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ABSTRACT: Disrupting the interaction between the p53 tumor suppressor and its regulator MDM2 is a promising therapeutic strategy in anticancer drug research. In our search for non peptide inhibitors of this protein-protein interaction, we have devised a ligand design concept exploiting the central position of Val 93 in the p53 binding pocket of MDM2. The design of molecules based on this concept has allowed us to rapidly identify compounds having a 3-imidazolyl indole core structure as the first representatives of a new class of potent inhibitors of the p53-MDM2 interaction.
Bioorganic & medicinal chemistry letters 03/2012; 22(10):3498-502. · 2.65 Impact Factor
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David Orain,
Silvio Ofner,
Manuel Koller,
David A Carcache,
Wolfgang Froestl,
Hans Allgeier,
Vittorio Rasetti,
Joachim Nozulak,
Henri Mattes,
Nicolas Soldermann,
Philipp Floersheim,
Sandrine Desrayaud, Joerg Kallen,
Kurt Lingenhoehl,
Stephan Urwyler
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ABSTRACT: A new set of quinazolinedione sulfonamide derivatives as competitive AMPA receptor antagonist with improved properties compared to 1 is disclosed. By modulating physico-chemical properties, compound 29 was identified with a low ED(50) of 5.5mg/kg in an animal model of anticonvulsant activity after oral dosage.
Bioorganic & medicinal chemistry letters 12/2011; 22(2):996-9. · 2.65 Impact Factor
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Joerg Berghausen,
Nicole Buschmann,
Pascal Furet,
Francois Gessier,
Lisztwan Hergovich Joanna,
Philipp Holzer,
Edgar Jacoby, Joerg Kallen,
Keiichi Masuya,
Soldermann Pissot Carole,
Haixia Ren,
Stefan. Stutz
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ABSTRACT: The invention relates to isoquinolinone and quinazolinone of formula I, which are MDM2 and MDM4 inhibitors and which are useful in the treatment of diseases mediated by the activity of MDM2 and/or MDM4. Compds. of formula I wherein Z is CH2 and NH and derivs.; X is halo; R6 is H, OH and derivs., and NH2 and derivs.; R7 is OH and derivs. and NH2 and derivs.; each R1 is independently halo, CN, NO2, C1-7 alkyl, etc.; R2 is substituted Ph, substituted 2-pyridyl, substituted 3-pyridyl, etc.; n is 0 to 2; with provisions; and tautomers, N-oxides and pharmaceutically acceptable salts thereof, are claimed. Example compd. II·TFA was prepd. by a multistep procedure (procedure given). All the invention compds. were evaluated for their MDM2 and MDM4 inhibitory activity (data given). [on SciFinder(R)]
Year: 06/2011
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Manuel Koller,
Kurt Lingenhoehl,
Markus Schmutz,
Ivan-Toma Vranesic, Joerg Kallen,
Yves P Auberson,
David A Carcache,
Henri Mattes,
Silvio Ofner,
David Orain,
Stephan Urwyler
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ABSTRACT: Quinazoline-2,4-diones with a sulfonamide group attached to the N(3) ring atom constitute a novel class of competitive AMPA receptor antagonists. One of the synthesized compounds, 28, shows nanomolar receptor affinity, whereas other examples of the series display oral anticonvulsant activity in animal models.
Bioorganic & medicinal chemistry letters 06/2011; 21(11):3358-61. · 2.65 Impact Factor
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ABSTRACT: The invention relates to tetrasubstituted heteroaryl compds. of formula I as MDM2 and/or MDM4 modulators. Such compds. are suitable for the treatment of a disorder or disease which is mediated by the activity of MDM2 and/or MDM4, or variants thereof. Compds. of formula I wherein each X are independently C and N; dashed bonds are single and double bonds; Y is CH, NH and N; provided that the total no. of ring nitrogens represented by each X and N is 1 or 2; A and B are independently Ph and pyridinyl, provided that the chlorine substituent is in the 3 or 4 position; R1 is CN, CNCH2, carboxy, aminocarbonyl, etc.; R4 is C2-7 alkyl, aryl, heteroaryl, etc.; R' and R'' are independently OH, C1-7 alkoxy, halo, C1-7 haloalkyl, etc.; and tautomers, N-oxides and pharmaceutically acceptable salts thereof, are claimed. Example compd. II was prepd. by amidation of 1-(5-chloro-2-methylphenyl)-5-(3-chlorophenyl)-2-(3,4-dimethylphenyl)-1H-imidazole-4-carboxylic acid with ethylamine. All the invention compds. were ev
Year: 03/2011
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ABSTRACT: Hu-antigen R (HuR) is a ubiquitous RNA-binding protein that comprises three RNA recognition motifs (RRMs). The first two tandem RRMs are known to bind to AU-rich elements (AREs) in the 3'-untranslated region of many mRNAs. The third RRM is connected to the second RRM through a basic hinge region that contains a localization signal termed HuR nucleocytoplasmic shuttling. Binding of HuR to the ARE in the 3'-untranslated region of mRNA leads to nuclear export, stabilization, and/or translational de-repression of the mRNA, resulting in upregulation of the encoded protein. Among the various ARE binding proteins known to date, HuR is still the only known ubiquitous antagonist of posttranscriptional gene silencing by AREs. Given the wide repertoire of known and suspected targets of HuR, it is considered to be a central node in the ARE pathway. Here, the x-ray crystal structure of the first RRM of HuR (amino acids 18-99) at 2.0 A resolution is presented. The overall fold consists of two alpha-helices and a four-stranded beta-sheet, with a beta1-alpha1-beta2-beta3-alpha2-beta4 topology and a beta-hairpin between alpha2 and beta4. The asymmetric unit consists of four chains. The large crystal contact interfaces observed between chains A/B and C/D contain hydrophobic residues located at the alpha-helix side of the fold, opposite to the RNA-binding interface. This hydrophobic region structurally resembles the protein-protein interaction site of RRM domains of other proteins. Because the nature of the assumed HuR homodimerization is mechanistically not well understood to date, we used site-directed mutagenesis, analytical size-exclusion chromatography and multiangle light scattering to investigate HuR interactions via the RRM hydrophobic region. Our data indicate that in vitro, HuR RRM1 and RRM1,2 homodimerization involves a disulfide bond at cysteine 13. This homodimerization mode may have a functional significance in redox modulation of HuR activity in response to oxidative stress. Because HuR is involved in many diseases (e.g., cancer, cachexia, and inflammatory bowel disease), the presented structure may provide a basis for rational drug design.
Journal of Molecular Biology 02/2010; 397(5):1231-44. · 4.00 Impact Factor
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ABSTRACT: p53 tumor suppressor activity is negatively regulated through binding to the oncogenic proteins Hdm2 and HdmX. The p53 residues Leu(26), Trp(23), and Phe(19) are crucial to mediate these interactions. Inhibiting p53 binding to both Hdm2 and HdmX should be a promising clinical approach to reactivate p53 in the cancer setting, but previous studies have suggested that the discovery of dual Hdm2/HdmX inhibitors will be difficult. We have determined the crystal structures at 1.3 A of the N-terminal domain of HdmX bound to two p53 peptidomimetics without and with a 6-chlorine substituent on the indole (which binds in the same subpocket as Trp(23) of p53). The latter compound is the most potent peptide-based antagonist of the p53-Hdm2 interaction yet to be described. The x-ray structures revealed surprising conformational changes of the binding cleft of HdmX, including an "open conformation" of Tyr(99) and unexpected "cross-talk" between the Trp and Leu pockets. Notably, the 6-chloro p53 peptidomimetic bound with high affinity to both HdmX and Hdm2 (K(d) values of 36 and 7 nm, respectively). Our results suggest that the development of potent dual inhibitors for HdmX and Hdm2 should be feasible. They also reveal possible conformational states of HdmX, which should lead to a better prediction of its interactions with potential biological partners.
Journal of Biological Chemistry 02/2009; 284(13):8812-21. · 4.77 Impact Factor
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Edgar Jacoby,
Andreas Boettcher,
Lorenz M Mayr,
Nathan Brown,
Jeremy L Jenkins, Joerg Kallen,
Caroline Engeloch,
Ulrich Schopfer,
Pascal Furet,
Keiichi Masuya,
Joanna Lisztwan
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ABSTRACT: Chemogenomics knowledge-based drug discovery approaches aim to extract the knowledge gained from one target and to apply it for the discovery of ligands and hopefully drugs of a new target which is related to the parent target by homology or conserved molecular recognition. Herein, we demonstrate the potential of knowledge-based virtual screening by applying it to the MDM4-p53 protein-protein interaction where the MDM2-p53 protein-protein interaction constitutes the parent reference system; both systems are potentially relevant to cancer therapy. We show that a combination of virtual screening methods, including homology based similarity searching, QSAR (Quantitative Structure-Activity Relationship) methods, HTD (High Throughput Docking), and UNITY pharmacophore searching provide a successful approach to the discovery of inhibitors. The virtual screening hit list is of the magnitude of 50,000 compounds picked from the corporate compound library of approximately 1.2 million compounds. Emphasis is placed on the facts that such campaigns are only feasible because of the now existing HTCP (High throughput Cherry-Picking) automation systems in combination with robust MTS (Medium Throughput Screening) fluorescence-based assays. Given that the MDM2-p53 system constitutes the reference system, it is not surprising that significantly more and stronger hits are found for this interaction compared to the MDM4-p53 system. Novel, selective and dual hits are discovered for both systems. A hit rate analysis will be provided compared to the full HTS (High-throughput Screening).
Methods in molecular biology (Clifton, N.J.) 02/2009; 575:173-94.
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ABSTRACT: The invention relates to 3-heterocyclyl indolyl compds. of formula I, which are capable of inhibiting the interaction between p53, or variants thereof, and MDM2 and/or MDM4, or variants thereof, resp. Due to their activity, the compds. are useful in the treatment of various disorders and diseases mediated by the activity of MDM2 and/or MDM4, or variants thereof. Compds. of formula I wherein R1 and R2 are independently (un)substituted alkyl, (un)substituted alkenyl, (un)substituted alkynyl, (un)substituted aryl and (un)substituted heterocyclyl; R3 is H, halo, (un)substituted alkyl, (un)substituted alkenyl, (un)substituted alkynyl, (un)substituted aryl, carboxy, cyano, etc.; RA is H, (un)substituted alkyl and acyl; X is H, C1-7 (halo)alkyl, C1-7 alkoxy, halo and CN; Y is C1-7 (halo)alkyl, C1-7 alkoxy, halo and CN; and their tautomers, N-oxides and salts thereof, are claimed. Example compd. II was prepd. by formylation of 6-chloro-1H-indole the resulting 6-chloro-1H-indole-3-carboxaldehyde underwent cy
Year: 10/2008
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ABSTRACT: Inverse agonists of the constitutively active human estrogen-related receptor alpha (ERRalpha, NR3B1) are of potential interest for several disease indications (e.g. breast cancer, metabolic diseases, or osteoporosis). ERRalpha is constitutively active, because its ligand binding pocket (LBP) is practically filled with side chains (in particular with Phe(328), which is replaced by Ala in ERRbeta and ERRgamma). We present here the crystal structure of the ligand binding domain of ERRalpha (containing the mutation C325S) in complex with the inverse agonist cyclohexylmethyl-(1-p-tolyl-1H-indol-3-ylmethyl)-amine (compound 1a), to a resolution of 2.3A(.) The structure reveals the dramatic multiple conformational changes in the LBP, which create the necessary space for the ligand. As a consequence of the new side chain conformation of Phe(328) (on helix H3), Phe(510)(H12) has to move away, and thus the activation helix H12 is displaced from its agonist position. This is a novel mechanism of H12 inactivation, different from ERRgamma, estrogen receptor (ER) alpha, and ERbeta. H12 binds (with a surprising binding mode) in the coactivator groove of its ligand binding domain, at a similar place as a coactivator peptide. This is in contrast to ERRgamma but resembles the situation for ERalpha (raloxifene or 4-hydroxytamoxifen complexes). Our results explain the novel molecular mechanism of an inverse agonist for ERRalpha and provide the basis for rational drug design to obtain isotype-specific inverse agonists of this potential new drug target. Despite a practically filled LBP, the finding that a suitable ligand can induce an opening of the cavity also has broad implications for other orphan nuclear hormone receptors (e.g. the NGFI-B subfamily).
Journal of Biological Chemistry 09/2007; 282(32):23231-9. · 4.77 Impact Factor
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ABSTRACT: Sanglifehrin A (SFA) is a novel immunosuppressant isolated from Streptomyces sp. that binds strongly to the human immunophilin cyclophilin A (CypA). SFA exerts its immunosuppressive activity through a mode of action different from that of all other known immunophilin-binding substances, namely cyclosporine A (CsA), FK506, and rapamycin. We have determined the crystal structure of human CypA in complex with SFA at 1.6 A resolution. The high resolution of the structure revealed the absolute configuration at all 17 chiral centers of SFA as well as the details of the CypA/SFA interactions. In particular, it was shown that the 22-membered macrocycle of SFA is deeply embedded in the same binding site as CsA and forms six direct hydrogen bonds with CypA. The effector domain of SFA, on the other hand, has a chemical and three-dimensional structure very different from CsA, already strongly suggesting different immunosuppressive mechanisms. Furthermore, two CypA.SFA complexes form a dimer in the crystal as well as in solution as shown by light scattering and size exclusion chromatography experiments. This observation raises the possibility that the dimer of CypA.SFA complexes is the molecular species mediating the immunosuppressive effect.
Journal of Biological Chemistry 07/2005; 280(23):21965-71. · 4.77 Impact Factor
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Sompong Wattanasin, Joerg Kallen,
Stewart Myers,
Qin Guo,
Michael Sabio,
Claus Ehrhardt,
Rainer Albert,
Ulrich Hommel,
Gisbert Weckbecker,
Karl Welzenbach,
Gabriele Weitz-Schmidt
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ABSTRACT: 1,4-Diazepane-2,5-diones (2) are found to be a new class of potent LFA-1 inhibitors. The synthesis, structure, and biological evaluation of these 1,4-diazepine-2,5-diones and related derivatives are described.
Bioorganic & Medicinal Chemistry Letters 03/2005; 15(4):1217-20. · 2.55 Impact Factor
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ABSTRACT: We disclose herein the discovery of estrogen receptor alpha (ERalpha) selective estrogen receptor modulators (SERMs) of the tetrahydroisoquinoline series that incorporate novel conformationally restricted side chains as replacement of the aminoethoxy residue typical of SERMs. Molecular modeling studies used in conjunction with the X-ray crystal structure of the ERalpha ligand binding domain (LBD) with raloxifene (7) suggested a diazadecaline moiety as a viable mimic of the SERM side chain. On the basis of this knowledge, the piperidinylethoxy moiety of our lead compound 60 was replaced by a diazadecaline subunit, providing the novel tetrahydroisoquinoline 29. In addition to exhibiting a binding affinity to ERalpha and antagonistic properties in the estrogen response element and MCF-7 assays similar to those of the parent compound 60, ligand 29 showed a reduced agonist behavior in the MCF-7 assay in the absence of 17beta-estradiol. These data point toward the fact that 29 may have a potential for breast cancer prevention/treatment in vivo, a feature which is particularly attractive in the quest for safe alternatives to hormone replacement therapy. In a pharmacokinetic experiment carried out in rats, 29 displayed an interesting profile, with a bioavailability of 49%. We also disclose the X-ray crystal structure of 29 in complex with ERalpha-LBD, which reveals the preferred configurations of 29 at the two chiral centers and the details of its interactions with the receptor. Finally, our structure-activity relationship studies show that other analogues bearing constrained side chains retain potency and antagonist activity and that a 3-OH substituted phenyl D-ring increases the selectivity of a set of piperazinyl-containing ligands in favor of ERalpha over ERbeta.
Journal of Medicinal Chemistry 02/2005; 48(2):364-79. · 5.25 Impact Factor
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ABSTRACT: The crystal structure of the ligand binding domain (LBD) of the estrogen-related receptor alpha (ERRalpha, NR3B1) complexed with a coactivator peptide from peroxisome proliferator-activated receptor coactivator-1alpha (PGC-1alpha) reveals a transcriptionally active conformation in the absence of a ligand. This is the first x-ray structure of ERRalpha LBD, solved to a resolution of 2.5 A, and the first structure of a PGC-1alpha complex. The putative ligand binding pocket (LBP) of ERRalpha is almost completely occupied by side chains, in particular with the bulky side chain of Phe328 (corresponding to Ala272 in ERRgamma and Ala350 in estrogen receptor alpha). Therefore, a ligand of a size equivalent to more than approximately 4 carbon atoms could only bind in the LBP, if ERRalpha would undergo a major conformational change (in particular the ligand would displace H12 from its agonist position). The x-ray structure thus provides strong evidence for ligand-independent transcriptional activation by ERRalpha. The interactions of PGC-1alpha with ERRalpha also reveal for the first time the atomic details of how a coactivator peptide containing an inverted LXXLL motif (namely a LLXYL motif) binds to a LBD. In addition, we show that a PGC-1alpha peptide containing this nuclear box motif from the L3 site binds ERRalpha LBD with a higher affinity than a peptide containing a steroid receptor coactivator-1 motif and that the affinity is further enhanced when all three leucine-rich regions of PGC-1alpha are present.
Journal of Biological Chemistry 12/2004; 279(47):49330-7. · 4.77 Impact Factor
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ABSTRACT: The integrin lymphocyte function-associated antigen-1 (LFA-1) (alphaLbeta2; CD11a/CD18) plays an important role in leukocyte migration and T cell activation. LFA-1 is inhibited by the cholesterol-lowering drug lovastatin, which binds to an allosteric site of the alphaL I domain termed the lovastatin site (L-site). Here we report for the first time the x-ray structures of the LFA-1 I domain complexed with derivatives of lovastatin optimized for LFA-1 inhibition. This analysis identified two new subpockets within the L-site occupied by chemical groups of the statin derivatives but not by lovastatin itself. Occupancy of these L-site subpockets led to distinct conformational changes in LFA-1, which were detectable by an epitope-monitoring assay. We utilized this assay to demonstrate improved LFA-1 inhibition in human blood in vitro and in blood samples from treated animals ex vivo. Moreover, we demonstrate that the novel lovastatin-derived LFA-1 inhibitor LFA878 exhibits potent anti-inflammatory effects in carrageenan-induced rat paw edema. In summary, the findings reported here extend the understanding of LFA-1 inhibition at the molecular level, allow for the identification and design of LFA-1 inhibitors of further enhanced potency, and support the expectation that LFA-1 inhibitors binding to the L-site will be of therapeutic value in treating inflammatory diseases.
Journal of Biological Chemistry 12/2004; 279(45):46764-71. · 4.77 Impact Factor
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ABSTRACT: The integrin lymphocyte function-associated antigen-1 (LFA-1) (αLβ2; CD11a/CD18) plays an important role in leukocyte migration and T cell activation. LFA-1 is inhibited by the cholesterol-lowering
drug lovastatin, which binds to an allosteric site of the αL I domain termed the lovastatin site (L-site). Here we report for the first time the x-ray structures of the LFA-1 I domain
complexed with derivatives of lovastatin optimized for LFA-1 inhibition. This analysis identified two new subpockets within
the L-site occupied by chemical groups of the statin derivatives but not by lovastatin itself. Occupancy of these L-site subpockets
led to distinct conformational changes in LFA-1, which were detectable by an epitope-monitoring assay. We utilized this assay
to demonstrate improved LFA-1 inhibition in human blood in vitro and in blood samples from treated animals ex vivo. Moreover, we demonstrate that the novel lovastatin-derived LFA-1 inhibitor LFA878 exhibits potent anti-inflammatory effects
in carrageenan-induced rat paw edema. In summary, the findings reported here extend the understanding of LFA-1 inhibition
at the molecular level, allow for the identification and design of LFA-1 inhibitors of further enhanced potency, and support
the expectation that LFA-1 inhibitors binding to the L-site will be of therapeutic value in treating inflammatory diseases.
Journal of Biological Chemistry 11/2004; 279(45):46764-46771. · 4.77 Impact Factor
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ABSTRACT: The retinoic acid-related orphan receptor alpha (RORalpha) is an orphan member of the subfamily 1 of nuclear hormone receptors. Our recent structural and functional studies have led to the hypothesis that cholesterol or a cholesterol derivative is the natural ligand of RORalpha. We have now solved the x-ray crystal structure of the ligand binding domain of RORalpha in complex with cholesterol-3-O-sulfate following a ligand exchange experiment. In contrast to the 3-hydroxyl of cholesterol, the 3-O-sulfate group makes additional direct hydrogen bonds with three residues of the RORalpha ligand binding domain, namely NH-Gln(289), NH-Tyr(290), and NH1-Arg(370). When compared with the complex with cholesterol, seven well ordered water molecules have been displaced, and the ligand is slightly shifted toward the hydrophilic part of the ligand binding pocket, which is ideally suited for interactions with a sulfate group. These additional ligand-protein interactions result in an increased affinity of cholesterol sulfate when compared with cholesterol, as shown by mass spectrometry analysis done under native conditions and differential scanning calorimetry. Moreover, mutational studies show that the higher binding affinity of cholesterol sulfate translates into an increased transcriptional activity of RORalpha. Our findings suggest that cholesterol sulfate could play a crucial role in the regulation of RORalpha in vivo.
Journal of Biological Chemistry 05/2004; 279(14):14033-8. · 4.77 Impact Factor
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ABSTRACT: The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORalpha-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization-mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography-mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D(3); 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORalpha-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.
Analytical Biochemistry 01/2004; 323(1):139-49. · 3.00 Impact Factor
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ABSTRACT: As part of a program aimed at the development of selective estrogen receptor modulators (SERMs), tetrahydroisoquinoline derivative 27 was discovered by high throughput screening. Successive replacements of the p-F substituent of 27 by an aminoethoxy side chain and of the 1-H of the tetrahydroisoquinoline core by a 1-Me group provided analogues 19 and 20. These compounds showed potencies in a cell-based reporter gene assay (ERE assay) varying between 0.6 and 20 nM and displayed antagonist behaviors in the MCF-7 human breast adenocarcinoma cell line with IC(50)s in the range of 2-36 nM. The effect of N-phenyl substituents on the activity and pharmacokinetic properties of tetrahydroisoquinoline analogues was explored. As a result of this investigation, two potent derivatives bearing a p-F N-aryl group, 19c and 20c, were discovered as candidates suitable for further profiling. To gain insight into the ligand-receptor interaction, the X-ray crystallographic structure of the 1-H tetrahydroisoquinoline derivative (R)-18a in complex with ERalpha-ligand binding domain (LBD)(301)(-)(553)/C-->S triple mutant was solved to 2.28 A. An overlay of this X-ray crystal structure with that reported for the complex of ERalpha-LBD(301)(-)(553)/carboxymethylated C and raloxifene (5) shows that both compounds bind to the same cleft of the receptor and display comparable binding modes, with differences being observed in the conformation of their "D-ring" phenyl groups.
Journal of Medicinal Chemistry 07/2003; 46(14):2945-57. · 5.25 Impact Factor
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ABSTRACT: The ligand-binding domain (LBD) of the human retinoic acid receptor-related orphan receptor (RORα-LBD), expressed in Sf9 cells, was purified and analyzed by electrospray ionization–mass spectrometry (ESI-MS). ESI-MS operated under native conditions showed the presence of a fortuitous ligand with molecular weight 386. Further analysis by gas chromatography–mass spectrometry (GC-MS) allowed the identification of the ligands bound to the LBD. Cholesterol (77%) and 7-dehydrocholesterol (provitamin D3; 18%) were shown to be the major ligands. A monohydroxylated cholesterol derivative was identified as a minor ligand. In addition, ligand exchange experiments monitored by ESI-MS showed that cholesterol sulfate has a higher affinity for RORα-LBD than cholesterol and 25-hydroxycholesterol. Binding of coactivator (CoA) peptide GRIP1P was shown to occur in a stoichiometric manner. Therefore, monitoring of binding of CoAs by mass spectrometry could be used for classification of the ligands as agonist or antagonist molecules.
Analytical Biochemistry.
