Research ArticleAntibiotics

Aminomethyl spectinomycins as therapeutics for drug-resistant respiratory tract and sexually transmitted bacterial infections

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Science Translational Medicine  20 May 2015:
Vol. 7, Issue 288, pp. 288ra75
DOI: 10.1126/scitranslmed.3010572
  • Fig. 1. Structure of spectinomycin and its analogs.

    (A) Spectinomycin, (B) trospectomycin, (C) spectinamide 1599, (D) a representative N-alkyl aminomethyl spectinomycin, and (E) compound 1, an example of N-benzyl aminomethyl spectinomycin explored in this study.

  • Fig. 2. Compound 1 modeled into the bacterial ribosome of S. pneumoniae, which shows the aryl side chain positioned in a side pocket adjacent to RpsE loop.

    (A) Structural variances between the E. coli and S. pneumoniae are highlighted in red within the loop of ribosomal protein RpsE, which contacts helix 34 of the 30S ribosomal spectinomycin binding site. (B) A magnified view of compound 1’s predicted interaction with the S. pneumoniae ribosome. Hydrogen bonds are highlighted in yellow dashed lines. (C) Compound 1’s predicted positioning within the S. pneumoniae RpsE loop.

  • Fig. 3. Activity against N. gonorrhoeae and C. trachomatis.

    (A) Zone of inhibition testing for N. gonorrhoeae. A representative image of disc diffusion assays for N. gonorrhoeae is shown. Compound (40 μg) dissolved in dimethyl sulfoxide (DMSO) was applied to the disc. (B and C) Representative images of mCherry-expressing C. trachomatis (orange) infected monolayers (green) treated with 12 μg/ml (B) spectinomycin or (C) compound 1.

  • Fig. 4. amSPCs mediate more effective protection than spectinomycin from invasive pneumococcal challenge.

    A dose of 5 mg/kg was administered twice daily beginning 18 hours after challenge with S. pneumoniae D39x. (A to C) Overall survival of mice receiving vehicle control or the indicated compounds (n = 5 mice per group). (D to F) Bacterial burden in the blood at 48 hours after challenge. (G to I) Representative bioluminescent images of mice at 72 hours after challenge. Statistical significance was determined using log-rank test (Mantel-Cox) for survival data and Mann-Whitney test for bacterial burden data. *P < 0.05 when compared to vehicle control group. **P < 0.05 when compared to spectinomycin control. P values were as follows: (A) *P = 0.0041, **P = 0.0023; (B) *P = 0.0021, **P = 0.0020; (C) *P = 0.023; (D) *P = 0.0117; (E) *P = 0.0097; (F) *P = 0.0208.

  • Table 2. Antipneumococcal activity of select amSPCs against drug-sensitive and drug-resistant isolates.

    MIC testing for amSPC activity against a panel of S. pneumoniae isolates.

    TreatmentMIC (μg/ml)
    Drug-sensitiveDrug-resistant
    R6T4XD39XBHN97xA66.1xOVA2BAA-1407Daw7Daw8Daw9Daw62Daw64Daw19Daw26Daw27Daw478249ATCC700904
    SPC1313136613132525132525255025252525
    1332223633333666636
    2663336136666613131313613
    33166322236633213
    Penicillin G≤0.2≤0.2≤0.2≤0.2≤0.2≤0.22332323333>2>2
    Cefotaxime130.4330.2
    Erythromycin≤0.2150≤0.2≤0.22≤0.2≤0.210.110.03>22
    Streptomycin13613131313502525>2002525>200>200
  • Table 3. Zone of inhibition testing against fastidious Gram-negative pathogens.

    Zone of inhibition testing for amSPCs against fastidious pathogens. Results presented are the range of two biologically independent experiments. Dashes indicate where values were not determined. Ng, Neisseria gonorrhoeae (ATCC 49226); Nm, Neisseria meningitidis (ATCC 13077); Hi, Haemophilus influenzae (ATCC 49247); Lp, Legionella pneumophila (ATCC 33153).

    CompoundNgNmHiLp
    Spectinomycin10–11.513–1511–12.50
    116–1919–2017–1932–33
    215.5–1616–21.515.540
    316–2018–191621
    420–2216–1815–1621–22
    5 (S)0000
    6 (S)8.2–8.88
  • Table 4. Pharmacokinetic parameters for select amSPCs.

    Parameters are expressed as mean. Values in parentheses indicate %CV. t1/2, half-life for metabolic degradation; T1/2, Pharmacokinetic half-life; Vd, volume of distribution; CL, clearance; fe, fraction excreted unchanged in urine.

    CompoundProtein bindingMicrosomal stabilityIntravenous pharmacokinetics (dose, 10 mg/kg)
    %BoundT1/2 (hours)t1/2 (hours)*Vd (liters/kg)CL (liters hour−1 kg−1)fe
    SPC13.0 (7.5)6.43 (0.13)0.75 (49.3)0.76 (45.2)0.60 (11.5)0.55 (27.0)
    143.0 (1.7)6.80 (0.56)1.12 (14.2)0.64 (39.1)0.56 (23.0)0.82 (12.4)
    362.6 (8.8)28.8 (2.19)1.99 (11.9)1.13 (32.7)0.53 (22.2)0.22 (28.1)
    457.6 (11.9)23.1 (1.64)1.74 (2.8)1.45 (12.7)0.58 (5.4)0.58 (4.7)

    *t1/2 is based on decline of plasma concentration in the therapeutically relevant concentration range.

    †Spectinomycin values are from (31) and included for comparison.

    Supplementary Materials

    • www.sciencetranslationalmedicine.org/cgi/content/full/7/288/288ra75/DC1

      Method S1. Chemical syntheses.

      Method S2. Chemical stability studies.

      Scheme S1. Synthesis of R-3′-aminomethyl-3′-hydroxy spectinomycins.

      Scheme S2. Synthesis of N-benzyl aminomethyl spectinomycin S-isomer controls.

      Fig. S1. Multiple sequence alignment of RpsE (protein S5).

      Fig. S2. In silico analysis of the spectinomycin binding pocket in amSPC-resistant mutants.

      Fig. S3. Computation analysis of the amSPC mutant binding site.

      Fig. S4. Susceptibility of L. pneumophila to spectinomycin and amSPCs.

      Fig. S5. Susceptibility of C. trachomatis to spectinomycin and amSPCs.

      Fig. S6. Chemical stability of spectinomycin and compound 1.

      Fig. S7. Efficacy in a mouse model of S. pneumoniae TIGR4 infection.

      Fig. S8. Efficacy trial comparing matched doses of compound 1 and ampicillin.

      Table S1. Activity against aerobic Gram-negative pathogens and M. tuberculosis.

      Table S2. Testing against mammalian ribosomes.

      Table S3. Lead profiling of compound 1.

      Table S4. Sensitivity of amSPC-resistant clones to various classes of antibiotics.

      Table S5. Antibacterial spectrum of activity for previously reported spectinomycins.

      Table S6. Chemical stability of spectinomycin and compound 1.

    • Supplementary Material for:

      Aminomethyl spectinomycins as therapeutics for drug-resistant respiratory tract and sexually transmitted bacterial infections

      David F. Bruhn, Samanthi L. Waidyarachchi, Dora B. Madhura, Dimitri Shcherbakov, Zhong Zheng, Jiuyu Liu, Yasser M. Abdelrahman, Aman P. Singh, Stefan Duscha, Chetan Rathi, Robin B. Lee, Robert J. Belland, Bernd Meibohm, Jason W. Rosch, Erik C. Böttger, Richard E. Lee*

      *Corresponding author. E-mail: richard.lee{at}stjude.org

      Published 20 May 2015, Sci. Transl. Med. 7, 288ra75 (2015)
      DOI: 10.1126/scitranslmed.3010572

      This PDF file includes:

      • Method S1. Chemical syntheses.
      • Method S2. Chemical stability studies.
      • Scheme S1. Synthesis of R-3′-aminomethyl-3′-hydroxy spectinomycins.
      • Scheme S2. Synthesis of N-benzyl aminomethyl spectinomycin S-isomer controls.
      • Fig. S1. Multiple sequence alignment of RpsE (protein S5).
      • Fig. S2. In silico analysis of the spectinomycin binding pocket in amSPC-resistant mutants.
      • Fig. S3. Computation analysis of the amSPC mutant binding site.
      • Fig. S4. Susceptibility of L. pneumophila to spectinomycin and amSPCs.
      • Fig. S5. Susceptibility of C. trachomatis to spectinomycin and amSPCs.
      • Fig. S6. Chemical stability of spectinomycin and compound 1.
      • Fig. S7. Efficacy in a mouse model of S. pneumoniae TIGR4 infection.
      • Fig. S8. Efficacy trial comparing matched doses of compound 1 and ampicillin.
      • Table S1. Activity against aerobic Gram-negative pathogens and M. tuberculosis.
      • Table S2. Testing against mammalian ribosomes.
      • Table S3. Lead profiling of compound 1.
      • Table S4. Sensitivity of amSPC-resistant clones to various classes of antibiotics.
      • Table S5. Antibacterial spectrum of activity for previously reported spectinomycins.
      • Table S6. Chemical stability of spectinomycin and compound 1.

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