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Modulating PCAF/GCN5 immune cell function through a PROTAC approach Zuni I. Bassi, Martin C. Fillmore, Afjal H. Miah, Trevor D. Chapman, Claire Maller, Emma J. Roberts, Lauren C. Davis, Darcy E. Lewis, Nicholas W. Galwey, Kirsty E. Waddington, Valentino Parravicini, Abigail L. MacMillan-Jones, Celine Gongora, Philip G Humphreys, Ian Churcher, Rab K. Prinjha, and David F Tough ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00705 • Publication Date (Web): 10 Sep 2018 Downloaded from http://pubs.acs.org on September 11, 2018
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ACS Chemical Biology
Modulating PCAF/GCN5 immune cell function through a PROTAC approach
Zuni I. Bassi1, Martin C. Fillmore2, Afjal H. Miah1, Trevor D. Chapman3, Claire Maller3, Emma J. Roberts3, Lauren C. Davis3, Darcy E. Lewis3, Nicholas W. Galwey4, Kirsty E. Waddington5, Valentino Parravicini6, Abigail L. MacMillan-Jones1, Celine Gongora7, Philip G. Humphreys3, Ian Churcher8, Rab K. Prinjha3 and David F. Tough3*
1
Protein Degradation DPU, Future Pipelines Discovery, GlaxoSmithKline, Medicines
Research Centre, Stevenage SG1 2NY, UK
2
NCE-MD Medicinal Chemistry UK Team, R&D Platform Technology & Science,
GlaxoSmithKline, Medicines Research Centre, Stevenage SG1 2NY, UK
3
Epigenetics DPU, Immuno-Inflammation and Oncology Therapy Area, GlaxoSmithKline,
Medicines Research Centre, Stevenage SG1 2NY, UK
4
Target Sciences Statistics, R&D Target Sciences, GlaxoSmithKline, Medicines Research
Centre, Stevenage SG1 2NY, UK 5
Current address: University College London, Division of Medicine, The Rayne Building, 5
University Street, London WC1E 6JF, UK 6
Current address: GammaDelta Therapeutics Limited, London Bioscience Innovation Centre,
2 Royal College Street, London NW1 0NH, UK
7
Institut de Recherche en Cancérologie de Montpellier, INSERM U1194, Université de
Montpellier, Montpellier F-34298, France
8
Current address: BenevolentBio, 40 Churchway, London NW1 1LW, UK
*e-mail:
[email protected] 1 ACS Paragon Plus Environment
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Abstract P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are closely-related epigenetic proteins, each containing an acetyltransferase domain and a bromodomain. Consistent with reported roles for these proteins in immune function, we find that PCAF-deficient macrophages exhibit a markedly reduced ability to produce cytokines upon stimulation with lipopolysaccharide (LPS). Investigating the potential to target this pathway pharmacologically, we show that chemical inhibition of the PCAF/GCN5 bromodomains is insufficient to recapitulate the diminished inflammatory response of PCAFdeficient immune cells. However, by generating the first PCAF/GCN5 proteolysis targeting chimera (PROTAC), we identify small molecules able to degrade PCAF/GCN5 and to potently modulate the expression of multiple inflammatory mediators in LPS-stimulated macrophages and dendritic cells. Our data illustrate the power of the PROTAC approach in the context of multi-domain proteins, revealing a novel anti-inflammatory therapeutic opportunity for targeting PCAF/GCN5.
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ACS Chemical Biology
Bromodomains are conserved modules which bind to acetyl-lysine residues, including those in histone tails.1 The acetyl-lysine binding sites in bromodomains are tractable to small molecule inhibition and compounds selectively targeting bromodomains in several different proteins have been described.2 Studies focussed on the bromodomain and extra-terminal (BET) family of proteins have shown that bromodomain inhibitors can block many of the functions of these proteins in cells, including those linked to inflammation.3-5 However, the contribution of bromodomains to the function of non-BET family proteins is much less understood. P300/CBP-associated factor (PCAF) and general control nonderepressible 5 (GCN5) are highly homologous multi-domain proteins, each containing an acetyltransferase domain and a bromodomain.6 Based on their possession of domains able to modify histones (acetyltransferase) and recognise modified histones (bromodomain), PCAF and GCN5 are classed as epigenetic proteins, linking their function to effects mediated at the chromatin level. PCAF and GCN5 are reported to contribute to several cellular pathways, including cell proliferation and differentiation, metabolic regulation, and DNA damage repair.6-9 In the immune system, PCAF has been implicated in the production of inflammatory cytokines through studies with knockout mice, which showed reduced production of TNF and IL-6 in lipopolysaccharide (LPS)-stimulated blood and bone marrow-derived macrophages from PCAF-/- mice versus controls.10-11 Given the proven efficacy of TNF- and IL-6-blocking approaches in various human inflammatory diseases,12-13 these findings suggested that PCAF might represent a potential therapeutic target for such diseases. Therefore, we investigated the potential to modulate inflammatory/immune cell function by targeting PCAF/GCN5 with a small molecule approach. We first sought to confirm the anti-inflammatory function of PCAF in bone marrowderived PCAF-/-14 mouse macrophages. Consistent with the previous reports, LPS-induced IL-6 and TNF production was significantly reduced in PCAF-/- versus wild type (WT) cells (Figure 1a). KC/GRO (IL-8) was also reduced in knockout macrophages, while no significant 3 ACS Paragon Plus Environment
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effect was observed for IL-12p70, IL-10 and IL-1β (Figure 1a). These results confirm and extend the previously identified role for PCAF in mediating LPS-induced production of cytokines. To investigate the potential to target this pathway pharmacologically, we evaluated whether GSK4027, a potent and selective small-molecule inhibitor targeting the bromodomains of PCAF and GCN5,15 could recapitulate the PCAF knockout phenotype in vitro. GSK4027 binds with similar potency to the bromodomains of PCAF and GCN5, but is highly selective over other bromodomain family members, and demonstrates an IC50 of 60 nM in a human cellular target engagement assay measuring displacement of full-length PCAF from histone H3.3.15 Mouse bone marrow cells were differentiated in vitro into macrophages in the presence or absence of GSK4027 and then stimulated with LPS. To mimic the conditions in knockout cells, GSK4027 was added to the culture media at day 0 and cells were incubated with compound throughout the differentiation period. Multiplex analysis of several different inflammatory cytokines surprisingly revealed no significant, concentration-dependent changes following treatment with GSK4027 versus controls (Figure 1b). To test the effect of GSK4027 on human macrophage function, CD14+ monocytes were differentiated in vitro into macrophages in the presence of GSK4027 and then stimulated with LPS. As observed for mouse macrophages, and in marked contrast to the findings in PCAF-/- cells, GSK4027 had little effect on LPS-induced cytokine production in human cells, although a small but significant decreasing trend was seen for IL-6 and IL-10 (Figure 1c). To investigate the biological effects of the compound more broadly, we profiled the activity of GSK4027 in the BioMAP Diversity PLUS panel (DiscoverX), a collection of primary human cell co-culture systems (including immune and adherent cell types) that model human inflammatory disease states. GSK4027 showed very limited activity across all assays, even when used at concentrations as high as 10 µM (Supplementary Figure 1).
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Altogether, these data suggest that inhibition of the PCAF/GCN5 bromodomains is insufficient to disrupt the immunomodulatory function of these proteins. Fig. 1a - WT vs PCAF KO Macrophages - pg/ml Log Mouse macrophages a
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ACS Chemical Biology
Figure 1. Inhibition of the PCAF/GCN5 bromodomains does not fully recapitulate the PCAF knockout phenotype. (a) Bone marrow cells from PCAF-/- and WT mice were differentiated in vitro into macrophages for 7 days and then stimulated with 10 ng/mL LPS for 24 hours. The levels of the indicated cytokines in the supernatants were measured by MSD. Bars represent mean ± SEM, dots represent individual mice (n=7 WT and n=9 PCAF -/- mice), **p
