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Biomedical subjects

K Drlica

Publications and source records attributed to K Drlica.

At least 19 recordsLinked to original sources
PubMed2002

Therapeutic options in an era of decreasing antimicrobial susceptibility.

Antimicrobial resistance among human pathogens has evolved to the multidrug-resistant phase. Consequently, preserving antimicrobial efficacy is now an important clinical consideration when selecting an agent for therapy. A new, in vitro measure of potency called the mutant prevention concentration (MPC) allows clinicians to select antibacterials on the basis of an agent's ability to restrict the selection of resistant mutants. With some pathogens, C-8-methoxy fluoroquinolones are expected to be quite effective at restricting the selection of resistance because the value of MPC is below the human serum drug concentration achieved with standard doses. Clinical trials are now needed to determine how well preclinical profiles of these drugs correlate with the prevention of resistance.

Anti-Bacterial Agents↗
PubMed2001

Restricting the selection of antibiotic-resistant mutants: a general strategy derived from fluoroquinolone studies.

Studies with fluoroquinolones have led to a general method for restricting the selection of antibiotic-resistant mutants. The strategy is based on the use of antibiotic concentrations that require cells to obtain 2 concurrent resistance mutations for growth. That concentration has been called the "mutant prevention concentration" (MPC) because no resistant colony is recovered even when >10(10) cells are plated. Resistant mutants are selected exclusively within a concentration range (mutant selection window) that extends from the point where growth inhibition begins, approximated by the minimal inhibitory concentration, up to the MPC. The dimensions of the mutant selection window can be reduced in a variety of ways, including adjustment of antibiotic structure and dosage regimens. The window can be closed to prevent mutant selection through combination therapy with > or =2 antimicrobial agents if their normalized pharmacokinetic profiles superimpose at concentrations that inhibit growth. Application of these principles could drastically restrict the selection of drug-resistant pathogens.

Animals↗
PubMed2001

Fluoroquinolone-resistant Streptococcus pneumoniae associated with levofloxacin therapy.

Fluoroquinolone-resistant cultures of Streptococcus pneumoniae were isolated from 2 patients who were treated for pneumonia with levofloxacin. Nucleotide sequence analysis of bacterial DNA showed that the isolates contained mutations in both parC (DNA topoisomerase IV) and gyrA (DNA gyrase), which were shown previously to confer fluoroquinolone resistance. With the resistant isolates, the MICs for ciprofloxacin, gatifloxacin, grepafloxacin, levofloxacin, and trovafloxacin were above the maximal serum drug concentrations reported for standard dosage regimens. In contrast, the MICs for gemifloxacin and moxifloxacin were below the maximal serum concentrations. Increased effectiveness at blocking the growth of resistant mutants should make gemifloxacin and moxifloxacin less likely to allow the enrichment of mutants within susceptible populations. Additional resistance mutations in the isolates were readily obtained by plating on gemifloxacin- or moxifloxacin-containing agar. Thus, neither compound is expected to halt further accumulation of resistance mutations once mutant enrichment has been initiated by less potent derivatives.

Aged↗
PubMed2001

Antibiotic resistance: can we beat the bugs?

The Discussion Forum provides a medium for airing your views on any issues related to the pharmaceutical industry and obtaining feedback and discussion on these views from others in the field. You can discuss issues that get you hot under the collar, practical problems at the bench, recently published literature, or just something bizarre or humorous that you wish to share. Publication of letters in this section is subject to editorial discretion and company-promotional letters will be rejected immediately. Furthermore, the views provided are those of the authors and are not intended to represent the views of the companies they work for. Moreover, these views do not reflect those of Elsevier, Drug Discovery Today or its editorial team. Please submit all letters to Rebecca Lawrence, News & Features Editor, Drug Discovery Today, e-mail: Rebecca.Lawrence@drugdiscoverytoday.com

Journal Article↗
PubMed2001

gyrB-225, a mutation of DNA gyrase that compensates for topoisomerase I deficiency: investigation of its low activity and quinolone hypersensitivity.

The B subunit of DNA gyrase (GyrB) consists of a 43 kDa N-terminal domain, containing the site of ATP binding and hydrolysis, and a 47 kDa C-terminal domain that is thought to play a role in interactions with GyrA and DNA. In cells containing a deletion of topA (the gene encoding DNA topoisomerase I) a compensatory mutation is found in gyrB. This mutation (gyrB-225) results in a two amino acid insertion in the N-terminal domain of GyrB. We found that cells containing this mutation are more sensitive than wild-type cells to quinolone drugs with respect to bacteriostatic and lethal action. We have characterised the mutant GyrB protein in vitro and found it to have reduced DNA supercoiling, relaxation, ATPase, and cleavage activities. The mutant enzyme is up to threefold more sensitive to quinolones than wild-type. The mutation also increases the affinity of GyrB for GyrA and DNA, while the affinity of quinolone for the enzyme-DNA complex is unaffected. We propose that the loss in activity is due to misfolding of the GyrB-225 protein, providing an example in which misfolding of one protein, DNA gyrase, suppresses a deficiency of another, topoisomerase I. The increased quinolone sensitivity is proposed to be a consequence of an altered conformation of the protein that renders quinolones better able to disrupt, rather than generate, gyrase-drug-DNA complexes.

Adenosine Triphosphatases↗
PubMed2001

Enhancement of fluoroquinolone activity by C-8 halogen and methoxy moieties: action against a gyrase resistance mutant of Mycobacterium smegmatis and a gyrase-topoisomerase IV double mutant of Staphylococcus aureus.

The increasing prevalence of antibiotic resistance among bacterial pathogens prompted a microbiological study of fluoroquinolone structure-activity relationships with resistant mutants. Bacteriostatic and bactericidal activities for 12 fluoroquinolones were examined with a gyrase mutant of Mycobacterium smegmatis and a gyrase-topoisomerase IV double mutant of Staphylococcus aureus. For both organisms C-8 halogen and C-8 methoxy groups enhanced activity. The MIC at which 99% of the isolates tested were inhibited (MIC(99)) was reduced three- to fivefold for the M. smegmatis mutant and seven- to eightfold for the S. aureus mutant by C-8 bromine, chlorine, and methoxy groups. With both organisms a smaller reduction in the MIC(99) (two- to threefold) was associated with a C-8 fluorine moiety. In most comparisons with M. smegmatis the response to a C-8 substituent was similar (within twofold) for wild-type and mutant cells. In contrast, mutant S. aureus was affected more than the wild type by the addition of a C-8 substituent. C-8 halogen and methoxy groups also improved the ability to kill the two mutants and the respective wild-type cells when measured with various fluoroquinolone concentrations during an incubation period equivalent to four to five doubling times. Collectively these data help define a group of fluoroquinolones that can serve (i) as a base for structure refinement and (ii) as test compounds for slowing the development of fluoroquinolone resistance during infection of vertebrate hosts.

Anti-Infective Agents↗
PubMed2001

Mutant prevention concentrations of fluoroquinolones for clinical isolates of Streptococcus pneumoniae.

The mutant prevention concentration (MPC) represents a threshold above which the selective proliferation of resistant mutants is expected to occur only rarely. A provisional MPC (MPC(pr)) was defined and measured for five fluoroquinolones with clinical isolates of Streptococcus pneumoniae. Based on their potential for restricting the selection of resistant mutants, the five fluoroquinolones, in descending order, were found to be moxifloxacin > trovafloxacin > gatifloxacin > grepafloxacin > levofloxacin. For several compounds, 90% of about 90 clinical isolates that lacked a known resistance mutation had a value of MPC(pr) that was close to or below the serum levels that could be attained with a dosing regimen recommended by the manufacturers. Since MPC(pr) overestimates MPC, these data identify moxifloxacin and gatifloxacin as good candidates for determining whether MPC(pr) can be used as a guide for choosing and eventually administering fluoroquinolones to significantly reduce the development of resistance.

Anti-Infective Agents↗
PubMed2000

Selection of antibiotic-resistant bacterial mutants: allelic diversity among fluoroquinolone-resistant mutations.

To obtain a general framework for understanding selection of antibiotic-resistant mutants, allelic diversity was examined with about 600 fluoroquinolone-resistant mutants of mycobacteria. Selection at low fluoroquinolone concentration produced many low-level resistance mutants. Some of these contained mutations that conferred unselected antibiotic resistance; none contained alterations in the quinolone-resistance-determining region of the GyrA protein, the principal drug target. As selection pressure increased, a variety of GyrA variants became prevalent. High concentrations of antibiotic reduced the variety to a few types, and eventually a concentration was reached at which no mutant was recovered. That concentration defined a threshold for preventing the selection of resistance. The pattern of variants selected, which was also strongly influenced by antibiotic structure, readily explained the variants present in clinical isolates. Thus, resistance arises from selection of mutants whose identity depends on drug concentration and structure, both of which can be manipulated to restrict selection.

Alleles↗
PubMed2000

Prokaryotic and eukaryotic chromosomes: what's the difference?

It is widely held that the profound differences in cellular architecture between prokaryotes and eukaryotes, in particular the housing of eukaryotic chromosomes within a nuclear membrane, also extends to the properties of their chromosomes. When chromosomal multiplicity, ploidy, linearity, transcriptional silencing, partitioning, and packaging are considered, no consistent association is found between any of these properties and the presence or absence of a nuclear membrane. Some of the perceived differences can be attributed to cytological limitations imposed by the small size of bacterial nucleoids and the arbitrary choice of representative organisms for comparison. We suggest that the criterion of nucleosome-based packaging of chromosomal DNA may be more useful than the prokaryote/eukaryote dichotomy for inferring the broadest phylogenetic relationships among organisms.

Animals↗
PubMed2000

Mutant prevention concentration as a measure of fluoroquinolone potency against mycobacteria.

Mutant prevention concentration (MPC) has been proposed as a new measure of antibiotic potency by which the ability to restrict selection of resistant mutants is evaluated. To determine whether MPC provides potency information unavailable from the more customary measurement of the MIC, 18 fluoroquinolones were examined for their ability to block the growth of Mycobacterium smegmatis and to select resistant mutants from wild-type populations. Both MPC and MIC were affected by changes in the moiety at the fluoroquinolone C-8 position and in alkyl groups attached to the C-7 piperazinyl ring. When eight resistant mutants, altered in the gyrase A protein, were tested with fluoroquinolones having either a methoxy or a hydrogen at the C-8 position, the MIC for the most resistant mutant correlated better with the MPC than did the MIC for wild-type cells. For C-8-fluorine derivatives, which were generally less active than the C-8-methoxy compounds but which were more active than C-8-hydrogen derivatives, the MICs for both the mutant and the wild type correlated well with the MPCs. Thus, measurement of the MICs for wild-type cells can reflect the ability of a quinolone to restrict the selection of resistance, but often it does not. With the present series of compounds, the most potent contained a C-8-methoxy and a small group attached to the C-7 ring.

Anti-Infective Agents↗
PubMed2000

Selective targeting of topoisomerase IV and DNA gyrase in Staphylococcus aureus: different patterns of quinolone-induced inhibition of DNA synthesis.

The effect of quinolones on the inhibition of DNA synthesis in Staphylococcus aureus was examined by using single resistance mutations in parC or gyrA to distinguish action against gyrase or topoisomerase IV, respectively. Norfloxacin preferentially attacked topoisomerase IV and blocked DNA synthesis slowly, while nalidixic acid targeted gyrase and inhibited replication rapidly. Ciprofloxacin exhibited an intermediate response, consistent with both enzymes being targeted. The absence of RecA had little influence on target choice by this assay, indicating that differences in rebound (repair) DNA synthesis were not responsible for the results. At saturating drug concentrations, norfloxacin and a gyrA mutant were used to show that topoisomerase IV-norfloxacin-cleaved DNA complexes are distributed on the S. aureus chromosome at intervals of about 30 kbp. If cleaved complexes block DNA replication, as indicated by previous work, such close spacing of topoisomerase-quinolone-DNA complexes should block replication rapidly (replication forks are likely to encounter a cleaved complex within a minute). Thus, the slow inhibition of DNA synthesis at growth-inhibitory concentrations suggests that a subset of more distantly distributed complexes is physiologically relevant for drug action and is unlikely to be located immediately in front of the DNA replication fork.

Anti-Infective Agents↗
PubMed2000

Mutant prevention concentration as a measure of antibiotic potency: studies with clinical isolates of Mycobacterium tuberculosis.

The mutant prevention concentration (MPC) of a C-8-methoxy fluoroquinolone exhibited a narrow distribution for 14 genetically diverse clinical isolates of Mycobacterium tuberculosis, indicating that results from single-isolate studies are likely to be representative. When one isolate was challenged with a variety of antituberculosis agents, C-8-methoxy fluoroquinolones were exceptional in having MPCs below the maximum concentration attained in serum by use of commonly recommended doses.

Anti-Infective Agents↗
PubMed2000

Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis.

Two genes with sequence homology to those encoding protein tyrosine phosphatases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. The calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17. 5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escherichia coli. The affinity-purified proteins dephosphorylated the phosphotyrosine residue of myelin basic protein (MBP), but they failed to dephosphorylate serine/threonine residues of MBP. The activity of these phosphatases was inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine/threonine phosphatases. Mutations at the catalytic site motif, cysteine 11 of MPtpA and cysteine 160 of MPtpB, abolished enzyme activity. Southern blot analysis revealed that, while mptpA is present in slow-growing mycobacterial species as well as fast-growing saprophytes, mptpB was restricted to members of the M. tuberculosis complex. These phosphatases were present in both whole-cell lysates and culture filtrates of M. tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.

Amino Acid Sequence↗
PubMed2000

The future of fluoroquinolones.

The mutant prevention concentration (MPC) is a new measure of antibiotic potency above which a microbe must attain two concurrent resistance mutations for growth. For some C-8-methoxy fluoroquinolone-pathogen combinations, the value of MPC is below human serum drug concentration achieved with standard doses. Although untested clinically, such a low value of MPC, coupled with high serum concentration, should allow these fluoroquinolones to restrict severely the selection of resistant mutants when used as monotherapy. Compounds that cannot meet the MPC-pharmacokinetic criterion will enrich resistant mutants unless they are a part of combination therapy. Separation of fluoroquinolones into groups suitable for monotherapy or for combination therapy, followed by appropriate adminstration, may help extend the lifespan of the fluoroquinolones.

Anti-Infective Agents↗
PubMed1999

Mechanism of fluoroquinolone action.

When fluoroquinolones bind to gyrase or topoisomerase IV in the presence of DNA, they alter protein conformation. DNA cleavage results with diminished religation, so the enzymes are trapped in ternary complexes with drug and cleaved DNA. Preferential localization of gyrase ahead of replication forks and topoisomerase IV behind them causes fluoroquinolone-mediated complexes with the two enzymes to have different physiological consequences.

Anti-Infective Agents↗