Abstract
The active form of transforming growth factor-β1 (TGF-β1) plays a key role in potentiating fibrosis. TGF-β1 is sequestered in an inactive state by a latency-associated glycopeptide (LAP). Sialidases (also called neuraminidases (NEU)) cleave terminal sialic acids from glycoconjugates. The sialidase NEU3 is upregulated in fibrosis, and mice lacking Neu3 show attenuated bleomycin-induced increases in active TGF-β1 in the lungs and attenuated pulmonary fibrosis. Here we observe that recombinant human NEU3 upregulates active human TGF-β1 by releasing active TGF-β1 from its latent inactive form by desialylating LAP. Based on the proposed mechanism of action of NEU3, we hypothesized that compounds with a ring structure resembling picolinic acid might be transition state analogs and thus possible NEU3 inhibitors. Some compounds in this class showed nanomolar IC50 for recombinant human NEU3 releasing active human TGF-β1 from the latent inactive form. The compounds given as daily 0.1–1-mg/kg injections starting at day 10 strongly attenuated lung inflammation, lung TGF-β1 upregulation, and pulmonary fibrosis at day 21 in a mouse bleomycin model of pulmonary fibrosis. These results suggest that NEU3 participates in fibrosis by desialylating LAP and releasing TGF-β1 and that the new class of NEU3 inhibitors are potential therapeutics for fibrosis.
SIGNIFICANCE STATEMENT The extracellular sialidase NEU3 appears to be a key driver of pulmonary fibrosis. The significance of this report is that 1) we show the mechanism (NEU3 desialylates the latency-associated glycopeptide protein that keeps the profibrotic cytokine transforming growth factor-β1 (TGF-β1) in an inactive state, causing active TGF-β1 release), 2) we then use the predicted NEU3 mechanism to identify nM IC50 NEU3 inhibitors, and 3) these new NEU3 inhibitors are potent therapeutics in a mouse model of pulmonary fibrosis.
Introduction
Fibrosis involves scar-like tissue forming in an internal organ leading to organ failure (Martinez et al., 2017). Pulmonary fibrosis has an incidence of 1 in 400 in the elderly, and there are no Food and Drug Administration–approved therapeutics that stop the progression of this disease (Raghu et al., 2011; Wynn and Ramalingam, 2012; Raghu and Selman, 2015). Transforming growth factor-β1 (TGF-β1) in its active mature form is a key factor that potentiates fibrosis (Fernandez and Eickelberg, 2012; Wolters et al., 2014). TGF-β1 is synthesized as a precursor protein made of latency-associated peptide (LAP) and the active TGF-β1 in a complex called latent TGF-β1 (L-TGF-β1) (Hinck et al., 2016; Robertson and Rifkin, 2016). Two dimerized TGF-β1 precursor proteins are processed to cleave active TGF-β1 from the LAP, with the LAP remaining essentially wrapped around and sequestering the TGF-β1 (Robertson and Rifkin, 2016). Like many other secreted proteins, LAP is glycosylated (Lewandrowski et al., 2006; Moremen et al., 2012; Travis and Sheppard, 2014; Hinck et al., 2016; Robertson and Rifkin, 2016).
Several things can release active TGF-β1 from sequestration by the LAP. Both the integrin αvβ6, which generates mechanical traction on the LAP, and thrombospondin-1 can release active TGF-β1 (Crawford et al., 1998; Munger et al., 1999). Proteases, such as plasmin, thrombin, matrix metalloproteinase-2, and matrix metalloproteinase-9, and reactive oxygen species–mediated conformational changes in LAP, can also release TGF-β1 from the LAP (Lyons et al., 1988; Pociask et al., 2004; Jobling et al., 2006; Jenkins, 2008) However, it is unclear whether these are the only mechanisms that cause upregulation of active TGF-β1 in fibrotic lesions.
Neuraminic acid (also known as sialic acid) is a sugar found at the terminal ends of some glycoproteins (Varki and Gagneux, 2012; Schwab and Nimmerjahn, 2013; Varki et al., 2015). The presence or absence of sialic acid on these glycoconjugates has a central role in various cellular regulatory mechanisms (Varki and Varki, 2007; Zhou et al., 2020). For instance, adding sialic acids to the glycan residues on IgG confers anti-inflammatory functions to IgG (Anthony and Ravetch, 2010). Conversely, the loss of sialic acid from the plasma glycoprotein serum amyloid P attenuates the potency of serum amyloid P to inhibit the differentiation of some human peripheral blood mononuclear cells (PBMCs) to fibrosis-associated fibroblast-like cells called fibrocytes (Cox et al., 2015).
Neuraminidases (also known as sialidases) are enzymes that catalyze a hydrolysis reaction that removes terminal sialic acids from glycoconjugates (Miyagi et al., 2012; Pshezhetsky and Ashmarina, 2013; Smutova et al., 2014). There are four known mammalian sialidases (Miyagi et al., 2012; Smutova et al., 2014). Of the four, the sialidase NEU3 is localized in endosomal structures and on the extracellular side of the plasma membrane and under some conditions can also be released by cells, and thus it can desialylate glycoproteins and glycolipids present outside the cell (Kopitz et al., 2001; Zanchetti et al., 2007; Miyagi and Yamaguchi, 2012; Mozzi et al., 2015; Rodriguez-Walker and Daniotti, 2017). Sialidases, especially NEU3, are upregulated in pulmonary fibrosis (Lambré et al., 1988; Karhadkar et al., 2017; van de Vlekkert et al., 2019). TGF-β1 upregulates NEU3 in human lung epithelial cells, pulmonary fibroblasts, small airway epithelial cells, and PBMCs (Karhadkar et al., 2017). In the epithelial cells, TGF-β1 increases levels of NEU3 by decreasing the NEU3 degradation rate and increasing NEU3 mRNA translation (Chen et al., 2020). Conversely, NEU3 upregulates TGF-β1 in human PBMCs and human pulmonary fibroblasts (Karhadkar et al., 2017). Intratracheal delivery of bleomycin upregulates TGF-β1 in the lungs (Cutroneo et al., 2007; Yue et al., 2010), and is a standard model of pulmonary fibrosis in mice (Moore et al., 2013; Blackwell et al., 2014). Mice lacking NEU3 or wild-type mice treated with sialidase inhibitors do not upregulate TGF-β1 in the lungs after bleomycin treatment and show attenuated bleomycin-induced pulmonary fibrosis (Karhadkar et al., 2017, 2020). Together, these observations suggested that a positive feedback loop in which TGF-β1 upregulates NEU3 and NEU3 upregulates TGF-β1 may potentiate fibrosis (Karhadkar et al., 2017, 2020). How NEU3 upregulates TGF-β1 is unknown.
Bacterial and viral sialidases can desialylate L-TGF-β1, leading to the release of active mature TGF-β1, possibly explaining why there are elevated levels of active TGF-β1 levels during infection with sialidase-expressing pathogens (Miyazono and Heldin, 1989; Miyazono et al., 1991; Schultz-Cherry and Hinshaw, 1996; Carlson et al., 2010; Gratz et al., 2017). There have been many previous attempts to develop inhibitors for viral (von Itzstein, 2007) or human (Cairo, 2014) sialidases. Examples of viral sialidase inhibitors include Tamiflu (oseltamivir) and Relenza (zanamivir), which can attenuate the progression of influenza in patients (Hayden et al., 1997; Monto et al., 1999; Mäkelä et al., 2000; Moscona, 2005). Potent inhibitors for viral neuraminidases exert poor inhibition for human neuraminidases (Hata et al., 2008; Albohy et al., 2011). There are a variety of inhibitors for human sialidases, but all have published IC50 of ∼10 µM for NEU3, making them relatively weak inhibitors (Potier et al., 1979; Magesh et al., 2006; Albohy et al., 2010; Zou et al., 2010; Cairo, 2014; Guo et al., 2018).
In this report, we find that NEU3 desialylates LAP to release active TGF-β1. We then identified a new class of potent NEU3 inhibitors, some with IC50 in the nM range. These compounds inhibit bleomycin-induced pulmonary fibrosis in mice, further supporting the idea that NEU3 inhibitors may be potential therapeutics for fibrosis.
Materials and Methods
Cell Isolation and Culture.
Human peripheral blood was collected from healthy volunteers who gave written consent with specific approval from the Texas A&M University human subjects review board. All methods were performed in accordance with the relevant guidelines and regulations. Blood collection, isolation of PBMCs, and cell culture were done as described previously (Pilling et al., 2009a,b).
Mouse NEU3 Expression in Human Embryonic Kidney 293 (HEK293) Freestyle Cells.
HEK293 freestyle cells (Life Technologies, Grand Island, NY) were cultured in FreeStyle 293 media (12338-018; Life Technologies). Cells (1 × 105) were mixed with 2 μg of 100 μg/ml of murine Neu3 expression clone (MR223297; Origene, Rockville, MD) in 100 μl PBS (GE Lifesciences, Marlborough, MA) and were transfected by electroporation using a 4D-Nucleofector System (Lonza, Walkersville, MD) following the manufacturer’s protocol. The transfected cells were kept at room temperature for 15 minutes for recovery, after which the cells were cultured in 25 ml Freestyle 293 media in a humidified incubator at 37°C with 5% CO2. After 24 hours, 400 μg/ml of G418 (345812; Calbiochem EMD, San Diego, CA) was added to select for transfected cells. After 10 days, the cells were isolated and lysed, and c-Myc–tagged recombinant mouse NEU3 was purified using a Myc-Trap_A kit (ytak-20; Chromotek, Hauppauge, NY) following the manufacturer’s protocol. The eluted protein was stored in 50 μl of 10% glycerol, 100 mM glycine, and 25 mM Tris-HCl, pH 7.3, at 4°C.
Human NEU3 Expression in Escherichia Coli.
The human NEU3 cDNA sequence was amplified from a human NEU3 open reading frame clone (RC216537; Origene). The amplified NEU3 sequence was inserted between the NdeI and EcoRI sites of the pMAL-c5X vector (N8108S; NEB, Ipswich, MA). The resulting construct encoding a fusion of maltose-binding protein (MBP) and NEU3 was transformed into chemically competent E. cloni cells (Lucigen, Middleton, WI) mixed in 37°C prewarmed LB broth (240230; BD, Franklin Lakes, NJ); grown with shaking at 37°C, 240 rpm for 1 hour; and plated on LB agar with ampicillin (100 μg/ml). Clones were selected and grown in 100 μg/ml ampicillin LB broth medium, and the plasmids were isolated using a GeneJET Plasmid Miniprep Kit (K0502; Thermo Scientific, Waltham, MA). Clones were verified by sequencing (Eton Bioscience, San Diego, CA). The sequence-verified clones were transformed into chemically competent BL21(DE3) E. coli cells (Lucigen); grown with shaking at 37°C, 240 rpm, for 1 hour in prewarmed LB broth; and plated on LB agar with 100 μg/ml ampicillin. Individual colonies were selected and added to 10 ml prewarmed LB broth with 100 μg/ml ampicillin and grown at 37°C, 240 rpm, for no longer than 16 hours. The culture was added to 1 l of LB broth and incubated at 37°C, 240 rpm, until (optical density measured at wavelength 600 nanometers) OD600 = ∼0.6. To induce protein expression, isopropyl β- d-1-thiogalactopyranoside (Goldbio, St. Louis, MO) solution was added to 0.5 mM and incubated at 20°C at 240 rpm overnight. Protein purification was done as described previously (Albohy et al., 2010) with the following modifications. Bacterial cells were harvested after 16–18 hours of induction by centrifugation at 4000g at 4°C. The pellet was resuspended in resuspension buffer (20 mM Tris, pH 7.2; 300 mM NaCl; 1 mM EDTA; and 0.1% Triton X-100) supplemented with 1× protease inhibitor (PIA32965; VWR, Radnor, PA). The lysate was sonicated three times for 3 minutes with the tube submerged in ice at setting 16 of a Microson ultrasonic cell disruptor (Misonix Incorporated, Farmingdale, NY) and then clarified by centrifugation at 10,000g for 20 minutes at 4°C. The supernatant was loaded onto an amylose column (New England Biolabs) equilibrated with 20 mM Tris/300 mM NaCl, pH 7.2. MBP-NEU3 was eluted with running buffer containing 10% glycerol (v/v) and 20 mM maltose.
Sialidase Effects on LAP Assay.
To determine the effect of sialidases on LAP, 400 ng/ml human recombinant LAP (246-LP/CF; R&D Systems, Minneapolis, MN) was incubated with and without 200 ng/ml of human recombinant sialidases NEU1 (TP300386; Origene), NEU2 (TP319858; Origene), NEU3 (TP316537; Origene), or NEU4 (TP303948; Origene) or Clostridium perfringens neuraminidase (N2876-2.5UN; Sigma-Aldrich, St. Louis, MO) in a total volume of 20 μl of 1× PBS (SH30256.01; GE Lifesciences), pH 6.9. The PBS was adjusted to pH 6.9 with 12N HCl (H613-05; Avantor Performance Materials, Radnor Township, PA). The reaction mixtures were incubated at 37°C for 2 hours. After incubation, 5 μl of 4× Laemmli’s buffer (GTX16355; GeneTex, Irvine, CA) containing 20 mM dithiothreitol was added to the reaction mixtures, and these were heated at 100°C for 5 minutes. Fifteen microliters of the heated reaction mixture was electrophorized on a 4–20% Tris/glycine polyacrylamide precast gel (12001-058; VWR) in Tris/glycine/SDS running buffer (13.5 g Tris base, 7.2 g glycine, 5 g SDS/l) at room temperature for 90–120 minutes.
Western blots and staining the blots with lectin were done as previously described (Cox et al., 2015), and immunostaining Western blots for NEU3 was done as previously described (Karhadkar et al., 2017, 2020). Silver staining of gels was done following (Morrissey, 1981). Staining intensity was quantified by Image Laboratory software (Bio Rad, Hercules, CA).
NEU3 Induced L-TGF-β1 Activation Assay.
To determine whether NEU3 can cause L-TGF-β1 to release active TGF-β1, 200 ng/ml recombinant human L-TGF-β1 (299-LT/CF; R&D Systems) was incubated with varying concentrations of sialidases with or without 1 mM calcium chloride in a total volume of 100 µl of PBS, pH 6.9, in a 96-well microplate. The microplates were covered with aluminum foil and incubated at 37°C for 2 hours. After incubation, the reaction mixtures were assayed using an active TGF-β1 ELISA kit (DY240; R&D Systems) following the manufacturer’s protocol with the exception that the reaction mixtures were not processed for acid treatment so as to measure only active TGF-β1 and not total TGF-β1. The absorbance was read with a SynergyMX plate reader (BioTek, Winooski, VT). To determine the enzyme kinetics of NEU3 with L-TGF-β1 as a substrate, varying concentrations of L-TGF-β1 were incubated with 100 ng/ml of recombinant human NEU3 (Origene) in a total volume of 100 μl, and the samples were processed as described above.
Sialidase Activity Assay with 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic Acid Sodium Salt Hydrate.
Sialidase assays were done following Potier et al. (1979) and Marathe et al. (2013) with the following modifications. PBS was adjusted to pH 6.9 with 12N HCl, and bovine serum albumin (BSA) (VWR) was added to a final concentration of 100 μg/ml (PBS-BSA). Varying concentrations of bacterially expressed recombinant human NEU3 were prepared in PBS-BSA. A final concentration of 200 μM 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (4MU-NANA) (M8639; Sigma-Aldrich) was added to the NEU3 in a total volume of 100 μl in a 96-well plate. When indicated, inhibitors were added for 30 minutes before adding the 4MU-NANA. The reactions were incubated at 37°C for 30 minutes, and the fluorescence was measured at 37°C in a prewarmed SynergyMX plate reader with excitation at 360 nm and emission at 440 nm. The fluorescence in the absence of sialidases was subtracted from all readings.
Inhibition of NEU3 Induced L-TGF-β1 Activation.
2,3-didehydro-2-deoxy-N-acetyl-neuraminic acid (DANA) (252926-10MG) was purchased from EMD-Millipore (Burlington, MA), Oseltamivir phosphate (Tamiflu) was purchased from (1479304) Sigma, 2-acetylpyridine (2AP) (sc-254121) was purchased from Santa Cruz Biotechnology (Dallas, TX), and methyl picolinate (MP) (sc-228575) was purchased from Santa Cruz. 4-Amino-1-methyl-2-piperidinecarboxylic acid (AMPCA) (A00285-13785-026) was synthesized by Sundia (Shanghai, China) following the scheme shown in Supplemental Fig. 1. These compounds were dissolved in water to 20 mM. All stocks were stored at 4°C and used within 2 weeks of preparation. Dilution series of the compounds were made in PBS, pH 6.9. Diluted compound (100 µl) was added to the well of a 96-well plate; then 50 µl of 400 ng/ml recombinant human sialidase NEU3 (TP316537; Origene) in PBS, pH 6.9, was added to each well (50 µl PBS, pH 6.9, for the control); and the plate was incubated for 30 minutes at 37°C. Fifty microliters of 800 ng/ml L-TGF-β1 (299-LT/CF; R&D Systems) in PBS, pH 6.9, was then added to the well. The plate was covered with aluminum foil and incubated for 2 hours at 37°C, and released active TGF-β1 was assayed as described above.
Inhibition of NEU3 Induced Interleukin-6 Accumulation.
Dilutions of the above compounds were made in serum-free medium prepared as previously described (Pilling et al., 2009b; Karhadkar et al., 2020), 50 µl of diluted compound was added to the well of a 96-well plate, then 50 µl of 400 ng/ml sialidase in serum-free medium was added to each well (50 µl serum-free medium for control), and the plate was incubated for 30 minutes at 37°C in a humidified incubator with 5% CO2. PBMCs (100 µl) at 1 × 105 cells/ml were then added to the well to make the total volume in a well 200 μl with 1 × 104 cells/well, and this was incubated at 37°C in a humidified incubator with 5% CO2 for 24 or 48 hours. Interleukin-6 (IL-6) in the culture supernatant was then assayed with an IL-6 ELISA kit (430501; BioLegend, San Diego, CA) following the manufacturer’s protocol, reading absorbance with a SynergyMX plate reader (BioTek). At least three different donors were used for each assay.
Mouse Model of Pulmonary Fibrosis.
To determine whether the NEU3 inhibitors affect pulmonary fibrosis in mice, 8–10-week-old 26–30 g male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were given an oropharyngeal aspiration of 3 U/kg bleomycin (2246-10; BioVision Incorporated, Milpitas, CA) in 50 µl of 0.9% saline to induce symptoms of pulmonary fibrosis or oropharyngeal saline alone as a control as previously described (Pilling and Gomer, 2014; Pilling et al., 2014; Cox et al., 2015). Starting 10 days after bleomycin had been administered, some of the bleomycin-treated mice were given daily intraperitoneal injections of 100 μl of PBS or 1 mg/kg 2-acetyl pyridine, 1 mg/kg methyl picolinate, or 0.1 mg/kg of 4-amino-1-methyl-2-piperidinecarboxylic acid in 100 µl PBS. All mice were weighed daily for 21 days after bleomycin was administered. At day 21, mice were euthanized by CO2 inhalation, and bronchoalveolar lavage (BAL) fluid and cell spots of BAL cells were obtained as previously described (Daubeuf and Frossard, 2012; Pilling and Gomer, 2014; Pilling et al., 2014; Cox et al., 2015). The liver, heart, kidneys, spleen, white fat tissue, and brown fat tissue were removed and weighed. The total cells from BAL cell spots were quantified as described previously (Daubeuf and Frossard, 2012; Pilling and Gomer, 2014). The lungs were removed and inflated with Surgipath frozen section compound (3801480; Leica, Buffalo Grove, IL) and preserved at −80°C. Cryosections of lungs (6–10 µm) were placed on glass slides (48311-703; VWR) and were air-dried for 48 hours before use. The mouse experiments were performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of National Institutes of Health. The Texas A&M University Animal Use and Care Committee approved the protocol (IACUC 2017-0414 (Institutional Animal Care And Use Committee 2017-0414)).
Histology, Immunohistochemistry, and BAL Analysis.
Air-dried cryosections were stained with hematoxylin and eosin or with Sirus red to detect collagen or with 1 μg/ml antiactive TGF-β1 antibodies (AB-100-NA; R&D Systems) as described previously (Pilling et al., 2007; Karhadkar et al., 2017). The staining was quantified with ImageJ (Schneider et al., 2012; Jensen, 2013). Immunohistochemistry on BAL cell spots and cryosections was performed as described previously (Pilling and Gomer, 2014; Pilling et al., 2014; Cox et al., 2015; Karhadkar et al., 2020) using anti-CD11b (101202, clone M1/70 BioLegend) to detect blood and inflammatory macrophages, anti-CD11c (M100-3, clone 223H7; MBL International, Woburn, MA) to detect alveolar macrophages and dendritic cells, anti-CD45 (103102, clone 30-F11; BioLegend) for total leukocytes, anti-Ly-6G (127602, clone 1A8; BioLegend) to detect neutrophils, and anti-Ly-6C (128001, clone HK1.4; BioLegend) to detect a variety of inflammatory immune system cells, with isotype-matched irrelevant antibodies (BioLegend) as controls. SDS-PAGE analysis of BAL and nanodrop protein assays were performed as described previously (Pilling et al., 2003; Karhadkar et al., 2017, 2020). Western blots stained for albumin were done as described previously (Karhadkar et al., 2020).
Hydroxyproline Assay.
Hydroxyproline assays were performed as described previously (Karhadkar et al., 2020) with the exception that approximately half of lobes of lungs frozen in optimal cutting temperature compound (OCT; Fisher) were cut off in three pieces, thawed, and washed three times with 0.5 ml PBS to remove optimal cutting temperature by centrifugation at 2000g for 5 minutes in preweighed Eppendorf tubes. After the last centrifugation, the tubes were kept inverted for 5 minutes to allow PBS to blot onto blotting paper, and the tissue was then weighed. Tissues were then processed using a hydroxyproline quantification kit (MAK008-1KT; Sigma-Aldrich) following the manufacturer’s directions.
Statistics.
Data were analyzed by t test or ANOVA using Prism 7 (GraphPad, La Jolla, CA). Significance was defined as P < 0.05. IC50 curves for enzyme assays were generated using the dose response–inhibition curve-fitting model with variable slope with Prism 7. Based on previously analyzed confidence intervals and historical data and using G*Power power analysis software (Faul et al., 2007, 2009), a sample size of three mice per group was determined sufficient to detect the effect sizes anticipated in the present experiments.
Results
Recombinant Human NEU3 Desialylates Recombinant Human LAP and Releases Active TGF-β1.
Neuraminidases from influenza virus and bacteria remove sialic acid from the LAP protein, which causes the LAP to release active TGF-β1 (Schultz-Cherry and Hinshaw, 1996; Carlson et al., 2010; Gratz et al., 2017). To determine the effect of recombinant human sialidases on recombinant human LAP (rhLAP), we incubated recombinant human sialidases (made in HEK293 cells) or C. perfringens neuraminidase (Cox et al., 2015) with rhLAP. Western blots of the rhLAP were stained with Sambucus nigra lectin to detect sialic acids (Shibuya et al., 1987; Feng et al., 2013). NEU3, NEU4, and C. perfringens neuraminidase significantly decreased sialic acid on rhLAP (Fig. 1, A and B).
Recombinant human NEU3 releases active TGF-β1 by desialylating recombinant human LAP. (A) Recombinant human LAP protein was incubated with or without recombinant human sialidases NEU1–4 or C. perfringens (C. per) neuraminidase, and a Western blot was stained with S. nigra lectin (SNA) (top panel) to detect sialic acids on glycoconjugates. Black bands are the LAP protein. Silver staining (bottom panel) was done as a loading control. Blots and silver staining are representative of three independent experiments. Degradation products from the sialidases are also visible in the silver-stained gel in addition to the LAP protein. (B) The Western blots from (A) were quantified for S. nigra staining of LAP protein. Values are mean ± S.E.M., n = 3. **P ≤ 0.01 (one-way ANOVA, Bonferroni’s test compared with control). (C) An ELISA assay, specific for active TGF-β1, was performed on rhL-TGF-β1 treated with the indicated concentrations of recombinant human NEU3. Values are mean ± S.E.M., n = 6. Purified bacterially expressed recombinant human MBP-NEU3 was assayed for effects on (D) rhL-TGF-β1 and (E) hydrolysis of the substrate 4MU-NANA to release fluorophore 4MU (4-Methylumbelliferone). Values are mean ± S.E.M., n ≥ 3. RFU - Relative fluorescence units. (F) Release of active TGF-β1 as a function of time by 1.94 nM recombinant human NEU3 from 3 nM rhL-TGF-β1. Values are mean ± S.E.M., n = 4. (G) Kinetics of recombinant human NEU3 activity in the TGF-β1 release assay. Values are mean ± S.E.M., n = 4.
We previously found that the addition of recombinant human NEU3 (rhNEU3) to human immune cells, pulmonary fibroblasts, and small airway epithelial cells causes them to upregulate extracellular and intracellular TGF-β1 (Karhadkar et al., 2017). To determine whether NEU3 causes recombinant human latent TGF-β1 (rhL-TGF-β1) to release active TGF-β1, we incubated rhL-TGF-β1 with rhNEU3 and measured released active TGF-β1. rhNEU3 did cause a release of active TGF-β (Fig. 1C). In the presence or absence of calcium [which potentiates the activity of some viral sialidases (Dimmock, 1971; Shtyrya et al., 2009)], rhNEU1 or rhNEU2 did not cause significant release of active TGF-β1 (Supplemental Fig. 2, A and B). rhNEU3 and C. perfringens neuraminidase released active TGF-β1, and NEU4 caused some release of TGF-β1 (Supplemental Fig. 2, A and B). Together, these results suggest that NEU3 removes sialic acid from the LAP protein and efficiently releases active TGF-β1 from rhL-TGF-β1.
The standard assay for sialidases is cleavage of the fluorogenic substrate 4MU-NANA (Potier et al., 1979). At concentrations of rhNEU3 up to 100 ng/ml (1.94 nM) and using 4MU-NANA with no enzyme as a control, we were unable to detect statistically significant activity of rhNEU3 on 4MU-NANA. Compared with commercially available HEK293 cell–synthesized rhNEU3, we were able to purify much higher amounts of a bacterially expressed fusion of the 42 kDa maltose-binding protein with the 51 kDa human NEU3 (brhNEU3; bacterially expressed recombinant human NEU3); Supplemental Fig. 3, A and B). The ∼93 kDa brhNEU3 contained a contaminant band that stained weakly for NEU3 on Western blots (Supplemental Fig. 3, A and B), so the concentration of bhrNEU3 was estimated by comparison of the ∼93-kDa band to BSA standards on silver-stained gels. The brhNEU3 caused a release of active TGF-β1 from rhL-TGF-β1, albeit with somewhat lower efficacy than rhNEU3 (Fig. 1, C and D). Compared with rhL-TGF-β1 as a substrate, a much higher concentration of brhNEU3 was required to obtain detectable cleavage of 4MU-NANA (Fig. 1, C–E).
A previous study with 4MU-NANA as the substrate for NEU3 reported a Km of 45 ± 3 μM and a kcat of 0.33 ± 0.02 minute−1 and thus a catalytic efficiency kcat/Km of 0.007 ± 0.006 µM−1 minute−1 (Albohy et al., 2010). Using rhL-TGF-β1 as the substrate for rhNEU3 and measuring the rate of TGF-β1 release for different concentrations of rhL-TGF-β1 (Fig. 1, F and G), we observed a Km of 1.5 ± 0.5 nM and a kcat of (4.0 ± 0.4) × 10−5 minute−1 (both mean ± S.E.M., n = 4) and thus a kcat/Km of 0.026 ± 0.008 µM−1 minute−1. The lower Km and higher kcat/Km for rhL-TGF-β1 compared with 4MU-NANA suggest a higher affinity of NEU3 for rhL-TGF-β1 compared with 4MU-NANA as a substrate. Together, these results suggest that rhL-TGF-β1 can be used as a better substrate than 4MU-NANA for a more sensitive assay to determine NEU3 activity.
Design of NEU3 Inhibitors.
We designed possible inhibitors of human NEU3 based in part on the structure of DANA (Fig. 2A), a known inhibitor of NEU3 (Ki = 30 μM) (Albohy et al., 2011). DANA contains a double bond, of which the sp2-hybridized carbon-2 (circled) may be a structural mimic of the putative oxacarbenium ion intermediate (OCI) (Fig. 2, A and B) of the chemical mechanism of enzymatic catalysis (Albohy et al., 2010). Because DANA lacks the positive charge of the OCI, a better mimic of this putative reaction intermediate is likely afforded by the tetrahydropyridine-1-ium-2-carboxylate (Fig. 2C), which provides the positive charge with retention of sp2-hybridized carbon-2. Consequently, we pursued substituted picolinic acids, which contain both a cationic nitrogen atom and sp2-hybridization at carbon-2 (Fig. 2D) as well as a substituted pipecolinic acid (compounds 3–5 in Table 1).
Schematic representation of the rationale for design of NEU3 inhibitors. Structures of (A) DANA, (B) the predicted OCI, (C) tetrahydropyridine-1-ium-2-carboxylate (THPC), and (D) a substituted picolinic acid analog of OCI (sPCA).
Compounds used in NEU3 activity assays
List of commercially available compounds used in the NEU3-induced release of active TGF-β1 assay and the cell-based NEU3-induced upregulation of IL-6 assay to determine IC50. The IC50 values are mean ± S.E.M., n ≥ 3.
Compounds 3–5 Inhibit NEU3 Release of TGF-β1.
To test the hypothesis that compounds 3–5 inhibit NEU3, we assayed their effects in the rhNEU3–rhL-TGF-β1 release of active TGF-β1 reaction (Supplemental Fig. 4; Table 1). Picolinates like 2AP and MP had higher potencies of inhibiting rhNEU3 activity compared with DANA and oseltamivir phosphate (Tamiflu) (Supplemental Fig. 4, A–D and F–I; Table 1), which supported our rationale for potential NEU3 inhibitors. The fully saturated pipecolinic acid compound, AMPCA, was also a very potent inhibitor of NEU3 (Supplemental Fig. 4, E and J; Table 1) despite its lack of an adjacent sp2-hybridized carbon as in the case of the picolinate inhibitors. These results suggest the cationic nitrogen and an adjacent keto or carboxylate group provided the essential structural elements to effect potent inhibition of rhNEU3, possibly via mimicry of the enzymatic transition state structure, and that these inhibitors are significantly improved over DANA and oseltamivir when rhL-TGF-β1 is the substrate.
Compounds 3–5 Inhibit NEU3-Induced Extracellular Accumulation of IL-6 by Human Immune Cells.
rhNEU3 upregulates extracellular accumulation of IL-6 from human PBMCs (Karhadkar et al., 2020). To determine whether other sialidases have an effect on IL-6 production by PBMCs, we cultured human PBMCs with recombinant human NEU1–4 and measured extracellular accumulation of IL-6 in media supernatants after 24 and 48 hours. At 48 hours, compared with controls only rhNEU3 significantly upregulated extracellular accumulation of IL-6 (Supplemental Fig. 5). We measured the effects of inhibitors in an NEU3-induced IL-6 release by human immune cells assay (Supplemental Fig. 6; Table 1). DANA, Tamiflu, 2AP, and MP all inhibited rhNEU3 in the cell-based NEU3-induced IL-6 release assay (Supplemental Fig. 6, A–D and F–I; Table 1). Compared with the other compounds, the pipecolinate AMPCA had an increased inhibition potency (Supplemental Fig. 6, E and J; Table 1). These results suggest that some picolinates and a pipecolinate can be used as inhibitors against NEU3 in cell-based or in vivo assays. The differences in inhibition potencies in the cell-based extracellular IL-6 accumulation assay and in vitro TGF-β1–based assay may be due to the presence or absence of factors that interact with NEU3 and/or the inhibitors in the cell-based IL-6 assay compared with the buffer-based TGF-β1 release assay.
NEU3 Inhibitors Inhibit Mouse NEU3.
To determine whether compounds 3–5 would be effective in mice, we tested them against recombinant mouse NEU3 in the NEU3-induced release of active TGF-β1 assay. The IC50 of 2AP was 0.8 ± 1.7 µM, MP was 3.2 ± 1.6 nM, and AMPCA was less than 1 nM (Supplemental Fig. 7, A–F). These results suggest that the three selected NEU3 inhibitors are inhibitors of mouse NEU3.
AMPCA Attenuates Weight Loss of Mice after Bleomycin Treatment.
To determine whether NEU3 inhibitors affect bleomycin-induced pulmonary fibrosis, C57BL/6 mice were treated with an oropharyngeal aspiration of saline or bleomycin, and then starting 10 days after saline or bleomycin, mice were given daily intraperitoneal injections of 1 mg/kg 2AP or MP or 0.1 mg/kg AMPCA. There was no discernable effect of any of the compounds on the appearance or behavior of the mice. As previously observed, compared with saline-treated control mice, bleomycin-treated control mice had lower body weights at day 21 after bleomycin aspiration (Fig. 3A). In saline-treated mice, 2AP, MP, and AMPCA did not significantly affect body weights. AMPCA attenuated the bleomycin-induced weight loss (Fig. 3A). 2AP, MP, and AMPCA did not significantly affect liver, heart, kidneys, spleen, white fat, or brown fat weights as a percent of total body weight (Supplemental Fig. 8, A–F). There was no discernable effect of any of the compounds on the appearance of any internal organs other than the lungs, which had large white patches with bleomycin alone and a normal pink color with the inhibitors without bleomycin. With bleomycin, the lungs of mice treated with 2AP or AMPCA were a normal pink color and showed occasional small white patches in mice treated with MP. Together these results suggest that AMPCA attenuates bleomycin-induced weight loss in mice.
AMPCA attenuates bleomycin-induced decline in body weight and protein increase in the BAL fluid at day 21 after bleomycin treatment. (A) Percent change in body weight after saline (S) or bleomycin (B) treatment at day 0 and then at day 10 starting daily intraperitoneal injections of PBS (Ctrl), 2AP, MP, or AMPCA. Values are mean ± S.E.M., n = 3. *P ≤ 0.05 (t test). BAL fluid was collected at day 21 after saline or bleomycin treatment and 2AP, MP, or AMPCA intraperitoneal injections. (B) BAL fluids from the indicated mice were analyzed by PAGE gels stained with Coomassie. Molecular mass markers in kDa are at left and in the center. BSA at 0.01, 0.1, 1.0, and 10.0 μg was loaded at right on each gel. (C) Quantification of protein by densitometry in lanes from (A), using the BSA band densities as standards and then for each mouse multiplying by the BAL volume to obtain total BAL protein. (D) Quantification of protein from BAL as in (B) using a nanodrop assay to obtain BAL protein concentrations and then for each mouse multiplying by the BAL volume to obtain total BAL protein. For (B and C), values are mean ± S.E.M., n = 3. *P < 0.05 (t test). (E) BAL volumes. Values are mean ± S.E.M., n = 3.
AMPCA Attenuates Protein Increase in the BAL Fluid at Day 21 after Bleomycin Treatment.
Bleomycin causes an increase in protein levels in the BAL fluid from mouse lungs (Parker and Townsley, 2004; Kulkarni et al., 2013; Pilling and Gomer, 2014). To determine whether inhibiting NEU3 affects these BAL protein levels, we measured the protein in the BAL as a measure of lung tissue damage post–bleomycin treatment. As observed previously (Parker and Townsley, 2004; Kulkarni et al., 2013; Pilling and Gomer, 2014; Karhadkar et al., 2020), at day 21, compared with saline-treated control mice, bleomycin-treated control mice showed upregulated BAL protein levels (Fig. 3, B–D). Compared with saline-treated control, the saline-treated 2AP-, MP-, or AMPCA-injected mice had no significant difference in the levels of proteins in the BAL as assayed by either gel densitometry (Fig. 3, B and C) or nanodrop (Fig. 3D). AMPCA treatment significantly attenuated the bleomycin-induced increase of protein levels in the BAL (Fig. 3, B–D). Although 2AP and MP seemed to decrease BAL protein levels in bleomycin-treated mice, the effect was not statistically significant. As reported previously (Parker and Townsley, 2004; Karhadkar et al., 2020), the bleomycin-induced protein in BAL appeared to be mainly albumin, which was confirmed by an immunoblot with anti-albumin antibodies (Supplemental Fig. 9, A and B). No significant differences were observed in the BAL volumes from the mice in the different groups (Fig. 3E). We previously observed that Tamiflu treatment but not DANA treatment attenuated bleomycin-induced increased protein levels in the BAL (Karhadkar et al., 2017). Together, these results suggest that some NEU3 inhibitors can attenuate bleomycin-induced upregulation of protein levels in the BAL.
NEU3 Inhibitors Attenuate the Increased Number of Inflammatory Cells in the BAL at Day 21 after Bleomycin Treatment.
Bleomycin aspiration also causes an upregulation of inflammatory cells in the BAL at day 21 (Murray et al., 2010; Pilling and Gomer, 2014; Pilling et al., 2014; Cox et al., 2015). Compared with saline-treated control mice, the bleomycin-treated control mice showed upregulated BAL cell counts (Fig. 4A). The saline-treated control mice and and saline-treated and 2AP-, MP-, or AMPCA-injected mice showed similar BAL cell counts (Fig. 4A). Compared with saline-treated mice, bleomycin-treated and MP- or AMPCA-injected mice showed upregulated BAL cell counts, but these were significantly less compared with bleomycin-treated control mice (Fig. 4A). In the BAL of bleomycin-treated control mice, as previously observed (Karhadkar et al., 2020), bleomycin increased the number of CD11b+ cells (Fig. 4 B), CD11c+ cells (Fig. 4C), CD45+ cells (Fig. 4D), and Ly6G positive cells (Fig. 4E). Significant differences were not observed for Ly6C positive cells (Fig. 4F). The BAL of saline-treated control mice and saline-treated and 2AP-, MP-, or AMPCA-treated mice showed similar counts of the above cell types (Fig. 4, B–F). Compared with BAL from saline-treated mice, bleomycin-treated 2AP- or AMPCA-injected mice showed upregulated counts of CD11b positive cells (Fig. 4B), CD11c positive cells (Fig. 4C), and CD45 positive cells (Fig. 4D) in the BAL, but these counts were significantly less compared with bleomycin-treated control mice. The MP-injected mice did not show significantly increased counts of the above cell types in the BAL of saline-treated or bleomycin-treated mice (Fig. 4, B–F). Together, these data suggest that NEU3 inhibitors attenuate the bleomycin-induced increase of inflammatory cell counts in the BAL.
2AP, MP, or AMPCA from day 10 after bleomycin treatment decrease BAL cells. (A) The total number of cells in mouse BAL after the indicated treatment. S indicates saline and B indicates bleomycin. Values are mean ± S.E.M., n ≥ 3 mice per group. **P < 0.01; ***P < 0.001; ****P < 0.0001 (one-way ANOVA, Bonferroni’s test). #P < 0.05; ##P < 0.01 (t test). BAL cell spots at day 21 were stained for the markers (B) CD11b, (C) CD11c, (D) CD45, (E) Ly6G, and (F) Ly6C. The percent of cells stained was determined in five randomly chosen fields of 100–150 cells, and the percentage was multiplied by the total number of BAL cells for that mouse to obtain the total number of BAL cells staining for the marker. Values are mean ± S.E.M., n = 3. *P < 0.05; ***P < 0.001; ****P < 0.0001 (one-way ANOVA, Bonferroni’s test). #P < 0.05; ##P < 0.01; ###P < 0.001 (t test). Ctrl, Control.
Lung sections were also stained to detect inflammatory cells. As previously observed (Karhadkar et al., 2020), bleomycin increased the counts of CD11b+, CD11c+, and CD45+ cells remaining in the lungs after BAL (Fig. 5, A–C) but did not affect the counts of Ly6G+ or Ly6C+ cells (Fig. 5, D–E). 2AP, MP, or AMPCA did not affect the counts of CD11b+ and CD11c+ cells (Fig. 5, A and B), but 2AP and MP significantly counteracted the increase in CD45+ cells in the lungs after BAL at day 21 (Fig. 5C). Together, these results suggest that some NEU3 inhibitors attenuate the upregulation of some leukocytes remaining in the lungs after BAL at day 21 post–bleomycin treatment.
Remaining immune cells in lungs post-BAL. Cryosections of mouse lungs were stained for (A) CD11b, (B) CD11c, (C) CD45, (D) Ly6G, and (E) Ly6C, and cells in five randomly chosen 0.45 mm–diameter fields of view were counted, and the number was then calculated to number per squared millimeter. Values are mean ± S.E.M., n = 3. *P < 0.05 (one-way ANOVA, Bonferroni’s test). #P < 0.05 (t test). B, bleomycin; S, saline
NEU3 Inhibitors Decrease Fibrosis.
To determine whether inhibiting NEU3 can decrease bleomycin-induced fibrosis, lung sections were stained with hematoxylin and eosin to detect tissue and picrosirius red to detect total collagen, and hydroxyproline levels were measured in the lung tissue lysates. As previously observed (Adamson and Bowden, 1974; Izbicki et al., 2002; Walters and Kleeberger, 2008; Moore et al., 2013), bleomycin treatment caused fibrosis in the mouse lungs at day 21 (Fig. 6, A–D and Supplemental Fig. 10). Compared with saline treatment, lungs from mice exposed to bleomycin and then treated with MP showed increased Sirius red staining and hydroxyproline levels at day 21 (Fig. 6, B–D). The Sirius red staining and hydroxyproline levels from bleomycin-treated and then 2AP-, MP-, or AMPCA-treated mice were significantly less than those from the bleomycin-treated control mice (Fig. 6, C and D; Supplemental Fig. 10). The bleomycin-treated and then 2AP-, MP-, or AMPCA-treated mice showed reduced staining for active TGF-β1 in the lung sections compared with bleomycin-treated control mice (Fig. 7, A and B). These results suggest that NEU3 inhibitors reduce bleomycin-induced fibrosis and TGF-β1 upregulation in mouse lungs.
2AP, MP, or AMPCA injections starting at day 10 after bleomycin (Bleo) attenuate fibrosis. Sections of lung tissue from mice treated with saline or bleomycin, then injected daily with control or 2AP or MP or AMPCA starting at day 10 after bleomycin, and then euthanized at day 21 were stained with (A) H&E and (B) picrosirius red to show collagen content. All images are representative of three mice per group. (C) Hydroxyproline in lungs. Values are mean ± S.E.M., n = 3. *P < 0.05; **P < 0.01 (one-way ANOVA, Bonferroni’s test). #P < 0.05 (t test). (D) Picrosirius red quantification analyzed by selecting five random fields of view of the sizes shown in (B) for each mouse, and the avg. percent area of the field of view showing staining for each mouse was measured. Values are mean ± S.E.M., n = 3. ****P < 0.0001 (one-way ANOVA, Bonferroni’s test). #P < 0.05 (t test). Ctrl, Control.
2AP, MP, or AMPCA injections starting at day 10 after bleomycin reduces TGF-β1 staining. (A) Sections of lung tissue from mice treated with saline or bleomycin, then injected daily with control or 2AP or MP or AMPCA starting at day 10 after bleomycin, and then euthanized at day 21 were stained with antibodies against TGF-β1. All images are representative of three mice per group. Pink is staining, blue is counterstain. (B) Quantification of staining analyzed by selecting three random fields of view of the sizes shown in (A) for each mouse, and the avg. percent area of the field of view showing staining for each mouse was measured. Values are mean ± S.E.M., n = 3. *P < 0.05 (t test). B, bleomycin; S, saline; Ctrl, Control.
Discussion
In this report, we observed that the human sialidase NEU3 desialylates human LAP and causes release of active TGF-β1. This is in agreement with the observation that LAP desialylation by viral and bacterial sialidases (Carlson et al., 2010; Gratz et al., 2017) causes a conformational modification of the LAP, which results in disruption of the interaction between LAP and active TGF-β1, releasing active TGF-β1 (Biernacka et al., 2011). Compared with the more conventional mechanism of a cell sensing a signal and activating a gene expression pathway to make and secrete a responding signal, the desialylation of LAP and release of active TGF-β1 are a much more rapid response. This then suggests that fibrosis is associated with a mechanism that originally evolved to rapidly respond to an insult, such as a sialidase-expressing pathogen, with the extracellular NEU3 mimicking and thus amplifying the pathogen signal.
NEU1 may also be involved in potentiating pulmonary fibrosis (Luzina et al., 2016), but this sialidase did not significantly desialylate LAP, cause TGF-β1 release, or induce IL-6 accumulation by PBMCs, suggesting that it may act by other mechanisms to promote fibrosis. NEU2 also did not significantly desialylate LAP, cause TGF-β1 release, or induce IL-6 accumulation by PBMCs. NEU4 did desialylate LAP and caused modest amounts of TGF-β1 release and IL-6 accumulation, but since this sialidase is present in lysosomes (Seyrantepe et al., 2004) or mitochondria and endoplasmic reticulum (Yamaguchi et al., 2005; Bigi et al., 2010), it either does not normally desialylate LAP or NEU4 from necrotic or otherwise lysed cells may normally desialylate LAP.
We used a predicted reaction intermediate from the NEU3 reaction mechanism (Albohy et al., 2010, 2011) to test the idea that picolinic acid–like compounds containing a cationic nitrogen replacing the putative positively charged oxygen of the reaction intermediate could act as NEU3 inhibitors. In both the TGF-β1 release from LAP and the IL-6 production by PBMC assays, we observed significantly improved inhibition potency of AMPCA compared with DANA or oseltamivir (Table 1). Since some tumors have upregulated NEU3 (Kakugawa et al., 2002; Nomura et al., 2006; Ueno et al., 2006; Kawamura et al., 2012) and since mice lacking Neu3 are resistant to colitis-associated colon tumor formation (Yamaguchi et al., 2012), AMPCA or similar picolinate compounds may useful to treat NEU3-associated tumors.
In support of the idea of using picolinate compounds against NEU3-associated tumors, a previous study found antiproliferative and therapeutic effects of picolinic acid on tumor cells (Ruffmann et al., 1987). Picolinic acid shows cytotoxic activity against virus-infected cells and also decreases viral replication (Fernandez-Pol and Johnson, 1977; Fernandez-Pol et al., 2001). Picolinic acid potentiates the antimicrobial activities of various fluoroquinolones by chelating metal ions (Cai et al., 2006; Shimizu and Tomioka, 2006). Picolinic acid functions as a second signal in enhancing interferon-γ–induced macrophage activation (Varesio et al., 1990; Melillo et al., 1994) and has neuroprotective, immune regulatory, and tumor suppression effects in human neurons (Guillemin et al., 2007). These reports, and our studies suggest that picolinic acid, a metabolite of tryptophan degradation (Varesio et al., 1990; Badawy, 2017), and picolinates may be useful as therapeutics.
NEU1 and NEU2 did not significantly desialylate LAP or release active TGF-β1 from rhL-TGF-β1, and thus this assay was incompatible for determining inhibition potencies of our compounds against NEU1 and NEU2. The extracellular pH of lesions in lung fibrosis ranges from 6.45 to 6.95 (Jones et al., 2015). Our repeated efforts to assay NEU1, NEU2, and NEU4 in this pH range with the substrate 4MU-NANA were unsuccessful. Thus, it is unclear whether our compounds inhibit sialidases other than NEU3. One test of the specificity of the inhibitors would be to test them for their ability to inhibit fibrosis in mice lacking NEU3, but since mice lacking NEU3 do not develop fibrosis in the bleomycin model (Karhadkar et al., 2020), this cannot be done, and we thus cannot rule out the possibility that the inhibitors inhibit fibrosis through some mechanism not involving NEU3.
As with AMPCA, DANA and Tamiflu attenuated bleomycin-induced protein upregulation in the BAL at day 21 (Karhadkar et al., 2017). However, 2AP or MP did not significantly attenuate bleomycin-induced protein upregulation in BAL at day 21. These disparate results may be due to dose effects or effects that DANA, Tamiflu, 2AP, MP, and AMPCA have on targets other than NEU3.
We previously observed that NEU3 is upregulated in the mouse bleomycin model and that inhibition of sialidases with DANA or oseltamivir or loss of NEU3 attenuates bleomycin-induced pulmonary fibrosis in mice (Karhadkar et al., 2017, 2020). The human and mouse NEU3 inhibitors 2AP, MP, and AMPCA all inhibited pulmonary inflammation, levels of active TGF-β1 in the lungs, and pulmonary fibrosis in the mouse bleomycin model. The UniProt sequences P01137 for human L-TGF-β1 and P04202 for mouse L-TGF-β1 show 90% amino acid sequence similarity and conserved glycosylation sites (Pruitt et al., 2007; Areström et al., 2012; Doğan et al., 2016; UniProt Consortium, 2019). A reasonable explanation for the ability of NEU3 inhibitors to decrease bleomycin-induced increased levels of active TGF-β1 is that mouse NEU3 also desialylates mouse LAP to release mouse TGF-β1. Together, these results support the hypothesis that a positive feedback loop in which NEU3 desialylates LAP to release active TGF-β1 and the active TGF-β1 causes lung epithelial cells to increase translation of NEU3 mRNA and decrease degradation of NEU3 protein (Chen et al., 2020) contributes to pulmonary fibrosis and suggest that NEU3 inhibitors are potential therapeutics for pulmonary fibrosis.
Acknowledgments
We thank Darrell Pilling for assistance with BAL sample collection and the LARR (Laboratory Animal Resources and Research) staff at Texas A&M for animal care. We also thank Kristen Consalvo, Wensheng Chen, and Darrell Pilling for comments on the manuscript.
Authorship Contributions
Participated in research design: Karhadkar, Meek, Gomer.
Conducted experiments: Karhadkar.
Performed data analysis: Karhadkar, Meek, Gomer.
Wrote or contributed to the writing of the manuscript: Karhadkar, Meek, Gomer.
Footnotes
- Received July 30, 2020.
- Accepted October 6, 2020.
This work was supported by National Institutes of Health National Heart, Lung, and Blood Institute [Grant R01 HL132919]. T.R.K., T.D.M., and R.H.G. are inventors on a patent application for the use of this class of sialidase inhibitors as therapeutics for a variety of diseases.
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This article has supplemental material available at jpet.aspetjournals.org.
Abbreviations
- AMPCA
- 4-amino-1-methyl-2-piperidinecarboxylic acid
- 2AP
- 2 acetylpyridine
- BAL
- bronchoalveolar lavage
- brhNEU3
- bacterially expressed recombinant human NEU3
- B SA
- bovine serum albumin
- DANA
- 2,3-didehydro-2-deoxy-N-acetyl-neuraminic acid
- HEK293
- human embryonic kidney 293
- IL-6
- interleukin-6
- LAP
- latency-associated glycopeptide
- L-TGF-β1
- latent TGF-β1
- MBP
- maltose-binding protein
- MP
- methyl picolinate
- 4MU-NANA
- 2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrat
- NEU
- neuraminidase
- OCI
- oxacarbenium ion intermediate
- PBMC
- peripheral blood mononuclear cell
- rhLAP
- recombinant human LAP
- rhL-TGF-β1
- recombinant human L-TGF-β1
- rhNEU
- recombinant human NEU
- TGF-β1
- transforming growth factor-β1
- Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics
