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S J Lippard

Publications and source records attributed to S J Lippard.

At least 55 records · Page 3Linked to original sources
PubMed2000

High-throughput synthesis and screening of platinum drug candidates.

Platinum drugs play an important role in the treatment of cancer, but there is room for improvement. Here we present a new platinum drug-discovery strategy to identify compounds having efficacy equivalent to that of cisplatin with the expectation that some may increase the spectrum of treatable tumors and/or reduce dose-limiting toxicity. Platinum drug candidates were generated through the use of automated synthesis, taking advantage either of the trans effect or by using silver chloride precipitation to activate the starting materials. Reaction products were screened for activity in a high-throughput transcription assay and the most promising candidates characterized. Over 3,600 reaction products were screened for their ability to inhibit transcription of beta-lactamase in the BlaM HeLa cell line by monitoring cleavage of a lactam ring linking the two halves of a fluorescent resonance energy transfer (FRET) dye, CCF2/AM. From this screen, three reactions produced good candidates, and four species were identified among these reaction products. Three of the compounds, cis-[(isopropylamine)2PtCl2], cis-[(cyclobutylamine)2PtCl2], and cis-[ammine(cyclobutylamine)PtCl2], have been previously determined to be active cisplatin analogs. The fourth compound, cis-[ammine(2-amino-3-picoline)PtCl2], represents a new kind of antitumor drug candidate similar to ZD0473, a recently reported analog. The discovery of these compounds represents an important proof of principle that platinum anticancer drug candidates can be rapidly prepared and screened in this manner.

Antineoplastic Agents↗
PubMed2000

Sequencing and analysis of the Mmethylococcus capsulatus (Bath) solublemethane monooxygenase genes.

The soluble methane monooxygenase (sMMO) hydroxylase is a prototypical member of the class of proteins with non-heme carboxylate-bridged diiron sites. The sMMO subclass of enzyme systems has several distinguishing characteristics, including the ability to catalyze hydroxylation or epoxidation chemistry, a multisubunit hydroxylase containing diiron centers in its alpha subunits, and the requirement of a coupling protein for optimal activity. Sequence homology alignment of known members of the sMMO family was performed in an effort to identify protein regions giving rise to these unique features. DNA sequencing of the Methylococcus capsulatus (Bath) sMMO genes confirmed previously identified sequencing errors and corrected two additional errors, each of which was confirmed by at least one independent method. Alignments of homologous proteins from sMMO, phenol hydroxylase, toluene 2-, 3-, and 4-monooxygenases, and alkene monooxygenase systems revealed an interesting set of absolutely conserved amino-acid residues, including previously unidentified residues located outside the diiron active site of the hydroxylase. By mapping these residues on to the M. capsulatus (Bath) sMMO hydroxylase crystal structure, functional and structural roles were proposed for the conserved regions. Analysis of the active site showed a highly conserved hydrogen-bonding network on one side of the diiron cluster but little homology on the opposite side, where substrates are presumed to bind. It is suggested that conserved residues on the hydroxylase surface may be important for protein-protein interactions with the reductase and coupling ancillary proteins and/or serve as part of an electron-transfer pathway. A possible way by which binding of the coupling protein at the surface of the hydroxylase might transfer information to the diiron active site at the interior is proposed.

Amino Acid Sequence↗
PubMed1999

Component interactions in the soluble methane monooxygenase system from Methylococcus capsulatus (Bath).

The soluble methane monooxygenase system of Methylococcus capsulatus (Bath) includes three protein components: a 251-kDa non-heme dinuclear iron hydroxylase (MMOH), a 39-kDa iron-sulfur- and FAD-containing reductase (MMOR), and a 16-kDa regulatory protein (MMOB). The thermodynamic stability and kinetics of formation of complexes between oxidized MMOH and MMOB or MMOR were measured by isothermal titration calorimetry and stopped-flow fluorescence spectroscopy at temperatures ranging from 3.3 to 45 degrees C. The results, in conjunction with data from equilibrium analytical ultracentrifugation studies of MMOR and MMOB, indicate that free MMOR and MMOB exist as monomers in solution and bind MMOH with 2:1 stoichiometry. The role of component interactions in the catalytic mechanism of sMMO was investigated through simultaneous measurement of oxidase and hydroxylase activities as a function of varied protein component concentrations during steady-state turnover. The partitioning of oxidase and hydroxylase activities of sMMO is highly dependent on both the MMOR concentration and the nature of the organic substrate. In particular, NADH oxidation is significantly uncoupled from methane hydroxylation at MMOR concentrations exceeding 20% of the hydroxylase concentration but remains tightly coupled to propylene epoxidation at MMOR concentrations ranging up to the MMOH concentration. The steady-state kinetic data were fit to numerical simulations of models that include both the oxidase activities of free MMOR and of MMOH/MMOR complexes and the hydroxylase activity of MMOH/MMOB complexes. The data were well described by a model in which MMOR and MMOB bind noncompetitively at distinct interacting sites on the hydroxylase. MMOB manifests its regulatory effects by differentially accelerating intermolecular electron transfer from MMOR to MMOH containing bound substrate and product in a manner consistent with its activating and inhibitory effects on the hydroxylase.

Catalysis↗
PubMed1999

Structure of the soluble methane monooxygenase regulatory protein B.

The soluble methane monooxygenase (sMMO; EC 1.14.13.25) from the pseudothermophile Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the selective oxidation of methane to methanol. We have used NMR spectroscopy to produce a highly refined structure of MMOB, the 16-kDa regulatory protein of this system. This structure has a unique and intricate fold containing seven beta-strands forming two beta-sheets oriented perpendicular to each other and bridged by three alpha-helices. The rate and efficiency of the methane hydroxylation by sMMO depend on dynamic binding interactions of the hydroxylase with the reductase and regulatory protein components during catalysis. We have monitored by NMR the binding of MMOB to the hydroxylase in the presence and absence of the reductase. The results of these studies provide structural insight into how the regulatory protein interacts with the hydroxylase.

Amino Acid Sequence↗
PubMed1999

Basis for recognition of cisplatin-modified DNA by high-mobility-group proteins.

The anticancer activity of cis-diamminedichloroplatinum(II) (cisplatin) arises from its ability to damage DNA, with the major adducts formed being intrastrand d(GpG) and d(ApG) crosslinks. These crosslinks bend and unwind the duplex, and the altered structure attracts high-mobility-group domain (HMG) and other proteins. This binding of HMG-domain proteins to cisplatin-modified DNA has been postulated to mediate the antitumour properties of the drug. Many HMG-domain proteins recognize altered DNA structures such as four-way junctions and cisplatin-modified DNA, but until now the molecular basis for this recognition was unknown. Here we describe mutagenesis, hydroxyl-radical footprinting and X-ray studies that elucidate the structure of a 1:1 cisplatin-modified DNA/HMG-domain complex. Domain A of the structure-specific HMG-domain protein HMG1 binds to the widened minor groove of a 16-base-pair DNA duplex containing a site-specific cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]] adduct. The DNA is strongly kinked at a hydrophobic notch created at the platinum-DNA crosslink and protein binding extends exclusively to the 3' side of the platinated strand. A phenylalanine residue at position 37 intercalates into a hydrophobic notch created at the platinum crosslinked d(GpG) site and binding of the domain is dramatically reduced in a mutant in which alanine is substituted for phenylalanine at this position.

Amino Acid Sequence↗
PubMed1999

Structural and kinetic studies of a cisplatin-modified DNA icosamer binding to HMG1 domain B.

The high mobility group (HMG) domain is a DNA-binding motif found in the non-histone chromosomal proteins, HMG1 and HMG2, and some transcription factors. Experimental evidence has demonstrated that HMG-domain proteins can play a role in sensitizing cells to the anticancer drug cisplatin. Fluorescence resonance energy transfer (FRET) experiments were performed in the present study to investigate structural changes that accompany complex formation between the HMG domain B of HMG1 and a cisplatin-modified, 20-base pair double-stranded DNA probe containing fluorescein and rhodamine tethered at its two ends. The binding affinity of HMG1 domain B for the cisplatin-modified DNA probe was investigated in fluorescence titration experiments, and a value of 60 +/- 30 nM was determined for the dissociation constant. Single photon counting methods were employed to measure the fluorescence lifetime of the fluorescein donor in the presence and absence of HMG1 domain B. These FRET experiments revealed a distance change that was used to estimate a bend angle of 80-95 degrees for the cisplatin-modified DNA upon protein binding. Stopped-flow fluorescence spectroscopic experiments afforded kinetic parameters for HMG1 domain B binding to the cisplatin-modified DNA probe, with kon = 1.1 +/- 0.1 x 10(9) M-1 s-1 and koff = 30 +/- 4 s-1.

Base Sequence↗
PubMed1999

Oxidation of ultrafast radical clock substrate probes by the soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenyl-cyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling.

Binding Sites↗
PubMed1999

Reactions of nitric oxide with the reduced non-heme diiron center of the soluble methane monooxygenase hydroxylase.

The soluble methane monooxygenase system from Methylococcus capsulatus (Bath) catalyzes the oxidation of methane to methanol and water utilizing dioxygen at a non-heme, carboxylate-bridged diiron center housed in the hydroxylase (H) component. To probe the nature of the reductive activation of dioxygen in this system, reactions of an analogous molecule, nitric oxide, with the diiron(II) form of the enzyme (Hred) were investigated by both continuous and discontinuous kinetics methodologies using optical, EPR, and Mössbauer spectroscopy. Reaction of NO with Hred affords a dinitrosyl species, designated Hdinitrosyl, with optical spectra (lambdamax = 450 and 620 nm) and Mössbauer parameters (delta = 0.72 mm/s, DeltaEQ = 1.55 mm/s) similar to those of synthetic dinitrosyl analogues and of the dinitrosyl adduct of the reduced ribonucleotide reductase R2 (RNR-R2) protein. The Hdinitrosyl species models features of the Hperoxo intermediate formed in the analogous dioxygen reaction. In the presence of protein B, Hdinitrosyl builds up with approximately the same rate constant as Hperoxo ( approximately 26 s-1) at 4 degrees C. In the absence of protein B, the kinetics of Hdinitrosyl formation were best fit with a biphasic A --> B --> C model, indicating the presence of an intermediate species between Hred and Hdinitrosyl. This result contrasts with the reaction of Hred with dioxygen, in which the Hperoxo intermediate forms in measurable quantities only in the presence of protein B. These findings suggest that protein B may alter the positioning but not the availability of coordination sites on iron for exogenous ligand binding and reactivity.

Bacterial Proteins↗
PubMed1999

An EPR study of the dinuclear iron site in the soluble methane monooxygenase from Methylococcus capsulatus (Bath) reduced by one electron at 77 K: the effects of component interactions and the binding of small molecules to the diiron(III) center.

Reduction of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) in frozen 4:1 buffer/glycerol solutions at 77 K by mobile electrons generated by gamma-irradiation produces an EPR-detectable, mixed-valent Fe(II)Fe(III) center. At this temperature the conformation of the enzyme remains essentially unaltered during reduction, so the mixed-valent EPR spectra serve to probe the active site structure of the EPR-silent, diiron(III) state. The EPR spectra of the cryoreduced samples reveal that the diiron(III) cluster of the resting hydroxylase has at least two chemically distinct forms, the structures of which differ from that of the equilibrium Fe(II)Fe(III) site. Their relative populations depend on pH, the presence of component B, and formation of the MMOH/MMOB complex by reoxidation of the reduced, diiron(II) hydroxylase. The formation of complexes between MMOB, MMOR, and the oxidized hydroxylase does not measurably affect the structure of the diiron(III) site. Cryogenic reduction in combination with EPR spectroscopy has also provided information about interaction of MMOH in the diiron(III) state with small molecules. The diiron(III) center binds methanol and phenols, whereas DMSO and methane have no measurable effect on the EPR properties of cryoreduced hydroxylase. Addition of component B favors the binding of some exogenous ligands, such as DMSO and glycerol, to the active site diiron(III) state and markedly perturbs the structure of the diiron(III) cluster complexed with methanol or phenol. The results reveal different reactivity of the Fe(III)Fe(III) and Fe(II)Fe(III) redox states of MMOH toward exogenous ligands. Moreover, unlike oxidized hydroxylase, the binding of exogenous ligands to the protein in the mixed-valent state is allosterically inhibited by MMOB. The differential reactivity of the hydroxylase in its diiron(III) and mixed-valent states toward small molecules, as well as the structural basis for the regulatory effects of component B, is interpreted in terms of a model involving carboxylate shifts of a flexible glutamate ligand at the Fe(II)Fe(III) center.

Binding Sites↗
PubMed1999

Single d(GpG)/cis-diammineplatinum(II) adduct-induced inhibition of DNA polymerization.

A 44 nucleotide DNA template containing a single site-specifically placed cisplatin adduct (cis-[Pt(NH3)2[d(GpG)-N7(1),-N7(2)]]) was annealed with a primer, positioning its 3'-end four bases before the adduct in the template strand. DNA polymerization in the presence of all four nucleotides revealed that both HIV-1 reverse transcriptase (RT) and T7 DNA polymerase strongly paused at one nucleotide preceding the first platinated guanine and at the positions opposite the two platinated guanines. Analysis of single nucleotide incorporation at each pause site showed that polymerization occurs with biphasic kinetics. A small percentage of DNA was bound productively, providing a small amplitude (1-3%) of a fast phase of polymerization, whereas most of the bound DNA (1-34%) was positioned at the pause site in a nonproductive manner and therefore elongated slowly (0.04-0.06 s-1). DNA substrates annealed to the cisplatin-modified template bind to HIV-1 RT with an affinity (10-20 nM) similar to that of unmodified substrates (6-9 nM). The cisplatin-DNA cross-link moderately weakened DNA binding to T7 DNA polymerase (12-115 nM) but significantly slowed the rate of incorporation of the next nucleotide (2-7 s-1 ), with larger effects closer to the cisplatin-DNA adduct. The crystal structure of the same cisplatin-DNA adduct [Takahara, P. M., Frederick, C. A., and Lippard, S. J. (1996) J. Am. Chem. Soc. 118, 12309-12321] reveals not only the bent DNA duplex but also the propeller twisted base pairs near the cisplatin-DNA adduct. The twisted base pairs may cause misalignment of the cisplatin-modified DNA at the binding cleft of T7 DNA polymerase and significantly slow the rate of the protein conformational change preceding polymerization, leading to the slight accumulation of intermediates within five base pairs of the adduct. The ground-state binding of the next correct nucleotide to the enzyme.DNA complex was weakened by the adduct with T7 DNA polymerase but unchanged with HIV-1 RT at sites other than the three strong pause sites. Nucleotide binding to both enzymes at the three strong pause sites was significantly weaker and less selective.

Bacteriophage T7↗
PubMed1999

Toxicity of platinum(II) amino acid (N,O) complexes parallels their binding to DNA as measured in a new solid phase assay involving a fluorescent HMG1 protein construct readout.

The compound [Pt(lysine)Cl2] (Kplatin) was previously identified in a study of platinum amino acid complexes as a potential antitumor drug candidate. The DNA binding properties, high mobility group (HMG)-domain protein affinity for the platinated DNA, and cytotoxicity against HeLa cells of Kplatin and three related (N,O) chelated platinum(II) amino acid complexes, [Pt(arginine)Cl2] (Rplatin), K[Pt(Nepsilon-acetyllysine)Cl2] (NacKplatin), and K[Pt(norleucine)Cl2] (Norplatin), are reported. The four complexes have identical PtCl2(N,O) coordination environments. A new solid phase screening methodology was devised in which platinated DNA probes are covalently attached to a nylon support and tested for their ability to bind a fluorescently labeled HMG-domain protein. The fluorescent HMG-domain protein was generated by expressing a fusion of the green fluorescent protein (GFP) with recombinant rat HMG1. Binding revealed by the solid phase method correlated well with the results of gel mobility shift and HeLa cytotoxicity assays. These results suggest that the net charge on the complex, rather than the nature of the side chain, is the most important factor underlying the DNA binding properties and toxicity of amino acid (N,O) chelated platinum complexes. This property explains why Kplatin was previously selected from the pool of platinum amino acid complexes based on the ability of its DNA adducts to bind HMG1.

Animals↗
PubMed1999

The reactivity of well defined diiron(III) peroxo complexes toward substrates: addition to electrophiles and hydrocarbon oxidation.

The reactivity of previously reported peroxo adducts [Fe(mu-O2)(mu-L)(O2CPhCy)2(1-Bu-Im)2] (1), and [Fe(mu-O2)(mu-L)(O2CPhCy)2(py)2] (2), where L is a dinucleating ligand based on the m-xylylenediamine bis(Kemp's triacid imide), toward a variety of substrates is described. These studies were performed to probe the electronic properties of 1 and 2 and evaluate their potential as selective hydrocarbon oxidants. Compound 1 is nucleophilic at -77 degrees C, reacting with phenols and carboxylic acids to liberate hydrogen peroxide, whereas the less electron-rich pyridine analogue 2 is unreactive toward both reagents. By contrast, neither reacts at -77 degrees C with electrophilic reagents such as olefins or triphenylphosphine, or with weak hydrogen atom donors such as dimethylbenzylamine. When solutions of 1 are warmed to room temperature in solvents such as THF, toluene, and cyclopentane, mixtures of alcohol and ketone products derived from the solvent are formed. A detailed investigation of cyclopentane oxidation strongly points to a radical autoxidation pathway. These results are discussed in the context of the selective hydroxylation chemistry that occurs at the carboxylate-bridged diiron centers in soluble methane monooxygenase.

Chromatography, Gas↗
PubMed1999

Mutational and structural analyses of the regulatory protein B of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

BACKGROUND: The soluble methane monooxygenase (sMMO) system in methanotrophic bacteria uses three protein components to catalyze the selective oxidation of methane to methanol. The coupling protein B (MMOB) both activates the carboxylate-bridged diiron center in the hydroxylase (MMOH) for substrate oxidation and couples the reaction to electron transfer from NADH through the sMMO reductase. Although the X-ray structure of the hydroxylase is known, little structural information is available regarding protein B. RESULTS: Wild-type protein B from Methylococcus capsulatus (Bath) is very susceptible to degradation. The triple mutant protein B, Gly10-->Ala, Gly13-->Gln, Gly16-->Ala is resistant to degradation. Analyzing wild-type and mutant forms of protein B using size exclusion chromatography and circular dichroism spectroscopy suggests that the amino terminus of MMOB (Ser1-Ala25) is responsible for the proteolytic sensitivity and unusual mobility of the protein. We used the stable triple glycine protein B mutant to generate an affinity column for the hydroxylase and investigated the interaction between MMOH and MMOB. These results suggest the interaction is dominated by hydrophobic contacts. CONCLUSIONS: A structural model is presented for protein B that explains both its proclivity for degradation and its anomalous behavior during size exclusion chromatography. The model is consistent with previously published biophysical data, including the NMR structure of the phenol hydroxylase regulatory protein P2. Furthermore, this model allows for detailed and testable predictions about the structure of protein B and the role of proposed recognition sites for the hydroxylase.

Amino Acid Sequence↗
PubMed1999

Rapid fluorescence-based reporter-gene assays to evaluate the cytotoxicity and antitumor drug potential of platinum complexes.

BACKGROUND: The need for new platinum antitumor drugs is underscored by the usefulness of cisplatin and carboplatin in chemotherapy and the resistance of many tumors to these compounds. Combinatorial chemistry could aid in the search for cisplatin analogs if fast, high-throughput assays were available. Our goal was to develop rapid cell-based assays suitable for high-throughput screening that accurately predict the cytotoxicity of platinum complexes. We examined the effects of platinum complexes and other agents on reporter-gene expression in cancer cells. RESULTS: HeLa Tet-On cells with inducible enhanced green fluorescent protein (EGFP) were prepared. Cisplatin and other cis-disubstituted platinum complexes inhibited EGFP expression, with a strong positive correlation between EGFP inhibition and cytotoxicity. By contrast, trans-[Pt(NH(3))(2)Cl(2)], other trans-platinum complexes, methyl methanesulfonate or heat shock stimulated EGFP expression. Northern and nuclear run-on analyses revealed that the changes in EGFP expression were at the level of transcription. In another reporter-gene assay in Jurkat cells, cisplatin, but not trans-[Pt(NH(3))(2)Cl(2)] or K(2)[PtCl(4)], inhibited beta-lactamase expression, as measured by hydrolysis of the fluorescent substrate CCF2. CONCLUSIONS: The EGFP results indicate that cytotoxic stress enhances transcription from the inducible promoter, whereas compounds able to form the 1,2-intrastrand platinum-DNA cross-links repress transcription. Both fluorescence-based reporter-gene assays afford promising new approaches to platinum anticancer drug discovery.

Antineoplastic Agents↗
PubMed1998

NMR solution structure of a DNA dodecamer duplex containing a cis-diammineplatinum(II) d(GpG) intrastrand cross-link, the major adduct of the anticancer drug cisplatin.

The structure of the DNA duplex dodecamer, d(CCTCTGGTCTCC. GGAGACCAGAGG), containing the cisplatin d(GpG) 1,2-intrastrand cross-link at the position denoted by asterisks, was determined in solution by high-resolution 2D NMR spectroscopy and restrained molecular dynamics refinement. The cis-[Pt(NH3)2¿d(GpG-N7(1), N7(2))¿] lesion causes the adjacent guanine bases to roll toward one another by 49 degrees, leading to an overall helix bend angle of 78 degrees. These features are more exaggerated than those observed in the X-ray crystal structure determined for the same platinated duplex [Takahara et al. (1995) Nature 377, 649-652]. A common property of the solution and crystal structures is the widening and flattening of the minor groove opposite the platinum adduct, affording geometric parameters resembling those found in A-form DNA. This deformation is especially noteworthy for the solution structure because its sugar puckers are primarily those of B-form DNA. The unwinding of the helix at the site of platination is 25 degrees. The curvature and shape of the platinated duplex are remarkably similar to those observed in DNA duplexes complexed by the HMG-domain proteins SRY and LEF-1. The structure reveals how cisplatin binding alters DNA in such a manner as to facilitate HMG-domain protein recognition.

Antineoplastic Agents↗
PubMed1998

p53-Dependent and -independent responses to cisplatin in mouse testicular teratocarcinoma cells.

Testicular cancers respond favorably to chemotherapy with the platinum-containing drug cis-diamminedichloroplatinum(II) (cisplatin). One factor that could explain the efficacy of cisplatin is the low frequency of p53 mutations observed in this tumor type. The present study examines the p53-mediated responses in murine testicular teratocarcinoma cells exposed to the drug. Cisplatin treatment of teratocarcinoma cells with a wild-type p53 gene resulted in accumulation of the p53 protein through posttranscriptional mechanisms; induction of p53-target genes was also observed. Drug treatment resulted in rapid apoptosis in p53-wild-type cells but not in p53(-/-) teratocarcinoma cells. In the latter cells, cisplatin exposure caused prolonged cell cycle arrest accompanied by induction of the p21 gene. Clonogenic assays demonstrated that the p53 mutation did not confer resistance to cisplatin. These experiments suggest that cisplatin inhibits cellular proliferation of testicular teratocarcinoma cells by two possible mechanisms, p53-dependent apoptosis and p53-independent cell cycle arrest.

Animals↗