Novel nonpeptidic inhibitors of peptide deformylase.

A novel series of nonpeptidic compounds structurally related to the known anticholesteremic thyropropic acid were found to inhibit Escherichia coli peptide deformylase (PDF), with IC50 values in the low-micromolar range. Kinetic analysis of [4-(4-hydroxyphenoxy)-3,5-diiodophenyl]acetic acid reveals competitive inhibition, with a Ki value of 0.66 +/- 0.007 microM. A structure-activity relationship study demonstrates that the carboxylate is required for activity, while the distal phenolic function can be methylated without significant effect. Either decreasing the number of iodine atoms on the molecule to one or increasing the number of iodine atoms to four results in the loss of an order of magnitude in potency. These compounds are the first nonpeptidic inhibitors disclosed and represent a template from which better inhibitors might be designed.

Escherichia coli requires the protease activity of FtsH for growth.

FtsH protease, the product of the essential ftsH gene, is a membrane-bound ATP-dependent metalloprotease of Escherichia coli that has been shown to be involved in the rapid turnover of key proteins, secretion of proteins into and through the membrane, and mRNA decay. The pleiotropic effects of ftsH mutants have led to the suggestion that FtsH possesses an ATP-dependent chaperone function that is independent of its protease function. When considering FtsH as a target for novel antibacterials, it is necessary to determine which of these functions is critical for the growth and survival of bacteria. To address this, we constructed the FtsH mutants E418Q, which retains significant ATPaseactivity but lacks protease activity, and K201N, which lacks both protease and ATPase activities. These mutants were introduced into an E. coli ftsH knockout strain which has wild-type FtsH supplied from a plasmid under control of the inducible araBAD promoter. Since neither mutant would complement the ftsH defect produced in the absence of arabinose, we conclude that the protease function of FtsH is required for bacterial growth.

Application of electrospray ionization mass spectrometry for studying human immunodeficiency virus protein complexes.

Mass spectrometry (MS) with electrospray ionization (ESI) has shown utility for studying noncovalent protein complexes, as it offers advantages in sensitivity, speed, and mass accuracy. The stoichiometry of the binding partners can be easily deduced from the molecular weight measurement. In many examples of protein complexes, the gas phase-based measurement is consistent with the expected solution phase binding characteristics. This quality suggests the utility of ESI-MS for investigating solution phase molecular interactions. Complexes composed of proteins from the human immunodeficiency virus (HIV) have been studied using ESI-MS. Multiply charged protein dimers from HIV integrase catalytic core (F185K) and HIV protease have been observed. Furthermore, the ternary complex between HIV protease dimer and inhibitor pepstatin A was studied as a function of solution pH. Zinc binding to zinc finger-containing nucleocapsid protein (NCp7) and the NCp7-psi RNA 1:1 stoichiometry complex was also studied by ESI-MS. No protein-RNA complex was observed in the absence of zinc, consistent with the role of the zinc finger motifs for RNA binding.

Biophysical characterization of zinc ejection from HIV nucleocapsid protein by anti-HIV 2,2'-dithiobis[benzamides] and benzisothiazolones.

HIV nucleocapsid protein (NCp7) has been suggested as a possible target for 2,2'-dithiobis-[benzamide] and benzisothiazolone agents that inhibit viral replication in infected cells (Rice et al. Science 1995, 270, 1194-1197). The solution behavior of these compounds and the mechanistic events leading to removal of Zn from HIV nucleocapsid protein in vitro has been studied by electrospray ionization mass spectrometry, 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy, and circular dichroism spectroscopy. We demonstrate that (1) Zn ejection is accompanied by formation of covalent complexes formed between the 2,2'-dithiobis[benzamide] monomers and Cys residues of Zn-depleted NCp7, (2) the rate of Zn ejection is faster for the C-terminal Zn finger and slower for the N-terminal finger, (3) Zn ejection results in a loss of structural integrity of the NCp7 protein, and (4) there is no appreciable interaction between a nonreactive isostere of the lead 2,2'-dithiobis[benzamide] and NCp7 in buffered aqueous solution. These findings are discussed in terms of the mechanism of action of Zn ejection by aromatic 2,2'-dithiobis[benzamides].

The in vitro ejection of zinc from human immunodeficiency virus (HIV) type 1 nucleocapsid protein by disulfide benzamides with cellular anti-HIV activity.

Several disulfide benzamides have been shown to possess wide-spectrum antiretroviral activity in cell culture at low micromolar to submicromolar concentrations, inhibiting human immunodeficiency virus (HIV) type 1 (HIV-1) clinical and drug-resistant strains along with HIV-2 and simian immunodeficiency virus [Rice, W. G., Supko, J. G., Malspeis, L., Buckheit, R. W., Jr., Clanton, D., Bu, M., Graham, L., Schaeffer, C. A., Turpin, J. A., Domagala, J., Gogliotti, R., Bader, J. P., Halliday, S. M., Coren, L., Sowder, R. C., II, Arthur, L. O. & Henderson, L. E. (1995) Science 270, 1194-1197]. Rice and coworkers have proposed that the compounds act by "attacking" the two zinc fingers of HIV nucleocapsid protein. Shown here is evidence that low micromolar concentrations of the anti-HIV disulfide benzamides eject zinc from HIV nucleocapsid protein (NCp7) in vitro, as monitored by the zinc-specific fluorescent probe N-(6-methoxy-8-quinoyl)-p-toluenesulfonamide (TSQ). Structurally similar disulfide benzamides that do not inhibit HIV-1 in culture do not eject zinc, nor do analogs of the antiviral compounds with the disulfide replaced with a methylene sulfide. The kinetics of NCp7 zinc ejection by disulfide benzamides were found to be nonsaturable and biexponential, with the rate of ejection from the C-terminal zinc finger 7-fold faster than that from the N-terminal. The antiviral compounds were found to inhibit the zinc-dependent binding of NCp7 to HIV psi RNA, as studied by gel-shift assays, and the data correlated well with the zinc ejection data. Anti-HIV disulfide benzamides specifically eject NCp7 zinc and abolish the protein's ability to bind psi RNA in vitro, providing evidence for a possible antiretroviral mechanism of action of these compounds. Congeners of this class are under advanced preclinical evaluation as a potential chemotherapy for acquired immunodeficiency syndrome.

HIV1 integrase expressed in Escherichia coli from a synthetic gene.

Human immunodeficiency virus type 1 (HIV1) integrase is cleaved from the gag-pol precursor by the HIV1 protease. The resulting 32-kDa protein is used by the infecting virus to insert a linear, double-stranded DNA copy of its genome, prepared by reverse transcription of viral RNA, into the host cell's chromosomal DNA. In order to achieve high levels of expression, to minimize an internal initiation problem and to facilitate mutagenesis, we have designed and synthesized a gene encoding the integrase from the infectious molecular clone, pNL4-3. Codon usage was optimized for expression in Escherichia coli and unique restriction sites were incorporated throughout the gene. A 905-bp cassette containing a ribosome-binding site, a start codon and the integrase-coding sequence, sandwiched between EcoRI and HindIII sites, was synthesized by overlap extension of nine long synthetic oligodeoxyribonucleotides [90-120 nucleotides (nt)] and subsequent amplification using two primers (28-30 nt). The cassette was subcloned into the vector pKK223-3 for expression under control of a tac promoter. The protein produced from this highly expressed gene has the expected N-terminal sequence and molecular mass, and displays the DNA processing, DNA joining and disintegration activities expected from recombinant integrase. These studies have demonstrated the utility of codon optimization, and lay the groundwork for structure-function studies of HIV1 integrase.

Interaction of C225SR1 mutant subunit of ribonucleotide reductase with R2 and nucleoside diphosphates: tales of a suicidal enzyme.

Ribonucleotide reductase (RDPR) from Escherichia coli is composed of two subunits, R1 and R2, both of which are required to catalyze the conversion of nucleotides to deoxynucleotides. This reduction process is accompanied by oxidation of two cysteines within the active site to a disulfide. One of these putative active site cysteines, C225, has been mutated to a serine, and the properties of this mutant (C225SR1) have been investigated in detail. Incubation of C225SR1 and R2 with [3'-3H,U-14C]UDP results in time-dependent inactivation of the enzyme! This inactivation is accompanied by production of 2.4 uracils, 3H2O, and 3H,14C-labeled protein with an absorbance change at 320 nm. There is an isotope effect (kH/k3H) on uracil production of 3.2. In addition, the tyrosyl radical on R2 is reduced. The observation of 3H2O, indicative of 3' carbon-hydrogen bond cleavage and loss of the tyrosyl radical, provides a direct test of our mechanistic hypothesis that cleavage of this bond occurs concomitantly with tyrosyl radical reduction. Incubation of [3'-2H]UDP with C225SR1 and R2 resulted in a V and V/K isotope effect on loss of the radical of 2.0 and 2.0, respectively. These studies provide the first direct evidence for protein radical involvement in catalysis. Reduction of the tyrosyl radical on R2 is accompanied by a stoichiometric cleavage of the R1 polypeptide into two new polypeptides of 26 and 61 kDa. The 26-kDa polypeptide is the N-terminus of R1, and hence cleavage of the polypeptide is occurring in the region of the mutation. The N-terminus of the 61-kDa polypeptide is blocked.(ABSTRACT TRUNCATED AT 250 WORDS)

A model for the role of multiple cysteine residues involved in ribonucleotide reduction: amazing and still confusing.

Ribonucleotide reductase from Escherichia coli catalyzes the conversion of nucleotides to deoxynucleotides. Multiple cysteins have been postulated to play a key role in this process. To test the role of various cysteines in nucleotide reduction, a variety of single and double mutants of the R1 subunit were prepared: C754S, C759S, C754-759S, C462S, C462A, C230S, and C292S. Due to the expression system, each mutant contains small amounts of contaminating wt-R1 (estimated to be 1.5-3% based on activity). An epitope tagging method in conjunction with anion exchange chromatography was used to partially resolve the mutant R1 from the wt-R1. The interaction of these mutants with the normal substrate was studied, which allowed a model to be proposed in which five cysteines of the R1 subunit of RDPR play a role in catalysis. C754S and C759S R1s catalyze CDP formation at rates similar to wt-R1 when DTT is used as a reductant. However, when thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH is used as reductant, the rates of dNDP production are similar to those expected for contaminating wt-R1 present as a heterodimer with the mutant. The impaired nature of these mutants with respect to reduction by TR suggests that their function is to transfer reducing equivalents from TR to the active site disulfide of R1 produced during NDP reduction. Single-turnover experiments, designed to avoid the problem of contaminating wt-R1, also support this role for C754 and C759. The double serine mutant of 754 and 759 has catalytic activity with DTT that is one-third the rate of wt-R1 with thioredoxin. C225 and C462 are thought to be the active site cysteines oxidized concomitantly with NDP reduction. Conversion of these cysteines to serines results in R1 mutants which convert the normal substrate into a mechanism-based inhibitor. C462SR1 upon incubation with R2 and [3'-3H,U-14C]UDP results in uracil release, 3H2O production, 3H,14C-labeled protein which has an absorbance change at 320 nm, and slow loss of the tyrosyl radical on R2. The isotope effect (kH/k3H) on 3' carbon-hydrogen bond cleavage is 1.7. This sequence of events is independent of the reductant, consistent with the postulate that C462 is an active site thiol. The C462AR1 has properties similar to C462SR1. Several additional mutant R1s, C230SR1, and C292SR1 were shown to have activities similar to wt-R1 with both TR/TRR/NADPH and DTT.

Ovothiols as free-radical scavengers and the mechanism of ovothiol-promoted NAD(P)H-O2 oxidoreductase activity.

Racemic ovothiol A [(+/-)-1a] and the ovothiol model compound 1,5-dimethyl-4-mercaptoimidazole (DMI, 2) were found to scavange the free radicals Fremy's salt (4) and Banfield' radical (5) much more rapidly than did the thiol antioxidant glutathione. Ovothiol A also scavenges the tyrosyl radical, with efficiency comparable to that of ascorbic acid and the tocopherol analogue trolox (3). The ovothiol model compound DMI was found to scavenge superoxide with a rate constant comparable to that of the reaction between superoxide and glutathione. These results suggest both a free-radical scavenging role for the ovothiols and a mechanism by which the ovothiols confer NAD(P)H-O2 oxidoreductase activity upon the enzyme ovoperoxidase. Investigation of this mechanism implicates the ovothiol thiyl radical and the NAD radical as key intermediates. The ovothiyl radical appears to be unreactive toward oxygen but highly reactive toward NADH. An estimate of the one-electron oxidation potential of the ovothiol anion is presented. The physical basis for the stability of the ovothiol free radical is discussed.

A qualitative fluorescence-based assay for tyrosyl radical scavenging activity: ovothiol A is an efficient scavenger.

A method for determining relative tyrosyl radical scavenging activity of antioxidants which requires only a standard fluorometer and commercially available materials is presented. Ultraviolet irradiation of aqueous tyrosine solutions containing superoxide dismutase and catalase produces fluorescent dityrosine residues via dimerization of photogenerated tyrosyl radicals. Added antioxidants suppress the buildup of fluorescence by scavenging the tyrosyl radicals. A correlation exists between the ability of a substance to suppress dityrosine formation and the substance's one-electron oxidation potential. This method demonstrates that ovothiol A scavenges tyrosyl radicals much more efficiently than glutathione or cysteine, resembling instead the known biological radical scavengers uric acid and ascorbic acid and the alpha-tocopherol analog trolox.