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Biomedical subjects

J H Toney

Publications and source records attributed to J H Toney.

18 recordsLinked to original sources
PubMed2001

Succinic acids as potent inhibitors of plasmid-borne IMP-1 metallo-beta-lactamase.

IMP-1 metallo-beta-lactamase (class B) is a plasmid-borne zinc metalloenzyme that efficiently hydrolyzes beta-lactam antibiotics, including carbapenems, rendering them ineffective. Because IMP-1 has been found in several clinically important carbapenem-resistant pathogens, there is a need for inhibitors of this enzyme that could protect broad spectrum antibiotics such as imipenem from hydrolysis and thus extend their utility. We have identified a series of 2,3-(S,S)-disubstituted succinic acids that are potent inhibitors of IMP-1. Determination of high resolution crystal structures and molecular modeling of succinic acid inhibitor complexes with IMP-1 has allowed an understanding of the potency, stereochemistry, and structure-activity relationships of these inhibitors.

Crystallography, X-Ray↗
PubMed2000

Inhibition of bacterial peptide deformylase by biaryl acid analogs.

Peptide deformylase is an essential eubacterial metalloenzyme involved in the maturation of proteins by cleaving the N-formyl group from N-blocked methionine polypeptides. Biaryl acid analogs containing tetrazole, acyl sulfonamide, or carboxylate pharmacophores were found to be potent inhibitors of recombinant Escherichia coli peptide deformylase. Two of these compounds, a biphenyl tetrazole, compound 1, and a biphenyl acyl sulfonamide, compound 4, were competitive inhibitors with K(i) values of 1.2 and 6.0 microM, respectively. By analogy to the binding of related compounds to other metalloenzymes such as Bacteroides fragilis metallo-beta-lactamase CcrA and human carbonic anhydrase, a mechanism of inhibition is proposed for these peptide deformylase inhibitors where the acidic moieties form direct ionic interactions with the active site metal cation.

Amidohydrolases↗
PubMed1999

Inhibition of IMP-1 metallo-beta-lactamase and sensitization of IMP-1-producing bacteria by thioester derivatives.

IMP-1 metallo-beta-lactamase is a transferable carbapenem-hydrolyzing enzyme found in some clinical isolates of Pseudomonas aeruginosa, Serratia marcescens and Klebsiella pneumoniae. Bacteria that express IMP-1 show significantly reduced sensitivity to carbapenems and other beta-lactam antibiotics. A series of thioester derivatives has been shown to competitively inhibit purified IMP-1. As substrates for IMP-1, the thioesters yielded thiol hydrolysis products which themselves were reversible competitive inhibitors. The thioesters also increased sensitivity to the carbapenem L-742,728 in an IMP-1-producing laboratory stain of Escherichia coli, but will need further modification to improve their activity in less permeable organisms such as Pseudomonas and Serratia. Nonetheless, the thioester IMP-1 inhibitors offer an encouraging start to overcoming metallo-beta-lactamase-mediated resistance in bacteria.

Bacteria↗
PubMed1999

Structure-activity relationships of biphenyl tetrazoles as metallo-beta-lactamase inhibitors.

Resistance to carbapenem antibiotics in gram-negative bacteria is due, in part, to expression of a wide spectrum metallo-beta-lactamase, which renders the drug inactive. Biphenyl tetrazoles containing 3-n-butyl-1-phenylpyrazole-5-carboxylates or the corresponding 5-ethyl esters were found to inhibit metallo-beta-lactamases as well as renal dehydropeptidase I to a lesser extent.

Bacteroides fragilis↗
PubMed1998

Unanticipated inhibition of the metallo-beta-lactamase from Bacteroides fragilis by 4-morpholineethanesulfonic acid (MES): a crystallographic study at 1.85-A resolution.

As part of a structure-aided effort to design clinically useful inhibitors of metallo-beta-lactamases, the X-ray crystal structure of a complex between the metallo-beta-lactamase from Bacteroides fragilis and 4-morpholinoethanesulfonic acid (MES) has been determined and a model for the structure has been refined to a crystallographic R-factor of 0.151 for data between 10.0- and 1.85-A resolution. Although the binding of MES was an adventitious result of the use of MES as a buffer in the crystallization mixture, MES was subsequently shown to be a competitive inhibitor of the enzyme, with a Ki of 23 +/- 5 mM. MES binds in the same fashion to both of the molecules in the crystallographic asymmetric unit; both direct and solvent-mediated hydrogen bonds to the protein and to the binuclear zinc cluster are observed, involving the oxygens of the sulfonic acid group and the nitrogen of the morpholino ring. In addition, there are hydrophobic interactions between the morpholino ring and residues in the flexible beta-strand of the enzyme between residues 26 and 36. Comparison of this structure with the previously reported unliganded structures of the same enzyme [Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J. -M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] reveals that although the overall conservation of structure in the three different crystal lattices is very high, binding of MES is correlated with a significant change in the conformation of this beta-strand. The flexibility of this beta-strand will be an important consideration in the design of inhibitors of the metallo-beta-lactamases.

Alkanesulfonic Acids↗
PubMed1998

Soluble penicillin-binding protein 2a: beta-lactam binding and inhibition by non-beta-lactams using a 96-well format.

High level methicillin resistance in Staphylococcus aureus is dependent upon the acquisition of the mecA gene encoding penicillin-binding protein 2a (PBP2a). PBP2a is a member of a family of peptidoglycan biosynthetic enzymes involved in assembly of the cell wall in bacteria and is poorly inactivated by beta-lactam antibiotics. We describe a 96-well-filter binding assay using recombinant, soluble PBP2a which allows for kinetic measurement of penicillin binding. The deacylation rate constant for the PBP2a-penicillin G covalent complex was found to be 5.7 +/- 1.0 x 10(-5) s-1 at 30 degrees C (half-life of approximately 200 min). For the PBP2a acylation reaction, the value of K(m) (penicillin G) = 0.5 +/- 0.1 mM and kcat = 1 x 10(-3) s-1, which yields a second-order rate constant (kcat/K(m)) for inactivation of 2.0 M-1 s-1. Using this assay, several non-beta-lactam inhibitors including Cibacron blue have been found which exhibit IC50 values between 10 and 30 microM. The binding affinities of several carbapenems and beta-lactams correlated well between the filter binding assay described in this report and an electrophoretic assay for PBP2a using membranes prepared form methicillin-resistant S. aureus.

Anti-Bacterial Agents↗
PubMed1998

Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-beta-lactamase.

BACKGROUND: High level resistance to carbapenem antibiotics in gram negative bacteria such as Bacteroides fragilis is caused, in part, by expression of a wide-spectrum metallo-beta-lactamase that hydrolyzes the drug to an inactive form. Co-administration of metallo-beta-lactamase inhibitors to resistant bacteria is expected to restore the antibacterial activity of carbapenems. RESULTS: Biphenyl tetrazoles (BPTs) are a structural class of potent competitive inhibitors of metallo-beta-lactamase identified through screening and predicted using molecular modeling of the enzyme structure. The X-ray crystal structure of the enzyme bound to the BPT L-159,061 shows that the tetrazole moiety of the inhibitor interacts directly with one of the two zinc atoms in the active site, replacing a metal-bound water molecule. Inhibition of metallo-beta-lactamase by BPTs in vitro correlates well with antibiotic sensitization of resistant B. fragilis. CONCLUSIONS: BPT inhibitors can sensitize a resistant B. fragilis clinical isolate expressing metallo-beta-lactamase to the antibiotics imipenem or penicillin G but not to rifampicin.

Bacteroides fragilis↗
PubMed1997

High-yield expression, purification, and characterization of active, soluble Bacteroides fragilis metallo-beta-lactamase, CcrA.

The gene from Bacteroides fragilis encoding a metallo-beta-lactamase, ccrA, was expressed in Escherichia coli BL21(DE3) containing the wild-type disulfide bond-catalyzing system dsb as an active, soluble enzyme in quantities exceeding 100 mg/liter using both rich and minimal media. Both the nonfusion and a glutathione S-transferase fusion enzyme lacking the periplasmic signal sequence were purified to homogeneity. Characteristics of the purified nonfusion enzyme are shown to be similar to those of the renatured enzyme previously reported. Thermal denaturation studies using circular dichroism and fluorescence spectroscopy show that CcrA undergoes a transition at approximately 50 degrees C which corresponds to the transition temperature of catalytic activity. The secondary structure of the protein and the catalytic apparatus are thus intimately linked.

Bacterial Proteins↗
PubMed1997

Non-steroidal L-245,976 acts as a classical antiandrogen in vitro.

Non-steroidal antiandrogens have been employed in the management of prostate cancer, but the mechanism of action is unclear due to a lack of good tissue culture models. The growth of a hamster ductus deferens cell line (DDT1) is highly dependent upon the addition of 10 nM testosterone to synthetic serum-free media. We describe a non-steroidal compound N-(4-chlorophenyl)-(Z,Z)-2,3-bis(-cyclopropylmethylene) cyclopentanecarboxamide (L-245976) which antagonizes the action of testosterone on DDT1 cells at 10 microM but exhibits little or no effect on cell growth by itself. This compound also blocks the binding of 3H-dihydrotestosterone (DHT) to the human androgen receptor (AR) with an IC50 of approximately 28 microM. In addition, L-245976 was found to antagonize DHT-dependent transactivation of the AR via the probasin gene promoter at comparable doses with no agonist activity.

Amides↗
PubMed1995

Tissue-specific pharmacology of testosterone and 5 alpha-dihydrotestosterone analogues: characterization of a novel canine liver androgen-binding protein.

The mechanism by which the hormones 5 alpha-dihydrotestosterone and testosterone differentially regulate such diverse functions as development of male internal and external genitalia and maintenance of prostatic growth via a single androgen receptor (AR) is not well understood. To search for potential AR isoforms, an extensive pharmacological survey of the binding of [3H]mibolerone (7 alpha,17 alpha-[3H]dimethyl-19-nortestosterone) in dog prostate, adrenal gland, testis, liver, kidney, brain, muscle, and spleen cytosolic extracts was carried out. The antagonist androst-4-en-3,17-dione (ATD), as well as a series of unsaturated analogues of testosterone, exhibited marked tissue specificity for binding to mibolerone-binding proteins (MBPs), with ATD having a 10-fold higher affinity for the MBPs present in liver than for those in prostate and testis. The difference in affinity was not due to tissue-specific metabolism of ATD. Competition binding profiles for ATD with mixtures of prostate and liver extracts were consistent with two distinct populations of binding sites. Both wild-type human AR-B and the recently discovered human AR-A isoform were expressed in COS cells and were found to exhibit pharmacology similar to that of the prostatic MBPs in dogs. Analogues of ATD or testosterone could prove to be useful probes for delineating the differential effects of 5 alpha-dihydrotestosterone and testosterone on the biological actions of the AR and related proteins.

Androgen-Binding Protein↗
PubMed1993

Conformational changes in chicken thyroid hormone receptor alpha 1 induced by binding to ligand or to DNA.

A classic model of steroid/thyroid hormone receptor activation postulates that a conformational change or "transformation" occurs upon ligand binding as a first step toward regulation of gene transcription. In order to test this model, physical studies have been carried out using purified full-length chicken thyroid hormone receptor alpha 1 (cT3R-alpha 1) expressed in Escherichia coli. Circular dichroism spectroscopic studies reveal that cT3R-alpha 1 adopts a different conformation upon specific binding to a cognate ligand triiodothyroacetic acid as well as to a thyroid hormone response element, an idealized inverted repeat AGGTCA TGACCT. These results suggest that cT3R-alpha 1 may adopt distinct conformations whether free or bound to ligand or to DNA. These states may reflect the changes in the conformation of steroid/thyroid hormone receptors in the signal transduction pathway.

Animals↗
PubMed1990

Characterization of a DNA damage-recognition protein from mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin.

A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, Kd, of the protein from cisplatin-modified DNA was estimated to be (1-20) X 10(-10) M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl2] (en, ethylenediamine), and [Pt(dach)Cl2] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein also does not bind to DNA containing UV-induced photoproducts. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin, as determined by gel mobility shifts with synthetic 110-bp duplex oligonucleotides; these modified oligomers contained five equally spaced adducts of either cis-[Pt(NH3)2d(GpG) or cis-[Pt(NH3)2d(ApG)]. Oligonucleotides containing the specific adducts cis-[Pt(NH3)2d(GpTpG)], trans-[Pt(NH3)2d(GpTpG)], or cis-[Pt(NH3)2(N3-cytosine)d(G)] were not recognized by the protein. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl2, CdCl2, CoCl2, or ZnCl2 and with 1 mM HgCl2.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1989

Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II)

DNA modified by the antitumor drug cis-diamminedichloroplatinum(II) (cis-DDP or cisplatin) was used to identify a factor in mammalian cells that binds to cis-DDP-damaged DNA and hence may play a role in repair. This factor selectively recognizes double-stranded DNA fragments modified by cis-DDP or [Pt(en)Cl2] (en, ethylenediamine). Little or no binding occurs to unmodified double-stranded DNA or to DNA modified with the clinically ineffective compounds trans-DDP and [Pt(dien)Cl]Cl (dien, diethylenetriamine). Low levels of binding to single-stranded DNA modified by cis-DDP are observed. The apparent molecular mass of the factor in a variety of mammalian cells is approximately 100 kDa, as determined by modified Western blotting. Two recombinant phage have been isolated from a human B-cell lambda gt11 library by using a cis-DDP-modified DNA restriction fragment as a probe. The two clones have insert sizes of 1.88 and 1.44 kilobases and are aligned at their 5' ends. The polypeptides encoded by the recombinant phage exhibit DNA binding properties similar to those of the cellular factor identified in crude extracts prepared from mammalian cells. Northern analysis with one of the clones revealed an mRNA of 2.8 kilobases that is conserved in humans and rodents. The methods used here should be applicable in studies of other damage-specific DNA binding proteins.

Bacteriophage lambda↗
PubMed1987

A highly conserved endonuclease activity present in Escherichia coli, bovine, and human cells recognizes oxidative DNA damage at sites of pyrimidines.

We have compared the sites of nucleotide incision on DNA damaged by oxidizing agents when cleavage is mediated by either Escherichia coli endonuclease III or an endonuclease present in bovine and human cells. E. coli endonuclease III, the bovine endonuclease isolated from calf thymus, and the human endonuclease partially purified from HeLa and CEM-C1 lymphoblastoid cells incised DNA damaged with osmium tetroxide, ionizing radiation, or high doses of UV light at sites of pyrimidines. For each damaging agent studied, regardless of whether the E. coli, bovine, or human endonuclease was used, the same sequence specificity of cleavage was observed. We detected this endonuclease activity in a variety of human fibroblasts derived from normal individuals as well as individuals with the DNA repair deficiency diseases ataxia telangiectasia and xeroderma pigmentosum. The highly conserved nature of such a DNA damage-specific endonuclease suggests that a common pathway exists in bacteria, humans, and other mammals for the reversal of certain types of oxidative DNA damage.

Animals↗
PubMed1985

Biodistribution and pharmacokinetics of vanadium following intraperitoneal administration of vanadocene dichloride to mice.

The biodistribution and pharmacokinetics of vanadium following i.p. administration of vanadocene dichloride (VDC), a representative of a new class of organometallic anticancer agents, is reported for Strain A mice. A convenient flameless atomic absorption spectroscopic assay is described and is used to determine kinetic profiles for vanadium in blood, kidney, liver, small intestine and brain tissue for times up to 24 h after administration. For a VDC dose of 80 mg/kg, vanadium concentration decreases rapidly from both the blood and small intestine, and the data can be fit to a phenomenological exponential function (blood: t1/2 = 118 +/- 43 min; small intestine: t1/2(alpha) = 18.10 +/- 0.14 min, t1/2(beta) = 341 +/- 45 min). In contrast, vanadium accumulates in both the kidney and liver up to a maximal concentration (1.12 +/- 0.06 mM and 0.56 +/- 0.06 mM after 12 and 8 h, respectively), and is then excreted with estimated half-lives of 7.9 +/- 0.7 and 12.1 +/- 0.1 h, respectively. No detectable levels of vanadium are found in the brain tissue over the temporal course of the experiment. These results are compared to previous mammalian studies with cis-dichlorodiammineplatinum(II) (CDDP) and related 'second generation' platinum derivatives; there are both qualitative similarities between the vanadium and platinum systems as well as important quantitative differences.

Animals↗
PubMed1979

Specific binding of 125I-labeled beta-hexosaminidase A to rat brain synaptosomes.

Purified human beta-hexosaminidase A (beta-N-acetylgulcosaminidase; 2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase, EC 3.2.1.30) has been labeled with 125I to high specific activity with the retention of 80% of its enzyme activity. The binding of this enzyme to sonicated synaptosomes from rat brain was shown to be a saturable and specific process. Glycoproteins containing a sialic acid-terminal oligosaccharide or a galactose-terminal oligosaccharide (i.e., alpha 1-acid glycoprotein and fetuin and their asialo derivatives) were strong inhibitors of the binding. In contrast, ovalbumin, which contains a mannose-rich oligosaccharide, and mannans were poor inhibitors of the binding. Of the monosaccharides tested, sialic acid, galactosamine, mannose, galactose, and lactose were inhibitory in decreasing potency of inhibition. Optimal binding occurred at pH 7.0 in the presence of 3 mM calcium ions. The binding was a linear function of synaptosomal protein concentration between 25 and 200 microgram of protein per assay and was directly proportional to time up to 3 hr, beyond which there was no further increase in specific binding. The data suggest a unique but complex mode of interaction of glycoproteins with receptors on synaptic membranes.

Animals↗
PubMedDate not supplied

Computer assisted small-scale Tay-Sachs carrier screening.

Fluorescence data from serum beta-hexosaminidase assays for Tay-Sachs heterozygote screening is processed by a program in BASIC for a Wang 2200 desk-top computer. An estimation of overall error in the results gives a measure of reliability. A preliminary classification is made on the clinical status of the unknown samples as a normal, heterozygous, homozygous, or inconclusive carrier/normal condition. This program allows a small-scale screening center convenient and rapid data processing and analysis.

Clinical Enzyme Tests↗