-Ligand Complex Conformations To Induce Affinity-Influencing Hydrogen Bonds.

Galectins play biological roles in immune regulation and tumor progression. Ligands with high affinity for the shallow, hydrophilic galectin-3 ligand binding site rely primarily on a galactose core with appended aryltriazole moieties, making hydrophobic interactions and π-stacking. We designed and synthesized phenyl sulfone, sulfoxide, and sulfide-triazolyl thiogalactoside derivatives to create affinity-enhancing hydrogen bonds, hydrophobic and π-interactions. Crystal structures and thermodynamic analyses revealed that the sulfoxide and sulfone ligands form hydrogen bonds while retaining π-interactions, resulting in improved affinities and unique binding poses. The sulfoxide, bearing one hydrogen bond acceptor, leads to an affinity decrease compared to the sulfide, whereas the corresponding sulfone forms three hydrogen bonds, two directly with Asn and Arg side chains and one water-mediated to an Asp side chain, respectively, which alters the complex structure and increases affinity. These findings highlight that the sulfur oxidation state influences both the interaction thermodynamics and structure.

Resistance to anti-PD-1/anti-PD-L1: galectin-3 inhibition with GB1211 reverses galectin-3-induced blockade of pembrolizumab and atezolizumab binding to PD-1/PD-L1.

Background: Galectin-3 (Gal-3) is a ß-galactoside-binding lectin that is highly expressed within the tumor microenvironment of aggressive cancers and has been suggested to predict a poor response to immune checkpoint therapy with the anti-PD-1 monoclonal antibody pembrolizumab. We aimed to assess if the effect of Gal-3 was a result of direct interaction with the immune checkpoint receptor. Methods: The ability of Gal-3 to interact with the PD-1/PD-L1 complex in the absence and presence of blocking antibodies was assessed in in vitro biochemical and cellular assays as well as in an in vivo syngeneic mouse cancer model. Results: Gal-3 reduced the binding of the checkpoint inhibitors pembrolizumab (anti-PD-1) and atezolizumab (anti-PD-L1), by potentiating the interaction between the PD-1/PD-L1 complex. In the presence of a highly selective Gal-3 small molecule inhibitor (GB1211) the binding of the anti-PD-1/anti-PD-L1 therapeutics was restored to control levels. This was observed in both a surface plasmon resonance assay measuring protein-protein interactions and via flow cytometry. Combination therapy with GB1211 and an anti-PD-L1 blocking antibody reduced tumor growth in an in vivo syngeneic model and increased the percentage of tumor infiltrating T lymphocytes. Conclusion: Our study suggests that Gal-3 can potentiate the PD-1/PD-L1 immune axis and potentially contribute to the immunosuppressive signalling mechanisms within the tumor microenvironment. In addition, Gal-3 prevents atezolizumab and pembrolizumab target engagement with their respective immune checkpoint receptors. Reversal of this effect with the clinical candidate GB1211 offers a potential enhancing combination therapeutic with anti-PD-1 and -PD-L1 blocking antibodies.

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.

Novel inhibitory effect of galectin-3 on the respiratory burst induced by Staphylococcus aureus in human neutrophils.

Among the responders to microbial invasion, neutrophils represent the earliest and perhaps the most important immune cells that contribute to host defense with the primary role to kill invading microbes using a plethora of stored anti-microbial molecules. One such process is the production of reactive oxygen species (ROS) by the neutrophil enzyme complex NADPH-oxidase, which can be assembled and active either extracellularly or intracellularly in phagosomes (during phagocytosis) and/or granules (in the absence of phagocytosis). One soluble factor modulating the interplay between immune cells and microbes is galectin-3 (gal-3), a carbohydrate-binding protein that regulates a wide variety of neutrophil functions. Gal-3 has been shown to potentiate neutrophil interaction with bacteria, including Staphylococcus aureus, and is also a potent activator of the neutrophil respiratory burst, inducing large amounts of granule-localized ROS in primed cells. Herein, the role of gal-3 in regulating S. aureus phagocytosis and S. aureus-induced intracellular ROS was analyzed by imaging flow cytometry and luminol-based chemiluminescence, respectively. Although gal-3 did not interfere with S. aureus phagocytosis per se, it potently inhibited phagocytosis-induced intracellular ROS production. Using the gal-3 inhibitor GB0139 (TD139) and carbohydrate recognition domain of gal-3 (gal-3C), we found that the gal-3-induced inhibitory effect on ROS production was dependent on the carbohydrate recognition domain of the lectin. In summary, this is the first report of an inhibitory role of gal-3 in regulating phagocytosis-induced ROS production.

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

PURPOSE: Galectin-3, a ß-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. METHODS: This 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. RESULTS: All 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. CONCLUSION: GB1211 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 REGISTRATION: The study was registered with ClinicalTrials.gov (identifier: NCT03809052).

Evaluating the affinity and kinetics of small molecule glycomimetics for human and mouse galectin-3 using surface plasmon resonance.

Galectin-3 is a beta-galactoside-binding mammalian lectin that is one of a 15-member galectin family that can bind several cell surface glycoproteins via its carbohydrate recognition domain (CRD). As a result, it can influence a range of cellular processes including cell activation, adhesion and apoptosis. Galectin-3 has been implicated in various diseases, including fibrotic disorders and cancer, and is now being therapeutically targeted by both small and large molecules. Historically, the screening and triaging of small molecule glycomimetics that bind to the galectin-3 CRD has been completed in fluorescence polarisation (FP) assays to determine KD values. Surface plasmon resonance (SPR) has not been widely used for compound screening and in this study it was used to compare human and mouse galectin-3 affinity measures between FP and SPR, as well as investigate compound kinetics. The KD estimates for a set of compounds selected from mono- and di-saccharides with affinities across a 550-fold range, correlated well between FP and SPR assay formats for both human and mouse galectin-3. Increases in affinity for compounds binding to human galectin-3 were driven by changes in both kon and koff whilst for mouse galectin-3 this was primarily due to kon. The reduction in affinity observed between human to mouse galectin-3 was also comparable between assay formats. SPR has been shown to be a viable alternative to FP for early drug discovery screening and determining KD values. In addition, it can also provide early kinetic characterisation of small molecule galectin-3 glycomimetics with robust kon and koff values generated in a high throughput manner.

N-BAR and F-BAR proteins-endophilin-A3 and PSTPIP1-control clathrin-independent endocytosis of L1CAM.

Recent advances in the field demonstrate the high diversity and complexity of endocytic pathways. In the current study, we focus on the endocytosis of L1CAM. This glycoprotein plays a major role in the development of the nervous system, and is involved in cancer development and is associated with metastases and poor prognosis. Two L1CAM isoforms are subject to endocytosis: isoform 1, described as a clathrin-mediated cargo; isoform 2, whose endocytosis has never been studied. Deciphering the molecular machinery of isoform 2 internalisation should contribute to a better understanding of its pathophysiological role. First, we demonstrated in our cellular context that both isoforms of L1CAM are mainly a clathrin-independent cargo, which was not expected for isoform 1. Second, the mechanism of L1CAM endocytosis is specifically mediated by the N-BAR domain protein endophilin-A3. Third, we discovered PSTPIP1, an F-BAR domain protein, as a novel actor in this endocytic process. Finally, we identified galectins as endocytic partners and negative regulators of L1CAM endocytosis. In summary, the interplay of the BAR proteins endophilin-A3 and PSTPIP1, and galectins fine tune the clathrin-independent endocytosis of L1CAM.

Galectin-8 Downmodulates TLR4 Activation and Impairs Bacterial Clearance in a Mouse Model of Pseudomonas aeruginosa Keratitis.

Pseudomonas aeruginosa provokes a painful, sight-threatening corneal infection. It progresses rapidly and is difficult to treat. In this study, using a mouse model of P. aeruginosa keratitis, we demonstrate the importance of a carbohydrate-binding protein, galectin-8 (Gal-8), in regulation of the innate immune response. First, using two distinct strains of P. aeruginosa, we established that Gal-8-/- mice are resistant to P. aeruginosa keratitis. In contrast, mice deficient in Gal-1, Gal-3, and Gal-9 were fully susceptible. Second, the addition of exogenous rGal-8 to LPS (TLR4 ligand)-stimulated bone marrow-derived macrophages suppressed 1) the activation of the NF-κB pathway, and 2) formation of the MD-2/TLR4 complex. Additionally, the expression level of reactive oxygen species was substantially higher in infected Gal-8-/- bone marrow neutrophils (BMNs) compared with the Gal-8+/+ BMNs, and the P. aeruginosa killing capacity of Gal-8-/- BMNs was considerably higher compared with that of Gal-8+/+ BMNs. In the bacterial killing assays, the addition of exogenous rGal-8 almost completely rescued the phenotype of Gal-8-/- BMNs. Finally, we demonstrate that a subconjunctival injection of a Gal-8 inhibitor markedly reduces the severity of infection in the mouse model of P. aeruginosa keratitis. These data lead us to conclude that Gal-8 downmodulates the innate immune response by suppressing activation of the TLR4 pathway and clearance of P. aeruginosa by neutrophils. These findings have broad implications for developing novel therapeutic strategies for treatment of conditions resulting from the hyperactive immune response both in ocular as well as nonocular tissues.

Sialic Acid 4-N-Piperazine and Piperidine Derivatives Bind with High Affinity to the P. mirabilis Sialic Acid Sodium Solute Symporter.

In search for novel antibacterial compounds, bacterial sialic acid uptake inhibition represents a promising strategy. Sialic acid plays a critical role for growth and colonisation of several pathogenic bacteria, and its uptake inhibition in bacteria was recently demonstrated to be a viable strategy by targeting the SiaT sodium solute symporters from Proteus mirabilis and Staphylococcus aureus. Here we report the design, synthesis and evaluation of potential sialic acid uptake inhibitors bearing 4-N-piperidine and piperazine moieties. The 4-N-derivatives were obtained via 4-N-functionalization with piperidine and piperazine nucleophiles in an efficient direct substitution of the 4-O-acetate of Neu5Ac. Evaluation for binding to bacterial transport proteins with nanoDSF and ITC revealed compounds possessing nanomolar affinity for the P. mirabilis SiaT symporter. Computational analyses indicate the engagement of a previously untargeted portion of the binding site.

Discovery and Optimization of the First Highly Effective and Orally Available Galectin-3 Inhibitors for Treatment of Fibrotic Disease.

Galectin-3 is a carbohydrate-binding protein central to regulating mechanisms of diseases such as fibrosis, cancer, metabolic, inflammatory, and heart disease. We recently found a high affinity (nM) thiodigalactoside GB0139 which currently is in clinical development (PhIIb) as an inhaled treatment of idiopathic pulmonary fibrosis. To enable treatment of systemically galectin-3 driven disease, we here present the first series of selective galectin-3 inhibitors combining high affinity (nM) with oral bioavailability. This was achieved by optimizing galectin-3 specificity and physical chemical parameters for a series of disubstituted monogalactosides. Further characterization showed that this class of compounds reduced profibrotic gene expression in liver myofibroblasts and displayed antifibrotic activity in CCl4-induced liver fibrosis and bleomycin-induced lung fibrosis mouse models. On the basis of the overall pharmacokinetic, pharmacodynamic, and safety profile, GB1211 was selected as the clinical candidate and is currently in phase IIa clinical trials as a potential therapy for liver cirrhosis and cancer.

Galectin-3 inhibitor GB0139 protects against acute lung injury by inhibiting neutrophil recruitment and activation.

Rationale: Galectin-3 (Gal-3) drives fibrosis during chronic lung injury, however, its role in acute lung injury (ALI) remains unknown. Effective pharmacological therapies available for ALI are limited; identifying novel concepts in treatment is essential. GB0139 is a Gal-3 inhibitor currently under clinical investigation for the treatment of idiopathic pulmonary fibrosis. We investigate the role of Gal-3 in ALI and evaluate whether its inhibition with GB0139 offers a protective role. The effect of GB0139 on ALI was explored in vivo and in vitro. Methods: The pharmacokinetic profile of intra-tracheal (i.t.) GB0139 was investigated in C57BL/6 mice to support the daily dosing regimen. GB0139 (1-30 µg) was then assessed following acute i.t. lipopolysaccharide (LPS) and bleomycin administration. Histology, broncho-alveolar lavage fluid (BALf) analysis, and flow cytometric analysis of lung digests and BALf were performed. The impact of GB0139 on cell activation and apoptosis was determined in vitro using neutrophils and THP-1, A549 and Jurkat E6 cell lines. Results: GB0139 decreased inflammation severity via a reduction in neutrophil and macrophage recruitment and neutrophil activation. GB0139 reduced LPS-mediated increases in interleukin (IL)-6, tumor necrosis factor alpha (TNFα) and macrophage inflammatory protein-1-alpha. In vitro, GB0139 inhibited Gal-3-induced neutrophil activation, monocyte IL-8 secretion, T cell apoptosis and the upregulation of pro-inflammatory genes encoding for IL-8, TNFα, IL-6 in alveolar epithelial cells in response to mechanical stretch. Conclusion: These data indicate that Gal-3 adopts a pro-inflammatory role following the early stages of lung injury and supports the development of GB0139, as a potential treatment approach in ALI.