Identification of compounds that potentiate CREB signaling as possible enhancers of long-term memory Menghang Xiaa, Ruili Huanga, Vicky Guob, Noel Southalla, Ming-Hsuang Choa, James Inglesea, Christopher P. Austina,1, and Marshall Nirenbergb,1 aNIH Chemical Genomics Center, bLaboratory of Biochemical Genetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892 Contributed by Marshall Nirenberg, December 29, 2008 (sent for review December 18, 2008) Many studies have implicated the cAMP Response Element Binding the CREB signaling pathway were able to improve some mea- (CREB) protein signaling pathway in long-term memory. To identify sures of memory formation. Inhibition of PKA activity by small molecule enhancers of CREB activation of gene expression, targeted delivery of a PKA inhibitory peptide to the nucleus we screened Ϸ73,000 compounds, each at 7–15 concentrations in resulted in a decrease in CREB phosphorylation and defects in a quantitative high-throughput screening (qHTS) format, for ac- long-term potentiation (16); similarly, the impairment of object tivity in cells by assaying CREB mediated ␤-lactamase reporter gene recognition long-term memory seen in heterozygous CBPϩ/Ϫ expression. We identified 1,800 compounds that potentiated CREB mice was rescued by administrating PDE4 inhibitors, and trans- mediated gene expression, with potencies as low as 16 nM, genic overexpression of a dominant active form of CREB comprising 96 structural series. Mechanisms of action were sys- partially reversed the late long-term potentiation deficits ob- tematically determined, and compounds that affect phosphodies- served in CBPϩ/Ϫ mice (17, 18). These results suggested that terase 4, protein kinase A, and cAMP production were identified, modulators of the CREB pathway may be clinically useful for as well as compounds that affect CREB signaling via apparently human disorders of memory and for ameliorating some of the unidentified mechanisms. qHTS folowed by interrogation of path- features of Rubinstein-Taybi Syndrome (OMIM 180849), a way targets is an efficient paradigm for lead generation for human genetic disorder caused by mutation in the gene encoding chemical genomics and drug development. CBP (19). High-throughput screening (HTS) has developed over the last memory enhancer ͉ phosphodiesterase inhibitor ͉ 2 decades as the principal method of lead identification in drug quantitative high-throughput screening discovery (20). With advances in molecular biology and genom- ics, target-based screens, which detect small modulators of he Cyclic-AMP Response Element Binding (CREB) protein proteins, have overtaken traditional phenotypic screens in HTS Tplays an important role in learning and long-term memory applications (21). However, the target-based screens may lead to (LTM) formation by coupling neuronal activity with changes in compounds being identified as active that sometimes do not have gene expression (1). CREB proteins comprise a family of activity in the physiological environment. Cell signaling, or transcription factors that regulate the transcription of genes ‘‘pathway’’ assays offer a potentially attractive assay format through binding to cAMP Responsive Elements (CRE), such as intermediate in complexity between isolated molecular target the nucleotide sequence, 5Ј-TGACGTCA, in DNA. CREB and phenotypic assays, operating in an intact cellular environ- signaling occurs via a multistep pathway in which binding of ment but with the readout dependent on signaling through a hormones or neurotransmitters to G protein coupled cell surface single pathway, which contains multiple components. receptors activates adenylate cyclase (AC) and catalyzes the We report here a panel of enhancers of CREB activity discov- Ͼ production of cAMP. Elevation of cAMP levels leads to activa- ered by screening 73,000 compounds in a CREB pathway assay. tion of protein kinase A (PKA) and translocation of the catalytic These compounds will serve as probes for known and novel CREB subunits of PKA to the nucleus, where they catalyze phosphor- pathway components, will be useful for the study of long-term ylation of CREB at Ser-133, which activates CREB (2, 3). memory, and potentially lead to new clinical memory enhancers for Phosphorylated CREB then binds to CRE nucleotide sequences widely prevalent disorders such as Alzheimer’s disease. in DNA as a dimer (4), and recruits cofactors such as CREB- Results Binding Protein (CBP) and p300 to form a larger transcriptional complex (5), which catalyzes histone acetylation and thereby Identification of Small-Molecule Enhancers of the CREB Pathway. regulates target genes. Negative regulators of the CREB path- Approximately 73,000 compounds were screened for CREB ␤ way include phosphodiesterases that degrade cAMP, calcium- enhancer activity using a cell-based CRE- -lactamase reporter calmodulin protein kinase II that catalyzes phosphorylation of gene assay (Fig. S1) in qHTS mode (22). Because qHTS tests CREB at Ser-142, thereby promoting dissociation of the CREB each compound at 7–15 concentrations, compound potencies dimer (6, 7), protein phosphatases 1 and 2A catalyze Ser-133 (EC50s) and efficacies are immediately available after the pri- dephosphorylation (8, 9) thereby reducing the binding of CREB to DNA, and histone deacetylase, which catalyzes the removal of Author contributions: M.X., J.I., C.A., and M.N. designed research; M.X., V.G., and M.-H.C. acetyl groups from histones. performed research; M.X., R.H., J.I., C.A., and M.N. analyzed data; and M.X., R.H., N.T.S., The role of CREB signaling in long-term memory initially was C.A., and M.N. wrote the paper. defined by Kandel and colleagues in Aplysia (10). Over- The authors declare no conflict of interest. expression of a dominant-negative CREB (11, 12) or a CREB Freely available online through the PNAS open access option. transcriptional repressor (dCREB2b) blocked the formation of Data deposition: The structures reported in this paper have been deposited in the Pubchem LTM in transgenic flies (13). These observations were subse- database, http://pubchem.ncbi.nlm.nih.gov (AID: 662, 905, 906, 907, 916). quently extended to mouse models; for example, mutant 1To whom correspondence may be addressed. E-mail: [email protected] or ␣␦ CREB mice exhibited impaired long-term memory in both [email protected]. spatial memory and contextual fear-conditioning tasks (14, 15), This article contains supporting information online at www.pnas.org/cgi/content/full/ accompanied by defects in long-term potentiation in hippocam- 0813020106/DCSupplemental. pal slices (14). At the same time, small molecule modulators of © 2009 by The National Academy of Sciences of the USA 2412–2417 ͉ PNAS ͉ February 17, 2009 ͉ vol. 106 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0813020106 Downloaded by guest on September 27, 2021 Table 1. Potency and number of compounds found that enhance share a common substructure or scaffold form a structural CREB activity series) were identified, 37 of which were selected for follow up Number of active compounds studies based on compound selectivity, potency/efficacy ranges, in curve classes and curve quality. Details on the procedure used for series prioritization can be found in Materials and Methods. Fig. 1A ␮ EC50 *, M 1.1 1.2 2.1 shows scaffolds of structural series containing compounds with 0.016–0.10 9 11 0 the greatest potencies and highest quality concentration- 0.10–1.00 52 65 7 response curves. These series are of particular interest as the 1.00–10.00 224 376 318 scaffolds have no previously reported pharmacological activity. 10.00–100.00 0 1 737 A large number of compounds with known pharmacological Total 1,800 activities are included in the NCGC screening collection, and many were active in the CREB qHTS (Fig. 1B). For example, *Concentration for half maximal increase in CREB activity. rolipram (23) and papaverine (24), which are known PDE inhibitors, enhanced CREB activity with EC50s of 200 nM and ␮ mary screen. A total of 1,800 compounds were classified as 1 M, respectively. Compounds with known activities relevant to potentiators of CREB activity having class 1.1, 1.2, and 2.1 CREB signaling aid both in determining mechanisms of action curves (see Fig. S2 for curve class definitions) and EC50s Ͻ100 of lead series, and in understanding the types of activities this ␮M, yielding a hit rate of 2.5%. The curve classes and potencies pathway assay is capable of detecting. of the 1,800 compounds are listed in Table 1. All data produced For the follow-up studies, 81 active compounds from 37 from this screen have been deposited in the PubChem database prioritized structural series (average 2-3 compounds from each (http://pubchem.ncbi.nlm.nih.gov), AID 662. The detailed series) were selected and again tested using the original CHO screen performance was described in SI Results (see Figs. S3 and CRE-␤-lactamase assay; of the 81 compounds that had been S4) and the most potent compounds identified from this primary tested in the primary screen, the activities of 80 compounds were screening were listed in Fig. S5. confirmed in the repeat assay, yielding a confirmation rate of The information-rich primary results from qHTS allowed us to perform a structure-activity relationship (SAR) analysis on the active compounds. Ninety-six structural series (compounds that NEUROSCIENCE Fig. 1. Scaffolds of enhancers of the CREB signaling pathway. (A) Ten of 30 scaffolds we identified are shown. Each scaffold is identified by a number. (B) Fig. 2. Confirmation study for compounds from series 41, using the CRE Six scaffolds of compounds with known biological activities are shown. In ␤-lactamase assay. (A) Concentration response curves of selected compounds parentheses are shown the number of active compounds found over the total from series 41. (B) Structures of these compounds are shown with NCGC number of compounds from that scaffold screened. compound IDs and EC50 values. Xia et al. PNAS ͉ February 17, 2009 ͉ vol.