Identification of small-molecule antagonists that inhibit an activator:coactivator interaction

Abstract

Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 in response to hormonal stimuli triggers cellular gene expression via the recruitment of the histone acetylase coactivator paralogs CREB binding protein (CBP) and p300 to the promoter. The NMR structure of the CREB:CBP complex, using relevant interaction domains called KID and KIX, respectively, reveals a shallow hydrophobic groove on the surface of KIX that accommodates an amphipathic helix in phospho (Ser-133) KID. Using an NMR-based screening approach on a preselected smallmolecule library, we identified several compounds that bind to different surfaces on KIX. One of these, KG-501 (2-naphthol-AS-Ephosphate), targeted a surface distal to the CREB binding groove that includes Arg-600, a residue that is required for the CREB:CBP interaction. When added to live cells, KG-501 disrupted the CREB: CBP complex and attenuated target gene induction in response to cAMP agonist. These results demonstrate the ability of small molecules to interfere with second-messenger signaling cascades by inhibiting specific protein-protein interactions in the nucleus.cAMP response element binding protein ͉ transcription P rotein-protein interactions often serve as key regulatory points for signal propagation in response to extracellular stimuli. The formation of protein-protein complexes is of particular interest in the field of transcriptional regulation, where multiple low-affinity interactions appear to contribute to the recruitment of the transcriptional apparatus to the promoter (1). Initially thought to result from low energy interactions between unstructured regions, the recognition of an activator by its coactivator cognate often involves discrete surface contacts between well-folded protein domains. Moreover, the interaction surfaces between such protein pairs often are modulated by covalent modifications at residues near the protein-protein interface.Phosphorylation of the cAMP response element binding protein (CREB) at Ser-133 stimulates its association with the coactivator paralogs CREB binding protein (CBP) and p300 via a direct mechanism (2); the Ser-133 phosphate forms a hydrogen bond with Tyr-658 and an ion pair with Lys-662 in the KIX domain (3). Binding of CREB to CBP͞p300 is further stabilized by a random coil-to-helix transition in KID that provides the majority of hydrophobic surface contacts with residues lining a shallow groove in KIX (4, 5). The KIX domain folds into a three-helix structure with helices ␣1 and ␣3 aligned in nearly parallel fashion to form a groove that accommodates hydrophobic residues in KID. Helix ␣2 does not participate directly in surface contacts with KID but appears to stabilize KIX structure.The CREB binding site in KIX also recognizes other activators, most notably the protooncogene c-Myb (6). Similar to phospho (Ser-133) KID, c-Myb binds to KIX via an amphipathic helix that forms numerous surface contacts with residues lining the shallow hydrophobic groove in KIX (7). Notably, surfaces distal to the CREB-bi...

HER-2/neu Mediates Oncogenic Transformation via Altered CREB Expression and Function

“…Recently, anticancer drugs targeting CREB have been developed, including the KID/KIX inhibitor KG-501, which blocks the KID-KIX interaction and has a similar activity when compared with the shCREB-silenced or lapatinib-treated cells (21,41). This is in line with data obtained in the present study demonstrating that CREB-specific shRNA, lapatinib, and KG501 treatment caused reversal of the transformed phenotype and growth characteristics of HER-2/neu þ cells.…”

“…Cells were treated for the indicated lengths of time with a range of signal transduction inhibitors, including: H89 (10 mmol/L; PKA inhibitor; Merck); LY294002 (5 mmol/L; PI3K inhibitor; Adipogen); PD98059 (10 mmol/L; MEK1 inhibitor; Axxora); RO31-8220 (5 mmol/L; PKC inhibitor; Cayman), the tyrosine kinase inhibitors (TKI) lapatinib (1.25-10 mmol/L), sunitinib (10 mmol/L), sorafenib (10 mmol/L), pazopanib (10 mmol/L) and axitinib (10 mmol/L; all from LC Laboratories); RAD001 (5 mmol/L; mTOR inhibitor); the chemotherapeutics paclitaxel (10 mmol/L; mitotic inhibitor; Sigma), doxorubicin (10 mmol/L; DNA synthase inhibitor; Sigma), and epoxomicin (10 mmol/L; proteasome inhibitor; Calbiochem); and the KID/KIX inhibitor KG-501 (2.5-10 mmol/L; 2-naphthol-AS-E-phosphate; Sigma), which blocks the CREB-CREB binding protein (CBP) interaction (21).…”

Colorectal Carcinogenesis: Connecting K-RAS–Induced Transformation and CREB Activity In Vitro and In Vivo

“…On the basis of the important role of tumor progression, CREB represents a promising target for cancer therapy. Therefore, several strategies have been developed to inhibit CREB phosphorylation, CREB-DNA, or CREB-CBP interaction, which are currently tested in CREB-overexpressing tumors (19,20).…”

Valproic acid inhibits neurosphere formation by adult subventricular cells by a lithium-sensitive mechanism

“…For co-treatment experiments, lithium/selective kinase inhibitor was added 30 min prior to the addition of valproic acid/sodium butyrate. The final concentrations of co-treated drugs in media were: (1) This article is a U.S. government work, and is not subject to copyright in the United States.…”

“…To test this hypothesis we measured mRNA levels of Egr1 in adult NSCs treated with VPA plus selective kinase inhibitors. We co-treated cells with PD98059 (30 M), a highly selective inhibitor of MEK1/2 activation and the MEK-ERK cascade [7], with 217505 (10 M) a small molecule shown to disrupt the interaction between CREB and CREB binding protein (CBP) [1] and, in view of the known inhibitory effects of lithium on GSK-3 activity [24], with the GSK-3 inhibitor SB 216763 (10 M). This analysis revealed inhibition of MEK1/2 but not CREB-CBP interaction or GSK-3 activity antagonized VPA-mediated increases in Egr1 transcription (Fig.…”