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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

Downregulation of the αvβ6 Integrin via RGD Engagement Is Affinity and Time Dependent

James A. Roper, Alex L. Wilkinson, Elaine Gower and Robert J. Slack
Journal of Pharmacology and Experimental Therapeutics February 2021, 376 (2) 273-280; DOI: https://doi.org/10.1124/jpet.120.000379

Abstract

The arginyl-glycinyl-aspartic acid (RGD) integrin alpha-v beta-6 (αvβ6) has been identified as playing a key role in the activation of transforming growth factor-β (TGFβ) that is hypothesized to be pivotal in the development of fibrosis and other diseases. In this study, αvβ6 small molecule inhibitors were characterized in a range of in vitro systems to determine affinity, kinetics, and duration of TGFβ inhibition. High αvβ6 binding affinity was shown to be correlated with slow dissociation kinetics. Compound 1 (high αvβ6 affinity, slow dissociation) and SC-68448 (low αvβ6 affinity, fast dissociation) induced concentration- and time-dependent internalization of αvβ6 in normal human bronchial epithelial (NHBE) cells. After washout, the αvβ6 cell surface repopulation was faster for SC-68448 compared with compound 1. In addition, αvβ6-dependent release of active TGFβ from NHBE cells was inhibited by compound 1 and SC-68448. After washout of SC-68448, release of active TGFβ was restored, whereas after washout of compound 1 the inhibition of TGFβ activation was maintained and only reversible in the presence of a lysosomal inhibitor (chloroquine). However, SC-68448 was able to reduce total levels of αvβ6 in NHBE cells if present continuously. These observations suggest αvβ6 can be degraded after high affinity RGD binding that sorts the integrin for lysosomal degradation after internalization, likely due to sustained engagement as a result of slow dissociation kinetics. In addition, the αvβ6 integrin can also be downregulated after sustained engagement of the RGD binding site with low affinity ligands that do not sort the integrin for immediate lysosomal degradation.

SIGNIFICANCE STATEMENT The fate of RGD integrin after ligand binding has not been widely investigated. Using the αvβ6 integrin as a case study, we have demonstrated that RGD-induced downregulation of αvβ6 is both affinity and time dependent. High affinity ligands induced downregulation via lysosomal degradation, likely due to slow dissociation, whereas sustained low affinity ligand engagement was only able to decrease αvβ6 expression over longer periods of time. Our study provides a potential unique mechanism for obtaining duration of action for drugs targeting integrins.

Introduction

The integrins are a family of transmembrane glycoprotein receptors that primarily act as signaling proteins in mammals (Hynes, 1987). These heterodimeric receptors are made up of an α- and β-subunit, of which in mammals there are 18 α-subunits and eight β-subunit variants that are able to form up to 24 heterodimers (Hynes, 2002). The alpha-v beta-6 (αvβ6) integrin is a member of the arginyl-glycinyl-aspartic acid (RGD) integrin subfamily (Busk et al., 1992; Hynes, 2002) that share an amino acid binding motif [arginine (R), glycine (G), and aspartic acid (D)] in their endogenous ligands, with selectivity for these ligands determined by their surrounding amino acid sequences (Ruoslahti, 1996). These endogenous RGD ligands include fibronectin, vitronectin, fibrinogen, von Willebrand factor, thrombospondin, laminin, tenascin, osteopontin, and the latency-associated peptide of the large latent transforming growth factor-β (TGFβ) complex. The αvβ6 integrin has been shown to be upregulated in fibrotic disease (Margadant and Sonnenberg, 2010) and hypothesized to drive fibrogenesis via activation of the potent profibrotic cytokine TGFβ (Munger et al., 1999). The αvβ6 integrin activates TGFβ from the constitutively expressed latent TGFβ after binding of the RGD sequence, located in the latency-associated peptide component of the TGFβ large latent complex, to the RGD binding site located in the interface between the αv and β6 integrin subunits (Goodwin and Jenkins, 2009). This interaction results in the β6 cytoplasmic domain binding to intracellular actin cytoskeleton that, when activated by mechanical stretch, induces a conformational change in the large latent complex and presentation of active TGFβ to its receptors, evoking canonical TGFβ signaling (Munger et al., 1999; Xu et al., 2009; Tatler and Jenkins, 2012).

Due to its potential as a therapeutic target in fibrosis, several αvβ6 drug discovery initiatives have been ongoing for the last decade. The furthest advanced of these is a selective non-RGD αvβ6 monoclonal antibody, BG00011 (Horan et al., 2008), that was until recently in a phase II idiopathic pulmonary fibrosis clinical study (NCT03573505). In addition, over recent years there has been a drive within the pharmaceutical industry to develop small molecule RGD-mimetics via both the inhaled (Maden et al., 2018) and oral routes (Hatley et al., 2018), targeting fibrotic disease and cancers within the lung and beyond.

As is the case for majority of the αv integrins, the relationships between αvβ6 RGD binding site affinity, receptor kinetics, and downregulation have not been fully explored. Although the internalization of the αvβ6 integrin has been well established after binding of ligands to the RGD binding site (Ramsay et al., 2007; Slack et al., 2016), less is known of the fate of the integrin in relation to the RGD binding site occupancy over time. The determination of these relationships is important to understand when designing drug candidates targeting αvβ6 in the clinic, as they can influence the duration of pharmacodynamic effect. Consequently, this will influence the dose required for efficacy and dosing frequency, which will factor into the likelihood of maintaining a therapeutic window over toxicological effects.

The aim of this present study is to investigate the role of αvβ6 integrin binding affinity and dissociation kinetics on the internalization and recycling kinetics of the integrin using two small molecule RGD-mimetics with different pharmacological profiles. In addition, the total cellular levels of αvβ6 were tracked after internalization to enable the impact of duration of RGD binding site engagement on αvβ6 expression, and therefore subsequent activation of TGFβ, to be determined.

Materials and Methods

Materials.

Compound 1 (Anderson et al., 2016), compound 2 (Rowedder et al., 2017), SC-68448 (Carron et al., 1998), and SB-525334 (Grygielko et al., 2005) (Fig. 1) were synthesized by the Fibrosis Discovery Performance Unit Medicinal Chemistry group at GlaxoSmithKline Medicines Research Centre (Stevenage, UK). Compound 2 was radiolabeled with [3H] by RC TRITEC Ltd. (Teufen, Switzerland) and had a specific activity of 16.1 Ci/mmol. All other chemicals and reagents were purchased from Sigma-Aldrich Co. Ltd. (Gillingham, UK) unless otherwise stated. Normal human bronchial epithelial (NHBE) cells, growth medium, and supplements were purchased from Lonza (Lonza Group Ltd., Basel, Switzerland) and maintained in culture as previously described (Slack et al., 2016). The epithelial transformed mink lung cell (TMLC) line (Abe et al., 1994) was obtained from Daniel Rifkin (New York University, NY) and maintained in culture as previously described (Weinreb et al., 2004). All other cell culture media and reagents were obtained from Invitrogen (Invitrogen Ltd., Paisley, UK). All tissue culture flasks and plates were purchased from Greiner Bio-One (Firckenhausen, Germany) unless otherwise stated. All antibodies used were commercially available and obtained from R&D Systems (Minneapolis, MN) unless otherwise stated and at a stock concentration of 10 µg/ml. These included, for flow cytometry, phycoerythrin (PE)-conjugated mouse IgG2B isotype control (IgG2B-PE) (product # IC0041P) and PE-conjugated mouse monoclonal non–function blocking, anti-human integrin beta-6 (product # FAB4155P) and, for high content screening (HCS), mouse monoclonal anti-human integrin beta-6 (product # MAB4155) and Alexa Fluor 488 goat IgG anti-mouse Invitrogen (product # A-11001; Invitrogen Ltd.). Cellular assays and radioligand binding assays were completed with a final DMSO concentration of 0.1% and 1%, respectively. Purified soluble protein for the human αvβ6 integrin (product # 3817-AV) was purchased from R&D Systems Inc.

Fig. 1.
Fig. 1.

The chemical structures of the small molecules used in this study.

Radioligand Binding Studies.

All radioligand binding experiments were performed via filtration binding in 96-well deep plates at 37°C as previously described (Rowedder et al., 2017). For determining the affinity of small molecules at the αvβ6 integrin, competition binding studies were completed by incubating integrin protein (0.3 nM) with a fixed concentration of [3H]compound 2 (∼KD) and increasing concentrations of unlabeled test ligand for 6 hours prior to filtration. To determine the receptor dissociation rates of unlabeled integrin ligands, 2 × KI concentrations of unlabeled small molecule RGD-mimetics were incubated with αvβ6 protein for 1 hour prior to addition of excess [3H]compound 2 (50 × KD = ∼50 nM) and then incubated at 37°C for varying times up to 48 hours prior to filtration.

Ligand-Induced αvβ6 Internalization and Recycling in NHBE Cells.

The measurement of surface and intracellular αvβ6 in NHBE cells was determined via flow cytometry using methods previously described (Slack et al., 2016). For HCS studies, supplement-starved NHBE cells in bronchial epithelial basal medium (BEBM) were plated in collagen I–coated 96-well imaging plates (20,000 cells/well) and treated with vehicle or test compound for up to 48 hours prior to β6 integrin staining with mouse anti-human integrin β6, then goat anti-mouse IgG Alexa Fluor 488 antibody (Invitrogen Ltd., Renfrewshire, UK). Image acquisition and analysis of stained cells were performed using the Arrayscan VTI High Content Reader (Thermo Fisher Scientific, MA), applying bioapplications to quantify β6 immunofluorescence. Nuclear staining was completed with Hoechst 33342 dye (Invitrogen Ltd.) to measure cell numbers, and if required cells were permeabilized with 0.2% w/v saponin (flow cytometry) or Triton X-100 (HCS) to determine total αvβ6 staining (membrane and intracellular).

Measurement of TGFβ Activation and Signaling.

For the measurement of concentration-dependent effects of test compounds on TGFβ signaling, supplement-starved NHBE cells in BEBM (20,000 cells/well) were plated in collagen I–coated 96-well plates and treated with vehicle (0.1% DMSO) or test compound for 24 hours. Cells were lysed in ice-cold cell lysis buffer [included in Milliplex TGFβ Signaling Magnetic Bead Panel 6 plex (Merck Millipore, Billerica, MA) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (100×); Life Technologies Ltd., Paisley, UK], then phospho mothers against decapentaplegic homolog 2 (pSmad2 )measured and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The Milliplex TGFβ Signaling Magnetic Bead Panel 6 plex (Merck Millipore) Luminex assay was used to quantify pSmad2 and GAPDH, according to the manufacturer’s instructions.

Activation of TGFβ via αvβ6 in NHBE cells was determined in a TMLC co-culture system as previously described (Xu et al., 2009). Briefly, supplement-starved NHBE cells in BEBM were plated in collagen I–coated 96-well plates (25,000 cells/well) and left to adhere for 24 hours prior to addition of TMLCs containing 20 μM lysophosphatidic acid (25,000 cells/well). Vehicle (0.1% DMSO) or test compound was added and incubated for 24 hours in the absence or presence of chloroquine prior to addition of cell lysis and detection reagents (Promega Corporation, Madison, WI). Plates were then incubated at ambient temperature (20–22°C) for 5 minutes before supernatants were transferred to a 96-well, white, solid bottom plates and luminescence read on a MicroBeta TriLux (PerkinElmer LAS UK Ltd., Beaconsfield, UK). For washout studies, vehicle or test compound in the absence or presence of 10 µM chloroquine was incubated with the NHBE cell/TMLC co-culture for 1 hour prior to washout. TGFβ was quantified by comparing values obtained under experimental conditions to readings obtained from a standard curve derived from increasing concentrations of active TGFβ added to the co-culture under identical conditions.

Quantification of β6 and Plasminogen Activator Inhibitor-1 mRNA Expression in NHBE Cells.

Cultured NHBE cells were seeded on to collagen I–coated 24-well plates (50,000 cells/well) and treated with vehicle (0.1% DMSO) or test compound for 24 hours. Cells were then washed with ice-cold PBS and stored at −80°C. Total RNA was isolated using the RNeasy mini kit (Qiagen, West Sussex, UK) with on-column DNase digestion as per the manufacturer’s instructions. Total RNA was reverse transcribed using the high-capacity complementary DNA reverse transcription kit (Life Technologies Ltd., Paisley, UK) as per the manufacturer’s instructions. Integrin β6, plasminogen activator inhibitor-1 (PAI-1), and GAPDH (housekeeping gene) gene expression were determined using the respective TaqMan real-time polymerase chain reaction primers ITGB6 (Hs00168458_m1), PAI-1 (Hs01126604_m1), and GAPDH (Hs99999905_m1) (Life Technologies Ltd., Paisley, UK) on a LightCycler 480 (Roche, Basil, Switzerland). This was completed using a LightCycler 480 Probes Master kit as per the manufacturer’s instructions with a preincubation at 95°C for 10 minutes, amplification at 95°C for 10 seconds followed by 60°C for 30 seconds (repeated for 45 cycles), and cooling at 4°C for 30 seconds. 2−ΔΔCt (fold increase in gene expression relative to comparator [control (0.3% DMSO) at t = 0 hours)] for integrin β6 and PAI-1 were calculated as described previously (Livak and Schmittgen, 2001) with gene expression normalized to the housekeeping gene (GAPDH).

Data Analysis.

Statistical analyses were completed using Prism 8.0 (GraphPad Software, San Diego, CA). Statistical significance between two data sets was tested using a Student’s unpaired t test. One-way ANOVA was used for comparison of more than two data sets and, when significance was observed, an appropriate post-test completed. For determining a statistical correlation between two data sets, a Pearson correlation was completed. Radioligand binding data were normalized to total (1% DMSO) and nonspecific binding (10 µM SC-68448). HCS internalization, TGFβ signaling, and TGFβ activation data were normalized to vehicle control (0.1% DMSO) and a control αvβ6 inhibitor (1 µM). Flow cytometry internalization data were normalized to human β6-PE staining in the absence of compound and background IgG2B-PE staining. Unless otherwise indicated, data shown graphically and in the text are either means ± S.D. or, where three or more data points/individual experiments have been completed, means ± S.E.M.

Results

Affinity and Dissociation Kinetics of αvβ6 Inhibitors.

To determine the affinity of unlabeled integrin ligands at the αvβ6 integrin, competition displacement binding curves were measured against [3H]compound 2. Compound 1 was shown to bind with high pM affinity to the αvβ6 integrin, whereas SC-68448 demonstrated an approximate 70-fold lower affinity (Fig. 2A; Table 1). The transforming growth factor beta receptor I (TGFβRI) inhibitor SB-525334 was shown not to bind to the αvβ6 integrin at concentrations up to 10 µM. To allow the ranking of unlabeled ligands in terms of rate of dissociation from the αvβ6 integrin, the association of [3H]compound 2 was used as surrogate for the dissociation of an unlabeled ligand from a preformed unlabeled ligand/integrin complex. Using this method, the dissociation profiles for compound 1 and SC-68448 were generated, with compound 1 demonstrating slow- and SC-68448 fast-αvβ6 dissociation kinetics (Fig. 2B; Table 1). The dissociation constant (koff) was calculated for compound 1, SC-68448, and a set of αvβ6 integrin ligands to allow correlation with affinity (Fig. 2C; Table 1). A significant correlation (Pearson correlation, P < 0.01) was observed between affinity and koff, with a high affinity engagement of the αvβ6 integrin resulting in a slow dissociation rate. From the set of molecules tested, compound 1 demonstrated the highest affinity and slowest off-rate, whereas SC-68448 was shown to have the lowest affinity and fastest off-rate.

Fig. 2.
Fig. 2.

Competition and dissociation binding profiles for small molecule molecules and the αvβ6 integrin. (A) Competition binding curves for compound 1, SC-68448, and SB-525334 against the αvβ6 integrin. (B) The dissociation profile of compound 1 and SC-68448 from the αvβ6 integrin. (C) Correlation of log koff vs. negative log10 of KI (pKI) for a set of αvβ6 RGD-mimetics with a range of affinities. Total and nonspecific binding values were measured at each time point in the presence of vehicle (1% DMSO) and 10 µM SC-68448, respectively, and were used to calculate the percent inhibition of radioligand bound to the αvβ6 integrin. Data shown are means ± S.E.M. (A and B) of at least four individual experiments carried out in singlicate or quadruplicate.

View this table:
TABLE 1

Pharmacological parameters for test compounds in assays measuring αvβ6 integrin binding, internalization, recycling, and functional inhibition of αvβ6-mediated TGFβ activation

Data shown are mean values ± S.E.M. for at least three individual determinations.

Ligand-Induced αvβ6 Internalization and Kinetics.

In high content cell imaging studies using NHBE cells, compound 1 and SC-68448 caused concentration-dependent αvβ6 internalization of surface integrins after a 24-hour incubation, with no effect on cell count (Fig. 3, A and B). To determine the rates of ligand-induced internalization, in flow cytometric studies the surface expression of αvβ6 was measured over time after the addition of a maximal concentration of test ligand, as determined in concentration-response curve studies. Both compound 1 and SC-68448 caused a rapid internalization of αvβ6 with comparable maximal loss of cell surface integrin observed over the 1-hour time course studied (Fig. 3C). The maximal effect observed was less than 100% compared with the HCS assay that was likely a result of the normalization to the isotype control in the flow cytometry system. The return of αvβ6 to the surface membrane after washout was ∼3-fold slower for the compound 1 compared with SC-68448 (Fig. 3C; Table 1).

Fig. 3.
Fig. 3.

RGD small molecule–induced internalization and surface repopulation kinetics of the αvβ6 integrin. (A) Internalization concentration response curve for the αvβ6 integrin in NHBE cells induced by compound 1 (cell count shown for comparison) determined by high content screening. Panel below shows example images of NHBE cell β6 immunofluorescence (green stain) and the effect of increasing concentrations of compound 1. Nuclear staining with Hoechst 33342 dye is also shown (blue stain). (B) Internalization concentration response curve for the αvβ6 integrin in NHBE cells induced by SC-68448 (cell count shown for comparison) determined by high content screening. (C) Internalization kinetics of the αvβ6 integrin post-treatment with compound 1 and SC-68448 followed by return of αvβ6 to cell surface after compound washout (1 hour after addition) determined by flow cytometry. Cell count (A and B) was determined by nuclear staining with Hoechst 33342 dye. Data shown are means ± S.E.M. of four individual experiments carried out in duplicate.

Concentration- and Time-Dependent Inhibition of TGFβ Activation and Signaling.

To demonstrate the inhibition of αvβ6-mediated TGFβ activation by compound 1 and SC-68448, Smad2 phosphorylation was measured in NHBE cells. Compound 1 and SC-68448 inhibited Smad2 phosphorylation in a concentration-dependent manner, as did the TGFβRI inhibitor SB-525334 (Fig. 4A; Table 1). To confirm if the differences in αvβ6 return half-life (t1/2) observed between compound 1 and SC-68448 resulted in a similar effect on TGFβ activation, washout studies were completed measuring TGFβ release from NHBE cells. After washout, the inhibitory effect of SC-68448 was completely lost, whereas compound 1 maintained an approximate 70% inhibition of TGFβ release (Fig. 4B). To determine the mechanism by which compound 1 caused the prolonged postwashout inhibition of αvβ6-mediated TGFβ release from NHBE cells, studies were also completed in the absence and presence of the lysosomal degradation inhibitor chloroquine. In the presence of chloroquine there was no statistical difference for levels of TGFβ activation inhibited by SC-68448 after washout compared with washout conditions in the absence of chloroquine (ANOVA, Bonferroni post-test, P > 0.05). This was not the case for compound 1, where the partial reversal of inhibition caused by washout in the absence of chloroquine was significantly further reversed in the presence of chloroquine (ANOVA, Bonferroni post-test, P < 0.01) (Fig. 4B).

Fig. 4.
Fig. 4.

Concentration- and time-dependent inhibition of αvβ6-mediated TGFβ activation. (A) Concentration-dependent inhibition of pSmad2 levels in NHBE cells by compound 1, SC-68448, and SB-525334 (TGFβRI inhibitor). (B) Duration of action of the inhibition of compound 1 on TGFβ-activation, and the effect of the lysosomal degradation inhibitor chloroquine, measured in an NHBE cell and TMLC (expressing the firefly luciferase under the control of a TGFβ-sensitive portion of the PAI-1 promoter) co-culture system (ANOVA, Bonferroni post-test). Data shown are means ± S.E.M. of at least four individual experiments carried out in duplicate or quadruplicate.

Time-Dependent Downregulation of Total Cell αvβ6.

To investigate the time course of downregulation of the αvβ6 integrin after continuous RGD binding site engagement, and for comparison with direct TGFβRI inhibition, the total expression in NHBE cells was monitored over time in the presence of a maximum concentration of compound 1, SC-68448, and SB-525334. Compound 1 and SC-68448 both caused downregulation of αvβ6 in a biphasic manner with an early phase over the first 8 hours and a late phase from 8 to 48 hours. Overall total αvβ6 expression was reduced to a greater extent by compound 1 compared with SC-68448 with significantly lower levels observed at 48 hours (ANOVA, Bonferroni post-test, P < 0.01) (Fig. 5A). SB-525334 only reduced αvβ6 expression at the 48 hour time point.

Fig. 5.
Fig. 5.

Effect of sustained αvβ6 inhibition on total integrin protein and mRNA expression of β6 and PAI-1 in NHBE cells. (A) Total levels of αvβ6 integrin determined by high content screening in NHBE cells after sustained inhibition with compound 1, SC-68448, and SB-525334 (**P < 0.01 compound 1 vs. SC-68448 and SB-525334, ANOVA, Bonferroni post-test). (B) The effect of the compound 1 and SB-525334 (TGFβRI inhibitor) on mRNA levels of β6 and PAI-1 in NHBE cells after a 24-hour incubation (ANOVA, Dunnett post-test). Data shown are means ± S.E.M. of at least three individual experiments carried out in duplicate or quadruplicate.

To further demonstrate the inhibition of αvβ6-mediated TGFβ activation and determine effects on the level of β6 integrin subunit in NHBE cells after compound addition, mRNA expression of PAI-1 (profibrotic mediator downstream of TGFβRI/II pathway activation) and the β6 integrin subunit were investigated. Both the activin receptor-like kinase 5 (ALK5 or TGFβRI) inhibitor SB-525334 and compound 1 caused a significant downregulation of β6 and PAI-1 mRNA (ANOVA, Dunnett post-test, with P < 0.01 for β6 and P < 0.05 for PAI-1) over a 24-hour incubation period compared with control (0.3% DMSO) (Fig. 5B).

Discussion

Although routinely completed for other transmembrane receptors, such as G protein–coupled receptors, the relationship between receptor binding kinetics and affinity has not been extensively studied in the context of integrins. Therefore, the initial aim of this study was to investigate the RGD binding site affinity and dissociation kinetics of a range of small molecule αvβ6 integrin RGD-mimetics. As it has been widely demonstrated that the binding of RGD ligands to αvβ6 induces internalization of the integrin (Hausner et al., 2009; Saha et al., 2010), it was also deemed of interest to examine the effect of small molecule RGD-mimetics with different affinity and receptor kinetics on the internalization and recycling kinetics of αvβ6. As a natural follow-on from this, the subsequent effect on the total cellular levels of αvβ6 were investigated after internalization. This would enable the impact of the duration of RGD binding site engagement on αvβ6 expression to be determined, as well as the functional consequences by measuring the activation of TGFβ.

The dissociation rates for a set of αvβ6 RGD ligands, with affinities across a 100-fold range, were measured, and an increase in affinity for the receptor was observed to be correlated with a slower koff. Generally, at steady state, the affinity (KD) of a ligand and its protein/receptor binding partner is defined by the ratio of the koff and association constant (kon) and is a measure of the likelihood of the ligand-receptor complex to dissociate (Hulme and Trevethick, 2010). Therefore, a high affinity ligand would be predicted to have a lower koff value and a slower dissociation from the protein/receptor. This was shown to be the case for the RGD binding interaction at the αvβ6 integrin. Using the highest (compound 1) and lowest (SC-68448) affinity αvβ6 tool small molecules, the internalization of the αvβ6 integrin was investigated. The potency observed for induction of internalization by compound 1 and SC-68448 tracked well with their αvβ6 affinity with a comparable difference between EC50 and KD values of ∼100-fold, as was the case for the IC50 values for functional inhibition of TGFβ production (i.e., pSmad2 levels as measure of the TGFβ canonical pathway). Both molecules were observed to internalize αvβ6 very quickly and with comparable t1/2 values that were measured within minutes of addition. The more interesting observations in this study were the rates of recycling or return of αvβ6 to the cell surface after washout of ligand. After washout of compound 1, αvβ6 returned to the cell surface ∼3-fold slower compared with SC-68448.

The rate of return of αvβ6 to the cell surface observed in these studies after washout of compound 1 compared with that of endogenous turnover of αvβ6 is considerably slower (Ramsay et al., 2007; Wang et al., 2011), suggesting that the internalized ligand-αvβ6 complex is potentially degraded and that returning αvβ6 is newly synthesized. To further develop this hypothesis, the lysosomal inhibitor chloroquine was tested in a system measuring the activation of TGFβ to observe its effect on compound 1. Chloroquine in its unprotonated form can freely diffuse across cell and organelle membranes; however, it becomes trapped in lysosomes once protonated due to the low pH of this environment (Solomon and Lee, 2009). In this form it has been shown to inhibit proteolytic processes and therefore has been used as a tool for determining mechanisms of receptor turnover (Dunmore et al., 2013). In washout experiments using NHBE cells investigating αvβ6-mediated inhibition of TGFβ activation, the sustained duration of inhibition observed with compound 1 was reversed in the presence of chloroquine. This observation suggests that after internalization of αvβ6 by compound 1, the integrin is sorted for degradation in lysosomes. Interestingly, for the low affinity αvβ6 ligand SC-68448 with a fast dissociation profile, the sustained duration of inhibition of TGFβ activation is not observed. Therefore, compound 1 causes a more prolonged downregulation of αvβ6 in this system compared with the lower affinity ligand SC-68448.

The high affinity of compound 1 for the αvβ6 integrin combined with its slow dissociation profile likely results in a prolonged activation of the integrin in intracellular vesicles. It could be hypothesized that the longer the integrin is engaged at the RGD site after internalization, as a result of slow dissociation kinetics, the more likely it is to be intracellularly designated for degradation, as has been observed with other small molecule/protein interactions (Long et al., 2012; Harling et al., 2013). The prolonged activation of the RGD integrin α5β1 has been shown to delay its recycling back to the cell surface of A2780 cells after internalization, as a result of sorting into lysosomes prior to transportation back to the plasma membrane (Dozynkiewicz et al., 2012). A similar mechanism is hypothesized here for degradation of αvβ6, where compound 1 binds with high affinity, activates and internalizes the integrin, and remains bound intracellularly, causing sustained activation and sorting of the integrin for lysosomal degradation rather than recycling. This then ultimately results in prolonged duration of inhibition of αvβ6-mediated TGFβ activation.

Many transmembrane based receptors self-regulate their expression on cell surfaces by downregulating upon continuous activation after sustained orthosteric binding of ligands (Kelly et al., 2008) as a homeostatic control of their signaling pathways. To assess if this was also the case for the αvβ6 integrin, total expression was tracked after continuous engagement over 48 hours with a maximal concentration of either compound 1 or SC-68448. Both molecules resulted in the reduction in the total amount of integrin but interestingly to different degrees, with compound 1 inducing significantly lower expression. This demonstrated that although low affinity engagement of the RGD site by SC-68448 for short periods of time did not induce downregulation, prolonged binding, activation, and internalization resulted in total expression levels being reduced. The difference between the effect observed with compound 1 and SC-68448 suggests that the high affinity/slow dissociation–induced downregulation combined with the continuous engagement is additive with respect to the effect on αvβ6 expression. In addition, blockade of the TGFβ pathway with SB-525334 also caused downregulation of αvβ6 but over a much longer timeframe. This demonstrated that the integrin is under transcriptional control by TGFβ in this system, as has been shown previously (Zambruno et al., 1995; Wang et al., 1996), and was confirmed by effects on β6 gene expression.

In conclusion, this study has demonstrated that high affinity binding to αvβ6 is driven by slow dissociation from the integrin. High affinity RGD ligand binding at the αvβ6 integrin induces downregulation, as a result of lysosomal degradation after integrin internalization, likely due to sustained engagement as a result of the slow dissociation kinetics. This novel relationship has not been demonstrated before and offers a unique mechanism for achieving sustained target engagement that can be driven by drug Cmax rather than slow clearance. In addition, a further novel finding from this study is that αvβ6 can be downregulated after continuous engagement of the RGD binding site with low affinity ligands, potentially as a homeostatic mechanism for controlling integrin activation.

Acknowledgments

The authors would like to acknowledge the Fibrosis Discovery Performance Unit (DPU) Medicinal Chemistry team at GlaxoSmithKline for the synthesis of compound 1, compound 2, SC-68448, and SB-525334. All studies were funded by GlaxoSmithKline.

Authorship Contributions

Participated in research design: Roper, Wilkinson, Gower, Slack.

Conducted experiments: Roper, Wilkinson, Gower, Slack.

Performed data analysis: Roper, Wilkinson, Gower, Slack.

Wrote or contributed to the writing of the manuscript: Roper, Slack.

Footnotes

    • Received October 16, 2020.
    • Accepted November 30, 2020.

  • This work received no external funding.

  • This work was previously presented at the following meeting: Gower E, Wilkinson A, and Slack RJ (2017) Downregulation of the αvβ6 integrin can occur via short engagement with high affinity ligands or long engagement with low affinity ligands. British Pharmacological Society (BPS) Pharmacology 2017; 2017 Dec 11–13; London, UK.

  • https://doi.org/10.1124/jpet.120.000379.

Abbreviations

BEBM
bronchial epithelial basal medium
GAPDH
glyceraldehyde-3-phosphate dehydrogenase
HCS
high content screening
NHBE
normal human bronchial epithelial
PAI-1
plasminogen activator inhibitor-1
PE
phycoerythrin
RGD
arginyl-glycinyl-aspartic acid
TGFβ
transforming growth factor-β
t1/2
half-life
TMLC
transformed mink lung cell
αvβ6
alpha-v beta-6

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Research ArticleGastrointestinal, Hepatic, Pulmonary, and Renal

RGD Engagement and Downregulation of αvβ6

James A. Roper, Alex L. Wilkinson, Elaine Gower and Robert J. Slack
Journal of Pharmacology and Experimental Therapeutics February 1, 2021, 376 (2) 273-280; DOI: https://doi.org/10.1124/jpet.120.000379
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