GSK137, a potent small-molecule BCL6 inhibitor with in vivo activity, suppresses antibody responses in mice
2021; Elsevier BV; Volume: 297; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2021.100928
ISSN1083-351X
AutoresAndrew C. Pearce, Mark J. Bamford, Ruth Barber, Angela Bridges, M.A. Convery, Constantinos Demetriou, Siân Evans, Thomas Gobbetti, David J. Hirst, Duncan S. Holmes, Jonathan P. Hutchinson, Sandrine Jayne, Larissa Lezina, Michael T. McCabe, Cassie Messenger, J Morley, Melissa C. Musso, Paul Scott‐Stevens, Ana Sousa Manso, Jennifer Schofield, Tom Slocombe, Don O. Somers, Ann L. Walker, Anastasia Wyce, Xiping Zhang, Simon D. Wagner,
Tópico(s)T-cell and B-cell Immunology
ResumoB-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB–POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB–POZ domain. The compound has good solubility (128 μg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response. B-cell lymphoma 6 (BCL6) is a zinc finger transcriptional repressor possessing a BTB–POZ (BR-C, ttk, and bab for BTB; pox virus and zinc finger for POZ) domain, which is required for homodimerization and association with corepressors. BCL6 has multiple roles in normal immunity, autoimmunity, and some types of lymphoma. Mice bearing disrupted BCL6 loci demonstrate suppressed high-affinity antibody responses to T-dependent antigens. The corepressor binding groove in the BTB–POZ domain is a potential target for small compound-mediated therapy. Several inhibitors targeting this binding groove have been described, but these compounds have limited or absent in vivo activity. Biophysical studies of a novel compound, GSK137, showed an in vitro pIC50 of 8 and a cellular pIC50 of 7.3 for blocking binding of a peptide derived from the corepressor silencing mediator for retinoid or thyroid hormone receptors to the BCL6 BTB–POZ domain. The compound has good solubility (128 μg/ml) and permeability (86 nM/s). GSK137 caused little change in cell viability or proliferation in four BCL6-expressing B-cell lymphoma lines, although there was modest dose-dependent accumulation of G1 phase cells. Pharmacokinetic studies in mice showed a profile compatible with achieving good levels of target engagement. GSK137, administered orally, suppressed immunoglobulin G responses and reduced numbers of germinal centers and germinal center B cells following immunization of mice with the hapten trinitrophenol. Overall, we report a novel small-molecule BCL6 inhibitor with in vivo activity that inhibits the T-dependent antigen immune response. B-cell lymphoma 6 (BCL6) is a transcriptional repressor that accomplishes its effects through binding of specific DNA recognition sequences by the C-terminal zinc fingers (1Hatzi K. Jiang Y. Huang C. Garrett-Bakelman F. Gearhart M.D. Giannopoulou E.G. Zumbo P. Kirouac K. Bhaskara S. Polo J.M. Kormaksson M. MacKerell Jr., A.D. Xue F. Mason C.E. 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We performed a high throughput screen to identify molecules capable of inhibiting the binding of a peptide derived from the SMRT co-repressor to the BCL6 BTB-POZ domain and identified hits with affinities in the 5 to 20 μM range. One of these hits was developed to produce the novel tool compound GSK137. We carried out a pharmacokinetic analysis of GSK137 and an in vivo study demonstrated reduction in specific antibody titers in response to immunization with T-dependent antigens at drug levels consistent with robust engagement with the BCL6 BTB-POZ domain target. To identify chemical starting points for inhibiting BCL6 consideration was given to the fact that the desired mechanism of action was specifically to antagonise the binding of BCL6 to its BTB-POZ domain binding co-repressors, preserving function of other parts of the protein. Further, given the targeted interaction was a protein-protein interaction rather than a classical catalytic pocket it was decided to screen the full diversity of the GSK compound collection (∼1.7 million compounds) in a cell-free assay (time resolved fluorescence resonance energy transfer, TR-FRET) to maximise the chance of identifying binders. Inhibition of binding of a peptide derived from the SMRT co-repressor to the BTB-POZ domain of BCL6, by small molecules from the compound library, was determined. Groups of hit compounds from the high throughput screen were refined by further binding assays including surface plasmon resonance and nuclear magnetic resonance to derive a hit compound, which was the starting point for the development of GSK137 (Fig. 1A), a compound with a pyrazolo-tetrahydropyrimidine core substituted by aminoquinoline and methylpyridine moieties. The synthetic route is presented in Figure S3. GSK137 displaced peptides derived from the human SMRT co-repressor from the BCL6 BTB-POZ domain in a TR-FRET assay with a pIC50 of 8 (Table 1). It also displaced peptides from human nuclear receptor co-repressor 1 and BCL6 co-repressor corepressors, which bind in the same position as SMRT (32Ghetu A.F. Corcoran C.M. Cerchietti L. Bardwell V.J. Melnick A. Privé G.G. Structure of a BCOR corepressor peptide in complex with the BCL6 BTB domain dimer.Mol. Cell. 2008; 29: 384-391Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), both with a pIC50 of 7.6. We assessed activity of GSK137 against mouse and rat BCL6 and SMRT and determined the pIC50 to be 7.8 and 7.6, respectively. The ability of GSK137 to inhibit binding of intracellular full-length human BCL6 to BCL6-binding domain of SMRT (residues 1292–1500) in live cells was measured by NanoBret assay, and pIC50 was determined to be 7.3 (Table 1). BI3802 activity was consistent with the previously reported potency in cell-free and cellular assays (41Kerres N. Steurer S. Schlager S. Bader G. Berger H. Caligiuri M. Dank C. Engen J.R. Ettmayer P. Fischerauer B. Flotzinger G. Gerlach D. Gerstberger T. Gmaschitz T. Greb P. et al.Chemically induced degradation of the oncogenic transcription factor BCL6.Cell Rep. 2017; 20: 2860-2875Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar).Table 1Key properties of GSK137pIC50Molecular weight (kD)CAD solubility (μg/ml)Chrom LogD/PFIHSA binding (%)Papp (nM/s)HumanMouse/ratTR-FRETNanoBret cellTR-FRETSMRTnuclear receptor co-repressor 1/BCL6 co-repressorSMRT87.6/7.67.37.8/7.63741282.3/6.39186pIC50 of the ability of the small molecule to inhibit binding of corepressor peptides from SMRT, nuclear receptor co-repressor 1, or BCL6 co-repressor and was measured by time-resolved FRET in vitro.Cellular pIC50 was determined by NanoBret assay comprising Nano Luciferase fused at the N terminus of full-length human BCL6 and Halo tag fused at the C terminus of the BCL6-binding domain (residues 1292–1500) of human SMRT corepressor protein. Solubility was determined by a GSK in-house kinetic solubility assay with the amount of solubilized material being measured by charged aerosol detector (CAD). Effective hydrophobicity (log D) was determined chromatographically with the measurement modified to account for the number of aromatic rings (PFI, property forecast index). Percentage binding to human serum albumin (HSA) and permeability (Papp) were also determined. Open table in a new tab pIC50 of the ability of the small molecule to inhibit binding of corepressor peptides from SMRT, nuclear receptor co-repressor 1, or BCL6 co-repressor and was measured by time-resolved FRET in vitro. Cellular pIC50 was determined by NanoBret assay comprising Nano Luciferase fused at the N terminus of full-length human BCL6 and Halo tag fused at the C terminus of the BCL6-binding domain (residues 1292–1500) of human SMRT corepressor protein. Solubility was determined by a GSK in-house kinetic solubility assay with the amount of solubilized material being measured by charged aerosol detector (CAD). Effective hydrophobicity (log D) was determined chromatographically with the measurement modified to account for the number of aromatic rings (PFI, property forecast index). Percentage binding to human serum albumin (HSA) and permeability (Papp) were also determined. The crystal structure of GSK137, in complex with the BCL6 BTB–POZ domain (residues 5–129) bearing changes Cys8Gln, Cys67Arg, and Cys84Asn to improve solubility of the protein (31Melnick A. Carlile G. Ahmad K.F. Kiang C.-L. Corcoran C. Bardwell V. Privé G.G. Licht J.D. Critical residues within the BTB domain of PLZF and Bcl-6 modulate interaction with corepressors.Mol. Cell Biol. 2002; 22: 1804-1818Crossref PubMed Scopus (169) Google Scholar), was obtained in order to explore the structure–activity relationship of BCL6 binding (Fig. 1, B and C). The 2.04 Å resolution crystal structure was generated from an apo BCL6 BTB–POZ domain crystal soaked with the compound. There was clear electron density for the ligand allowing the binding mode and absolute stereochemistry ([R]-chiral center at C6 and [S] at C8) to be determined. The compound binds in the lateral grooves in an equivalent manner on both sides of the interface utilizing the same key interactions with both protein chains of the BTB–POZ domain homodimer. Key interactions are observed between the three principal moieties of the compound and the homodimeric protein at both binding sites: (i) The aminoquinoline moiety is partially surface exposed but makes dual hydrogen bonds to the side chain of Glu115 residue and packs against a miniloop from Cys53 to Gly55, between two small helices of one monomer (monomer A in Fig. 1, B and C). (ii) The pyrazolo-tetrahydropyrimidine core lies deeper in the binding pocket, pi stacking against the aromatic side chain of Tyr58 and hydrogen bonding to Met51 main chain carbonyl, of monomer A, and Arg24 side chain of monomer B (Fig. 1C). Arg24 also hydrogen bonds to the Tyr58 hydroxyl (of monomer A [Fig. 1B]) to further stabilize the complex. (iii) The methylpyridine moi
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