Oxindole-based inhibitors of cyclin-dependent kinase 2 (CDK2): design, synthesis, enzymatic activities, and X-ray crystallographic analysis.

Two closely related classes of oxindole-based compounds, 1H-indole-2,3-dione 3-phenylhydrazones and 3-(anilinomethylene)-1,3-dihydro-2H-indol-2-ones, were shown to potently inhibit cyclin-dependent kinase 2 (CDK2). The initial lead compound was prepared as a homologue of the 3-benzylidene-1,3-dihydro-2H-indol-2-one class of kinase inhibitor. Crystallographic analysis of the lead compound bound to CDK2 provided the basis for analogue design. A semiautomated method of ligand docking was used to select compounds for synthesis, and a number of compounds with low nanomolar inhibitory activity versus CDK2 were identified. Enzyme binding determinants for several analogues were evaluated by X-ray crystallography. Compounds in this series inhibited CDK2 with a potency approximately 10-fold greater than that for CDK1. Members of this class of inhibitor cause an arrest of the cell cycle and have shown potential utility in the prevention of chemotherapy-induced alopecia.

Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors.

The peroxisome proliferator-activated receptors (PPARs) are transcriptional regulators of glucose, lipid, and cholesterol metabolism. We report the x-ray crystal structure of the ligand binding domain of PPAR alpha (NR1C1) as a complex with the agonist ligand GW409544 and a coactivator motif from the steroid receptor coactivator 1. Through comparison of the crystal structures of the ligand binding domains of the three human PPARs, we have identified molecular determinants of subtype selectivity. A single amino acid, which is tyrosine in PPAR alpha and histidine in PPAR gamma, imparts subtype selectivity for both thiazolidinedione and nonthiazolidinedione ligands. The availability of high-resolution cocrystal structures of the three PPAR subtypes will aid the design of drugs for the treatments of metabolic and cardiovascular diseases.

Prevention of chemotherapy-induced alopecia in rats by CDK inhibitors.

Most traditional cytotoxic anticancer agents ablate the rapidly dividing epithelium of the hair follicle and induce alopecia (hair loss). Inhibition of cyclin-dependent kinase 2 (CDK2), a positive regulator of eukaryotic cell cycle progression, may represent a therapeutic strategy for prevention of chemotherapy-induced alopecia (CIA) by arresting the cell cycle and reducing the sensitivity of the epithelium to many cell cycle-active antitumor agents. Potent small-molecule inhibitors of CDK2 were developed using structure-based methods. Topical application of these compounds in a neonatal rat model of CIA reduced hair loss at the site of application in 33 to 50% of the animals. Thus, inhibition of CDK2 represents a potentially useful approach for the prevention of CIA in cancer patients.

Structural basis for autorepression of retinoid X receptor by tetramer formation and the AF-2 helix.

The 9-cis-retinoic acid receptors (RXRalpha, RXRbeta, and RXRgamma) are nuclear receptors that play key roles in multiple hormone-signaling pathways. Biochemical data indicate that, in the absence of ligand, RXR can exist as an inactive tetramer and that its dissociation, induced by ligand, is important for receptor activation. In this article we report the inactivated tetramer structures of the RXRalpha ligand-binding domain (LBD), either in the absence of or in the presence of a nonactivating ligand. These structures reveal that the RXR LBD tetramer forms a compact, disc-shaped complex, consisting of two symmetric dimers that are packed along helices 3 and 11. In each monomer, the AF-2 helix protrudes away from the core domain and spans into the coactivator binding site in the adjacent monomer of the symmetric dimer. In this configuration, the AF-2 helix physically excludes the binding of coactivators and suggests an autorepression mechanism that is mediated by the AF-2 helix within the tetramer. The RXR-tetramer interface is assembled from amino acids that are conserved across several closely related receptors, including the HNF4s and COUP transcription factors, and may therefore provide a model for understanding structure and regulation of this subfamily of nuclear receptors.

Asymmetry in the PPARgamma/RXRalpha crystal structure reveals the molecular basis of heterodimerization among nuclear receptors.

The nuclear receptor PPARgamma/RXRalpha heterodimer regulates glucose and lipid homeostasis and is the target for the antidiabetic drugs GI262570 and the thiazolidinediones (TZDs). We report the crystal structures of the PPARgamma and RXRalpha LBDs complexed to the RXR ligand 9-cis-retinoic acid (9cRA), the PPARgamma agonist rosiglitazone or GI262570, and coactivator peptides. The PPARgamma/RXRalpha heterodimer is asymmetric, with each LBD deviated approximately 10 degrees from the C2 symmetry, allowing the PPARgamma AF-2 helix to interact with helices 7 and 10 of RXRalpha. The heterodimer interface is composed of conserved motifs in PPARgamma and RXRalpha that form a coiled coil along helix 10 with additional charge interactions from helices 7 and 9. The structures provide a molecular understanding of the ability of RXR to heterodimerize with many nuclear receptors and of the permissive activation of the PPARgamma/RXRbeta heterodimer by 9cRA.

The formation of a covalent complex between a dipeptide ligand and the src SH2 domain.

The X-ray crystal structure of the src SH2 domain revealed the presence of a thiol residue (Cys 188) located proximal to the phosphotyrosine portion of a dipeptide ligand. An aldehyde bearing ligand (1) was designed to position an electrophilic carbonyl group in the vicinity of the thiol. X-ray crystallographic and NMR examination of the complex formed between (1) and the src SH2 domain revealed a hemithioacetal formed by addition of the thiol to the aldehyde group with an additional stabilizing hydrogen bond between the acetal hydroxyl and a backbone carbonyl.

Catalytic activity of the SH2 domain of human pp60c-src; evidence from NMR, mass spectrometry, site-directed mutagenesis and kinetic studies for an inherent phosphatase activity.

During solution structural studies it was apparent that the human recombinant pp60c-src SH2 domain (srcSH2, residues 144-249) possessed an inherent phosphatase (Pase) activity. Complexes of U[13C,15N]srcSH2 with unlabeled Ac-pYEEIE (I) were examined using 31P and 1H-detected isotope filtered NMR methods. The presence of a high-affinity complex in equimolar solutions of I and U[13C, 15N]-srcSH2 was demonstrated by chemical shift perturbations, line broadening, and the observation of intermolecular nuclear Overhauser effects from the pY and Ile side-chain protons of I to protons on amino acid residues present in the binding pocket of srcSH2. Solutions containing excess I relative to srcSH2 revealed a slow hydrolysis of I to produce Ac-YEEIE and inorganic phosphate. The hydrolysis rate determined from NMR and HPLC-electrospray ionization mass spectrometry data at 30 degrees C for solutions containing excess I was 0.002-0.003 h-1. srcSH2 also catalyzed the hydrolysis of p-nitrophenyl phosphate (pNPP). Isoelectric focusing gels of a number of mutant srcSH2s demonstrated that this activity comigrated with srcSH2. Km, kcat, and kcat/Km were 3.7 +/- 0.4 mM, 3.1 +/- 0.2 x 10(-2) min-1, and 8.4 +/- 0.4 M-1 min-1, respectively, toward pNPP. The C188A mutant of the srcSH2 domain displayed 15% of the activity displayed by wild-type srcSH2, demonstrating that this residue is not absolutely required for activity. Two additional mutations in the known pY binding site, R178K and R158K, also resulted in decreased pNPPase activity, suggesting that the activity resides in or near this site. The inhibitor profile and pH dependence suggest that this is a novel protein Pase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Solution structure of the human pp60c-src SH2 domain complexed with a phosphorylated tyrosine pentapeptide.

Human pp60c-src is a cellular nonreceptor tyrosine kinase that participates in cytosolic signal transduction and has been implicated in the development of malignant tumors in the human breast and colon. Signal transduction is mediated by highly specific interactions between the SH2 domain and receptor phosphorylated tyrosine binding motifs. To elucidate the molecular conformation and interactions in solution, a family of highly resolved nuclear magnetic resonance (NMR) structures was determined for the src SH2 domain complexed with a high-affinity phosphorylated pentapeptide, acetyl-p YEEIE-OH. The 23 structures, generated with a distance geometry (DG) and a dynamical simulated annealing (SA) procedure, satisfied 2072 experimental restraints derived from a variety of multifrequency/multidimensional and isotope-filtered NMR data. Superimposition of residues 143-245 upon the mean coordinate set yielded an atomic rmsd of 0.58 +/- 0.09 A for the N, C alpha, C' atoms and 1.04 +/- 0.08 for all the non-hydrogen atoms. Residues in the ordered secondary structure regions superimpose to 0.29 +/- 0.04 A for the N, C alpha, C' and 0.73 +/- 0.08 A for all the non-hydrogen atoms. The angular order parameter calculated for the phi, psi angles was > 0.9 for 81 of the 106 protein residues. The main protein conformational features are three antiparallel beta-strands that traverse a compact core with an alpha-helix on each side of the core near the N- and C-termini. The observed intermolecular nuclear Overhauser effects (NOE) from the pY, +1E, and +3I residues positioned the ligand in an extended conformation across the SH2 domain surface with the pY and +3I side chains inserted into the protein binding pockets. In general, the protein conformation is consistent with previously reported structures of different SH2 domain complexes determined by X-ray crystallography. However, inter- or intramolecular interactions involving the guanidinium side chains of the solvated R alpha A2 or the buried R beta B5 were not observed at pH = 5.5 or 7.0. If such interactions exist in solution, the absence of any confirming data probably arises from rapid exchange with solvent and/or undetermined dynamic components. Thus, the unrestrained R alpha A2 side chain did not show an amino-aromatic interaction or a hydrogen bond to the -1 carbonyl oxygen as observed in the crystal structures. This result is consistent with the solution structure of a different SH2 domain complex. A more detailed comparison between the crystal structure and the NMR-derived solution structures of the same src SH2 domain complex is presented.(ABSTRACT TRUNCATED AT 400 WORDS)

NMR studies of [U-13C]cyclosporin A bound to cyclophilin: bound conformation and portions of cyclosporin involved in binding.

Cyclosporin A (CsA), a potent immunosuppressant, is known to bind with high specificity to cyclophilin (CyP), a 17.7 kDa protein with peptidyl-prolyl isomerase activity. In order to investigate the three-dimensional structure of the CsA/CyP complex, we have applied a variety of multidimensional NMR methods in the study of uniformly 13C-labeled CsA bound to cyclophilin. The 1H and 13C NMR signals of cyclosporin A in the bound state have been assigned, and from a quantitative interpretation of the 3D NOE data, the bound conformation of CsA has been determined. Three-dimensional structures of CsA calculated from the NOE data by using a distance geometry/simulated appealing protocol were found to be very different from previously determined crystalline and solution conformations of uncomplexed CsA. In addition, from CsA/CyP NOEs, the portions of CsA that interact with cyclophilin were identified. For the most part, those CsA residues with NOEs to cyclophilin were the same residues important for cyclophilin binding and immunosuppressive activity as determined from structure/activity relationships. The structural information derived in this study together with the known structure/activity relationships for CsA analogues may prove useful in the design of improved immunosuppressants. Moreover, the approach that is described for obtaining the structural information is widely applicable to the study of small molecule/large molecule interactions.

Isotope-edited NMR of cyclosporin A bound to cyclophilin: evidence for a trans 9,10 amide bond.

The binding of a 13C-labeled cyclosporin A (CsA) analog to cyclophilin (peptidyl prolyl isomerase) was examined by means of isotope-edited nuclear magnetic resonance (NMR) techniques. A trans 9,10 peptide bond was adopted when CsA was bound to cyclophilin, in contrast to the cis 9,10 peptide bond found in the crystalline and solution conformations of CsA. Furthermore, nuclear Overhauser effects (NOEs) were observed between the zeta 3 and epsilon 3 protons of the methylleucine (MeLeu) residue at position 9 of CsA and tryptophan121 (Trp121) and phenylalanine (Phe) protons of cyclophilin, suggesting that the MeLeu9 residue of CsA interacts with cyclophilin. These results illustrate the power of isotope-edited NMR techniques for rapidly providing useful information about the conformations and active site environment of inhibitors bound to their target enzymes.

NMR methods for determining the structures of enzyme/inhibitor complexes as an aid in drug design.

Four approaches are described for providing detailed structural information on large enzyme/inhibitor complexes to aid in the design of improved enzyme inhibitors. In one approach, proton NMR spectra are simplified by isotope-editing procedures in which only those protons that are attached to isotopically labeled nuclei (e.g. 13C or 15N) and their scalar or dipolar coupled partners are observed. Using this strategy, the conformation of an inhibitor bound to porcine pepsin can be determined and structural information on the active site obtained. In another approach, two-dimensional nuclear Overhauser effect (2D NOE) difference spectra are obtained by subtracting NOE spectra of two enzyme/inhibitor complexes prepared with either a protonated or a deuterated inhibitor. Only NOEs arising from protons of the inhibitor substituted with deuterium appear in the 2D NOE difference spectra as illustrated for a pepsin/inhibitor complex. In a third strategy, deuterated enzymes are employed to eliminate the many proton NMR signals of the enzyme and allow the selective detection of the resonances corresponding to the bound ligand as demonstrated for CTP bound to CMP-3-deoxy D-manno-octulosonic acid (KDO) synthetase. Finally, a fourth approach is described using heteronuclear three-dimensional NMR spectroscopy in which homonuclear 2D NMR spectra are edited with respect to the heteronuclear chemical shifts. Using these methods the complete three-dimensional structures of large enzyme/inhibitor complexes can potentially be obtained. Examples of the spectral simplification that can be achieved using 3D NMR are given for 15N-labeled CMP-KDO synthetase complexed with an inhibitor and CTP.

Heteronuclear three-dimensional NMR spectroscopy applied to CMP-KDO synthetase (27.5 kD).

A heteronuclear three-dimensional NMR experiment has been applied to uniformly 15N-labeled CMP-KDO synthetase (CTP:3-deoxy-D-manno-octulosonate cytidylyl transferase; E.C. 2.7.7.38) complexed with an inhibitor and CTP. Using this 3D technique, the 2D NOE spectrum of the ternary complex was dramatically simplified by editing with respect to the 15N frequencies of the labeled enzyme. This 3D NMR method is a useful tool for resolving spectral overlap and is particularly well-suited for NMR studies of large molecules which are difficult to study by conventional 2D NMR techniques.

NMR study of the solution conformation of rat atrial natriuretic factor 7-23 in sodium dodecyl sulfate micelles.

The conformation of the cyclic portion (7-23) of naturally occurring rat atrial natriuretic factor, ANF(1-28), has been examined in sodium dodecyl sulfate (SDS) micelles using high-resolution NMR techniques. Evidence is presented which shows that ANF(7-23) has several regions of definable structure in SDS micelles which were not observed in earlier studies in bulk solvents. The 1H NMR resonances of ANF(7-23) in SDS micelles were assigned using sequential assignment techniques, and the conformational properties were analyzed primarily from proton-proton distances obtained from the quantitative analysis of two-dimensional nuclear Overhauser effect spectra. Three-dimensional structures consistent with the NMR data were generated by using distance geometry and constrained minimization/dynamics. Several similar but not identical structures were found which adequately satisfied the NMR constraints. Although none of the structures adopted a standard secondary structure, the conformations of three different sections of the peptide, 8-13, 14-17, and 18-21, were nearly identical in all of the predicted structures when individually superimposed.