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Cited by 162Antibiotic Resistance in BacteriaAntimicrobial Resistance in StaphylococcusAntibiotics Pharmacokinetics and Efficacy

Target- and resistance-based mechanistic studies with TP-434, a novel fluorocycline antibiotic

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Grossman, Trudy H., Starosta, Agata L., Fyfe, Corey, O'Brien, William, Rothstein, David M., Mikolajka, Aleksandra, Wilson, Daniel N., Sutcliffe, Joyce A.·2012·Antimicrobial Agents and Chemotherapy
DOI 10.1128/aac.06187-11PMC PMC3346605
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Abstract

TP-434 is a novel, broad-spectrum fluorocycline antibiotic with activity against bacteria expressing major antibiotic resistance mechanisms, including tetracycline-specific efflux and ribosomal protection. The mechanism of action of TP-434 was assessed using both cell-based and in vitro assays. In Escherichia coli cells expressing recombinant tetracycline resistance genes, the MIC of TP-434 (0.063 μg/ml) was unaffected by tet (M), tet (K), and tet (B) and increased to 0.25 and 4 μg/ml in the presence of tet (A) and tet (X), respectively. Tetracycline, in contrast, was significantly less potent (MIC ≥ 128 μg/ml) against E. coli cells when any of these resistance mechanisms were present. TP-434 showed potent inhibition in E. coli in vitro transcription/translation (50% inhibitory concentration [IC 50 ] = 0.29 ± 0.09 μg/ml) and [ 3 H]tetracycline ribosome-binding competition (IC 50 = 0.22 ± 0.07 μM) assays. The antibacterial potencies of TP-434 and all other tetracycline class antibiotics tested were reduced by 4- to 16-fold, compared to that of the wild-type control strain, against Propionibacterium acnes strains carrying a 16S rRNA mutation, G1058C, a modification that changes the conformation of the primary binding site of tetracycline in the ribosome. Taken together, the findings support the idea that TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis and that this activity is largely unaffected by the common tetracycline resistance mechanisms.

AI snapshot

Written from the paper’s full text.

TL;DR

TP-434 is a novel fluorocycline that inhibits E. coli translation and binds the ribosome, with activity largely unaffected by common tetracycline resistance mechanisms. However, TP-434 MIC still rises with tet(A), tet(X), and a 16S rRNA G1058C mutation.

Key findings

  • In an E. coli-coupled in vitro transcription/translation assay, TP-434 had an IC50 of 0.29 ± 0.09 μg/ml, while tetracycline had an IC50 of 1.26 ± 0.48 μg/ml. Addition of purified Tet(M) did not affect TP-434 (0.27 ± 0.09 μg/ml) but increased tetracycline’s IC50 5-fold to 6.50 ± 3.3 μg/ml.
  • TP-434 competed with [3H]tetracycline for 70S ribosome binding with an IC50 of 0.22 ± 0.07 μM; tigecycline had a similar IC50 (0.22 ± 0.08 μM), and tetracycline was ~14-fold less potent (IC50 = 3.00 ± 1.15 μM). Erythromycin up to 100 μM did not compete.
  • Against isogenic E. coli DH10B expressing resistance genes, TP-434 MIC was 0.063 μg/ml for lacZ, tet(M), and tet(B); 0.031 μg/ml for tet(K); 0.25 μg/ml for tet(A); and 4 μg/ml for tet(X). Tetracycline MIC was ≥128 μg/ml with each resistance mechanism.
  • In P. acnes, strains SW101T and P:413 carrying the 16S rRNA G1058C mutation had TP-434 MICs of 1 μg/ml versus 0.063 μg/ml for wild-type ATCC 6919; all tetracycline-class antibiotics tested showed 4- to 16-fold reduced potency against the mutant strains.

Methods

  • Mechanistic assays: E. coli-coupled in vitro transcription/translation with a firefly luciferase readout, with and without purified Tet(M) at 2.75 μM; [3H]tetracycline ribosome-binding competition using 0.4 μM E. coli 70S ribosomes and 8 μM [3H]tetracycline.
  • Susceptibility testing: MIC assays against E. coli DH10B expressing recombinant tet(A), tet(B), tet(K), tet(M), tet(X), or lacZ control, performed by CLSI methods with ampicillin and arabinose induction; P. acnes strains ATCC 6919, SW101 CDA, SW101T, and P:413 tested anaerobically in Wilkins-Chalgren broth.
  • Analysis: IC50s were averaged from at least two independent experiments; MIC assays were performed at least twice, with representative results reported.

Limitations

  • Not stated in the paper (no explicit limitations section).
  • Evident from the text: mechanistic assays use E. coli in vitro translation and purified ribosomes, and resistance testing uses recombinant isogenic E. coli plus a small set of P. acnes strains, so clinical generalizability is limited.
  • Evident from the text: TP-434 activity is still reduced by tet(A) (4-fold MIC increase to 0.25 μg/ml), tet(X) (MIC 4 μg/ml), and the G1058C mutation (MIC 1 μg/ml), so resistance is not fully overcome.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1128/aac.06187-11

Cites

44 of 44 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 15 cited sources checked · 0 supported · 0 flagged

  • Findingown data
    § RESULTS AND DISCUSSION

    Tigecycline susceptibility (MIC = 0.063 μg/ml) was unaffected by tet(M), tet(K), and tet(B).

    “Susceptibility to tigecycline (MIC = 0.063 μg/ml) was unaffected by tet(M), tet(K), and tet(B)”
    Quote in the paperNot a substantial claim
  • Findingown data
    § RESULTS AND DISCUSSION

    The MIC of ceftriaxone remained unaffected by overexpression of any of the tetracycline resistance mechanisms.

    “The MIC of ceftriaxone, a nontetracycline control, remained unaffected by the overexpression of any of the tetracycline resistance mechanisms.”
    Quote in the paperNot a substantial claim
  • Findingcitation
    § RESULTS AND DISCUSSION

    The tet(A) gene expressed in the present study had the same variations as the Tn1721-associated tet(A) gene, with amino acids Ala-201, Ser-202, and Phe-203 rather than Ser-201, Phe-202, and Val-203 of the RP1 tet(A) sequence.

    “The tet(A) gene expressed in the present study had the same variations as the Tn1721-associated tet(A) gene, where the amino acids are Ala-201, Ser-202, and Phe-203 rather than those of the tet(A) sequence from E. coli plasmid RP1, Ser-201, Phe-202, and Val-203”
    Quote in the paperSubstantial claim
  • Findingown data
    § Abstract

    In E. coli cells expressing recombinant tet(A), the MIC of TP-434 increased to 0.25 μg/ml (4-fold increase).

    “increased to 0.25 and 4 μg/ml in the presence of tet(A) and tet(X), respectively.”
    • Also stated in: § RESULTS AND DISCUSSION · plain
      “a 4-fold increase in TP-434 MIC (0.25 μg/ml) was observed in the presence of tet(A) expression”
    Quote in the paperSubstantial claim
  • Findingown data
    § Abstract

    Tetracycline was significantly less potent (MIC ≥ 128 μg/ml) against E. coli cells when any of these resistance mechanisms were present.

    “Tetracycline, in contrast, was significantly less potent (MIC ≥ 128 μg/ml) against E. coli cells when any of these resistance mechanisms were present.”
    Quote in the paperSubstantial claim
  • May combine two claims. 11
  • Classified as probably not a substantial claim. 7

  • May combine two claims.

    Findingown data
    § Abstract

    TP-434 competed with [3H]tetracycline for ribosome binding with IC50 = 0.22 ± 0.07 μM.

    “[3H]tetracycline ribosome-binding competition (IC50 = 0.22 ± 0.07 μM) assays.”
    • Also stated in: § RESULTS AND DISCUSSION · plain
      “TP-434 competed with [3H]tetracycline for ribosome binding (Fig. 3), with an IC50 of 0.22 ± 0.07 μM.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data
    § RESULTS AND DISCUSSION

    When purified Tet(M) was added to the transcription/translation reaction, the IC50s of TP-434 and tigecycline were unaffected (0.27 ± 0.09 and 0.09 ± 0.04 μg/ml), whereas the IC50 for tetracycline increased 5-fold (6.50 ± 3.3 μg/ml).

    “When purified Tet(M) was added to the transcription/translation reaction, the IC50s of both TP-434 and tigecycline were unaffected (0.27 ± 0.09 μg/ml and 0.09 ± 0.04 μg/ml, respectively), whereas the IC50 for tetracycline increased 5-fold (6.50 ± 3.3 μg/ml).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation
    § INTRODUCTION

    Mutations within helices h31 and h34 of 16S rRNA confer tetracycline resistance in H. pylori and P. acnes.

    “mutations within helices h31 and h34 of 16S rRNA have been reported to confer tetracycline resistance in Helicobacter pylori and Propionibacterium acnes (31, 40).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [31] 31. Ross JI, Eady EA, Cove JH, Cunliffe WJ. 1998. 16S rRNA mutation associated with tetracycline resistance in a Gram-positive bacterium. Antimicrob. Agents Chemother. 42:1702–1705 [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Not checked in the free run.

    • cites [40] 40. Trieber CA, Taylor DE. 2002. Mutations in the 16S rRNA genes of Helicobacter pylori mediate resistance to tetracycline. J. Bacteriol. 184:2131–2140 [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data
    § RESULTS AND DISCUSSION

    TP-434 has improved activity against major tetracycline resistance mechanisms and is 4-fold more potent than tigecycline in E. coli expressing Tn1721-associated tet(A).

    “Further, TP-434 has improved activity against major tetracycline resistance mechanisms and is 4-fold more potent than tigecycline in E. coli expressing a widespread tetracycline efflux pump, Tn1721-associated tet(A).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § MATERIALS AND METHODS

    Results in transcription/translation and binding assays are averages from at least two independent experiments.

    “Results were reported as averages from at least two independent experiments.”
    • Also stated in: § MATERIALS AND METHODS · plain
      “Results presented are averages from at least two independent experiments.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data
    § RESULTS AND DISCUSSION

    The RPP mechanism is specific to tetracycline, as linezolid IC50 was unaffected by Tet(M).

    “The IC50 for linezolid, a nontetracycline control, was unaffected by the addition of Tet(M), substantiating that the RPP mechanism was specific to tetracycline.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data
    § MATERIALS AND METHODS

    Optimal preinduction conditions producing tetracycline MIC values of ≥128 μg/ml were identified for each strain in preliminary experiments.

    “Optimal preinduction conditions producing tetracycline MIC values of ≥128 μg/ml were identified for each strain in preliminary experiments, in which temperatures and l-arabinose concentrations were varied.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductown data
    § MATERIALS AND METHODS

    MIC assays were performed at least twice, and results from a representative experiment are reported.

    “Assays were performed at least twice, and results from a representative experiment are reported in Table 3.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation
    § INTRODUCTION

    TP-434 contains the tetracyclic core scaffold common to other tetracycline antibiotics, with a fluorine atom at C-7 and a pyrrolidinoacetamido group at C-9 on the tetracyclic D ring.

    “TP-434 contains the tetracyclic core scaffold common to other tetracycline antibiotics, with two unique modifications: a fluorine atom at position C-7 and a pyrrolidinoacetamido group at C-9 on the tetracyclic D ring (Fig. 1).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [8] 8. Clark RB, et al. 2012. Fluorocyclines. 2. Optimization of the C-9 side-chain for antibacterial activity and oral efficacy. J. Med. Chem. 55:606–622 [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [18] 18. Hunt D, et al. 2010. TP-434 is a novel broad-spectrum Fluorocycline, abstr F1-2157. Abstr. 50th Intersci. Conf. Antimicrob. Agents Chemother American Society for Microbiology, Washington, DC [Google Scholar]

      Not checked in the free run.

    • cites [44] 44. Xiao XY, et al. 2012. Fluorocyclines. 1. 7-Fluoro-9-pyrrolidinoacetamido-6-demethyl-6-deoxytetracycline: a potent, broad spectrum antibacterial agent. J. Med. Chem. 55:597–605 [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation
    § INTRODUCTION

    Tigecycline has improved activity against both tetracycline-specific efflux and RPP mechanisms and is a more potent inhibitor of protein synthesis.

    “The parenteral glycylcycline tigecycline, another semisynthetic derivative, was developed to have improved activity against both tetracycline-specific efflux and RPP mechanisms (26) and is a more potent inhibitor of protein synthesis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [26] 26. Peterson LR. 2008. A review of tigecycline—the first glycylcycline. Int. J. Antimicrob. Agents 32(S4):S215–S222 [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

  • May combine two claims.

    Study design and conductown data
    § MATERIALS AND METHODS

    The constant presence of ampicillin and l-arabinose had no effect on MIC assay results; MIC values for all antibiotics tested against DH10B lacZ plasmid versus DH10B without ampicillin/l-arabinose were within 2-fold.

    “To verify that the constant presence of ampicillin and l-arabinose had no effect on assay results, all antibiotics used in this study were tested against DH10B cells containing the lacZ plasmid (in the presence of 50 μg/ml ampicillin plus 1% l-arabinose) versus DH10B cells (without ampicillin and l-arabinose); MIC values obtained under both conditions were within 2-fold for all antibiotics tested,”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data
    § Abstract

    TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis.

    “Taken together, the findings support the idea that TP-434, like other tetracyclines, binds the ribosome and inhibits protein synthesis”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation
    § INTRODUCTION

    Semisynthetic tetracycline derivatives doxycycline and minocycline show improved activity versus tetracycline against tetracycline-specific efflux but remain susceptible to resistance by RPPs.

    “Semisynthetic tetracycline derivatives, such as doxycycline and minocycline (Fig. 1), show improved activity versus tetracycline against tetracycline-specific efflux but remain susceptible to resistance by RPPs (39).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [39] 39. Thaker M, Spanogiannopoulos P, Wright GD. 2010. The tetracycline resistome. Cell. Mol. Life Sci. 67:419–431 [DOI] [PMC free article] [PubMed] [Google Scholar]

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation
    § INTRODUCTION

    Tetracycline antibiotics bind to the bacterial 30S ribosomal subunit and prevent chain elongation by blocking aminoacyl-tRNA binding to the A site.

    “Tetracycline antibiotics bind to the bacterial 30S ribosomal subunit and prevent chain elongation by blocking aminoacyl-tRNA binding to the A site (3, 21, 27).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [3] 3. Brodersen DE, et al. 2000. The structural basis for the action of the antibiotics tetracycline, pactamycin and hygromycin B on the 30S ribosomal subunit. Cell 103:1143–1154 [DOI] [PubMed] [Google Scholar] source

      Not checked in the free run.

    • cites [21] 21. Maxwell IH. 1967. Partial removal of bound transfer RNA from polysomes engaged in protein synthesis in vitro after addition of tetracycline. Biochim. Biophys. Acta 138:337–346 [DOI] [PubMed] [Google Scholar]

      Not checked in the free run.

    • cites [27] 27. Pioletti M, et al. 2001. Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J. 20:1829–1839 [DOI] [PMC free article] [PubMed] [Google Scholar] source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Study design and conductown data
    § MATERIALS AND METHODS

    Cloned inserts were verified by sequencing and comparing with reported sequences in GenBank.

    “Cloned inserts were verified by sequencing and comparing with reported sequences in GenBank”
    Quote in the paperSubstantial claim
15 of 15

  • The paper relies on this without support.

    The E. coli in vitro transcription/translation and ribosome-binding assays reflect TP-434's mechanism of action in other bacterial species.

    “The mechanism of action of TP-434 was assessed using both cell-based and in vitro assays.”
    “TP-434 inhibits translation and is unaffected by Tet(M).”

  • The paper relies on this without support.

    The recombinant resistance genes expressed in E. coli DH10B reproduce the effects of the same resistance mechanisms in clinical pathogens.

    “To determine the impact of specific tetracycline resistance genes in isogenic E. coli strains”
    “These properties impart to TP-434 a broad spectrum of activity against multidrug-resistant Gram-positive and Gram-negative pathogens”
2 of 2

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Third-generation tetracycline derivatives such as tigecycline display enhanced antimicrobial activity, overcoming efflux and ribosome protection mechanisms.

    “The third generation of tetracycline derivatives, such as tigecycline (Tgc), display enhanced antimicrobial activity, overcoming efflux and ribosome protection mechanisms (6)(7)(8).”

    From Structural basis for TetM-mediated tetracycline resistance (2012) · cites this paper as [8]

  • The attached C9-glycyl side chain of tigecycline would prevent access of the residues of loop III of TetM, and this steric hindrance contributes, together with the increased affinity of tigecycline, to explaining how tigecycline overcomes TetM-mediated resistance whereas tetracycline cannot.

    “In contrast, the attached C9-glycyl side chain of Tgc would prevent access of the residues of loop III of TetM (Fig. 3 E and F), leading us to suggest that this steric hindrance contributes, together with the increased affinity of Tgc (7,8,27), to explaining how Tgc overcomes TetM-mediated resistance whereas tetracycline cannot (8).”

    From Structural basis for TetM-mediated tetracycline resistance (2012) · cites this paper as [8]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • TP-434 IC50 standard deviation differs between text and Table 1

    Minor

    For the TP-434 IC50 of 0.27 μg/ml, the results text reports a standard deviation of 0.09 μg/ml, while Table 1 reports 0.16 μg/ml.

  • Table 1 reverses the Tet(M) condition labels

    Minor

    Table 1 labels the 1.26 μg/ml tetracycline IC50 as measured with Tet(M) and the 6.50 μg/ml value as measured without it. The results text says the opposite: adding Tet(M) increased the IC50 to 6.50 μg/ml. The condition labels appear to be reversed for the table.

  • Reported MICs exceed the stated assay concentration range

    Minor

    The methods state that MIC assay drug concentrations went only up to 32 μg/ml, but Table 3 reports MICs of 64 and 128 μg/ml and values greater than 128 μg/ml. Those values cannot be determined from the stated range.

3 of 3

Cites

Flagged first, then by citations

Research works printed but not linked: 4

  1. FyfeC GrossmanT O'BrienW AchornC SutcliffeJ. 2011. The novel broad-spectrum fluorocycline TP-434 is active against MDR Gram-negative pathogens, abstr P1149. Abstr. 21st Eur. Congr. Clin. Microbiol. Infect. Dis.
  2. HuntD. 2010. TP-434 is a novel broad-spectrum Fluorocycline, abstr F1-2157. Abstr. 50th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.
  3. MurphyT SleeA SutcliffeJ. 2010. TP-434 is highly efficacious in animal models of infection, abstr F1-2161. Abstr. 50th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.
  4. SutcliffeJ. 2010. In vitro activity of fluorocycline TP-434 against panels of recent bacterial clinical isolates, abstr F1-2158. Abstr. 50th Intersci. Conf. Antimicrob. Agents Chemother. American Society for Microbiology, Washington, DC.

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Authors
Grossman, T. H., Starosta, A. L., Fyfe, C., O'Brien, W., Rothstein, D. M., Mikolajka, A., Wilson, D. N., & Sutcliffe, J. A.
Affiliations
  • Tetraphase Pharmaceuticals (United States) (US)
  • Ludwig-Maximilians-Universität München (DE)
  • Center for Integrated Protein Science Munich (DE)
ORCID
Venue
Antimicrobial Agents and Chemotherapy
Publisher
American Society for Microbiology
Published
2012-05
Volume
56
Issue
5
Pages
2559-2564
ISSN
  • 0066-4804 (ISSN-L)
  • 1098-6596
DOI
10.1128/aac.06187-11
PMC
PMC3346605
OpenAlex
W2163177484
Semantic Scholar
b655674a6d32982275d3dbbd3f3ff85d545567c6
Cited by
162
References
44
Citations by year
2026: 8 · 2025: 8 · 2024: 13 · 2023: 9 · 2022: 5 · 2021: 14 · 2020: 14 · 2019: 19 · 2018: 13 · 2017: 5 · 2016: 16 · 2015: 6 · 2014: 8 · 2013: 18 · 2012: 5
FWCI
5.46
Type
journal article
Language
en
Update notices
correction (2015-08-14)
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Antimicrobial Agents and Chemotherapy (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Medicine › Antibiotic Resistance in Bacteria
  • Health Sciences › Medicine › Infectious Diseases › Antimicrobial Resistance in Staphylococcus
  • Health Sciences › Medicine › Pharmacology › Antibiotics Pharmacokinetics and Efficacy
Keywords
tetracycline resistance, tetracycline resistance genes, tetM, tetK, tetB, tetA, tetX, Escherichia coli, Propionibacterium acnes, ribosome binding, protein synthesis inhibition, mechanism of action, antibiotic resistance
MeSH terms
Anti-Bacterial Agents, Bacterial Proteins, Binding, Competitive, Escherichia coli, Microbial Sensitivity Tests, Mutation, Propionibacterium acnes, Ribosomes, RNA, Ribosomal, 16S, Tetracycline, Tetracycline Resistance, Tetracyclines, Transformation, Bacterial, Protein Biosynthesis, Gene Expression

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