An Overview of the Aminoglycosides Drug Class
Jay Kaplan
*Content in blue text is novel information not presented in class.
Discovery & Controversy
1939: Rene Dubos discovers bactericidal effect of a soil bacillus extract on Gram Positive
Bacteria in 1939.
1940: Selman Waksman and H.B. Woodruff discover Actinomycin I which is produced by
Streptomyces antibioticus. This compound exhibits high toxicity as it binds to DNA by
intercalating its phenoxazone ring at a GpC sites thereby disrupting translation and
DNA replication. Actinomycin D is now used as a potent anti-cancer drug.
1942: Waksman and Woodruff discover Streptothrycin, a water-soluble antimicrobial
produced by Streptomyces lavendulae.
1944: Waksman and Schatz discover that Streptomyces produces an antibacterial agent
active against Gram negative bacteria. This substance produced by the Actinomyces,
Streptomyces griseus, was Streptomycin and became the first successful treatment against
TB. Waksman & Schatz filed a patent as co-inventors of Streptomycin, but Waksman later
claimed to be the sole inventor reaping the benefits and winning the 1952 Nobel Prize.
* Clear zones indicate
absence of bacterial growth
Production Method
Streptomycin is produced by fermentation of S. griseus. Spores are transferred to an
inoculum medium and when sufficient growth is maintained they are moved to a
fermentation tank for aquatic subculture production.
Structure & Synthesis
Aminoglycosides consist of an amino sugars connected via glycosidic links.
Streptomycin is a trisaccharide of a streptidine, a streptose, and an N-methyl-glucosamine.
Biosynthetic Gene Cluster:
• The BGC is activated by a cascade initiated by slowing cell growth which increases
production of factor A causing accumulation of factor A.
• A Factor binds to ArpA dimer enabling RNA Polymerase to initiate transcription of the
adpA gene. This transcriptional promoter along with StrR is the pathway-specific
transcriptional activator for the whole gene cluster (BGC).
CBE/MOL 411
Mechanisms & Targets
Streptomycin binds to the phosphate backbone of the bacterial 16S rRNA subunit through
connections between four different parts of the molecule and the ribosome via salt
bridges and hydrogen bonds.
• The binding stabilizes the ribosomal RAM state which has a higher affinity for non-
cognate tRNAs. This inhibits ribosome action and produces specific misreads during
protein synthesis.
• Misread proteins are deposited into the cell membrane which creates membrane
channels that facilitate the uptake of extracellular streptomycin via leakage. This creates
a positive feedback mechanism which continues until a critical intracellular
concentration of antibiotic is reached. This completely halts protein synthesis and leads
to an irreversible translation blockade.
Structural analysis of the bacterial ribosome and antibiotic interactions have
shown that mutations in the S12 ribosomal protein result in a hyperaccurate
phenotype as S12 stabilizes the same region in the ribosome as streptomycin.
• A weak hyperaccurate phenotype manifests as streptomycin resistance. In these
resistance mutations the RAM state is sufficiently destabilized so that any
additional streptomycin-induced stabilization does not trap the ribosome in this
state. A strong hyperaccurate phenotype manifests as streptomycin dependence.
• In the dependent mutants, the ribosome is so destabilized
that the binding action of streptomycin is necessary to
move into the ram state.
• As can be seen in the figure on the right, the
streptomycin binding site facilitates interaction between
the antibiotic and the S12 ribosomal protein.
Chemical Diversity
The aminoglycoside class all consist of an amino group bond to a glycoside:
• Streptomycin 1944 (Streptomyces griseus)
• Neomycin 1949 (Streptomyces fradiae)
• Kanamycin 1957 (Streptomyces kanamyceticus)
• Gentamycin 1963 (Micromonospora)
• Tobramycin 1967 (Streptomyces tenebrarius)
• Amikacin 1972 (Semi-synthetic form of Kanamycin)
• Plazomicin 2009 (Semi-synthetic form of Sisomicin)
Side Effects:
The aminoglycosides have varying degrees of toxicity resulting in ototoxicity,
nephrotoxicity, and neuropathy. The peripheral neuropathy, encephalopathy, and
neuromuscular blockades are caused by inhibition of quantal release of
acetylcholine in the neuromuscular junction pre-synaptically and a post-junctional
binding of of aminoglycosides to the acetylcholine receptor complex.
Amikacin in-Depth
• Developed by Bristol-Banyu Research in 1972.
• The first semi-synthetic derivative of the Aminoglycosides.
• Kanamycin was acylated at the C-1 amino group of the deoxystreptamine group
with L-(-)-!-amino-"-hydroxybutyric acid.
This modification prevents degradation by bacterial resistance enzymes and
reduces cross-resistance between Amikacin and other aminoglycosides. However,
there has been a rise in resistance with the primary mechanism being acetylation of
the 6’-N position by the AAC(6’)-I enzymes.
• Recent research has found numerous compounds capable of inhibiting this class
of enzymes that could be used in conjunction with aminoglycosides. The three
most promising small molecules are found below:
Resistance
Resistance Modalities of Streptomycin:
Modification of Antibiotic: Bacterial enzymes catalyze acetylation, adenylation, or
phosphorylation of aminoglycoside hydroxyl groups.
Modification of Primary Target: A site-specific mutagenesis changes the C912 à
U192 in the 16S rRNA Gene. A mutation in ribosomal protein S12 results in a
weak hyperaccuracy resistant phenotype.
Exclusion or Expulsion of Antibiotic:
Stages of Uptake:
I ) Aminoglycoside adsorbs to cell surface based on charge.
II ) A small amount is taken up dependent on transmembrane potential.
III) Incorporation of misfolded proteins disrupt cell membrane integrity rapidly
increase uptake of antibiotic..
• Electron transport chain reduces uptake of drugs into cells by decreasing the
membrane potential, as seen in Figure 3 where a decrease in membrane
potential significantly reduces MIC.
• Mutations in the outer membrane such as increased expression of H1 protein
reduce streptomycin uptake. As can be seen in Figure 1, rate at which H181
over-producing mutants were killed by gentamycin was significantly lower
than that of the wild-type.
• The development of efflux pumps that expel streptomycin such as the 11B
transposon mutant of the MexXY gene. As can be seen in Figure 2, high efflux
pump expression was correlated with a marked increase in Tobramycin MIC.
Fig. 1 Fig. 2 Fig. 3
Tolerance
Tolerant populations can outlive exposure to raised antibiotic concentrations without
modification of the MIC by slowing down essential bacterial processes.
• This enables prolonged survival under antibiotic exposure. The addition of metabolites
have been shown to re-sensitize aminoglycoside tolerant mutants.
Factors that Impair Aminoglycoside Activity by introducing Tolerance:
The absence of metabolites causes the cell to enter a non-growing phase. As such, the cell
slows translation which reduces the uptake of aminoglycosides which leads tolerance.
• Low extracellular pH increases membrane potential which reduces aminoglycoside
uptake into the cell.
• Anaerobic conditions result in a very low membrane potential which prevents uptake of
aminoglycosides and essentially makes anaerobic bacteria immune to their action.
• Certain types of translational initiation such as chloramphenicol which blocks peptide
chain elongation.
Re-sensitization of Tolerant Cells:
• Membrane targeting agents such as Rhamnolipids which enable PMF-independent
cellular uptake of aminoglycosides and cell killing.
• A study found the addition of silver nitrate was found to reduce the MIC of
aminoglycosides by over ten-fold and was PMF-independent.
References
CBE/MOL411 Lecture Slides 2022 by M.P. Brynildsen
Bryan, L. E., & Kwan, S. (1983). Antimicrobial Agents and Chemotherapy, 23(6), 835–845.
BARKER, E. P. Et al., (1992). Journal of General Microbiology, 138(3), 551–561.
Ezraty, B. Et al., (2017). Molecular Microbiology, 105(1), 115–126.
Häussler, S. et al., (2020). Antimicrobial Agents and Chemotherapy, 65(1).
Ramakrishnan, V. Et al., (2000). Nature, 407(6802), 340–348.
Tolmasky, M. Et al., (2017). Molecules, 22(12), 2267.
Wiela-Hojeńska, A. Et al., (2021). Molecules, 26(24), 7456.
Wright, G. E. Et al., (1974). Antimicrobial Agents and Chemotherapy, 5(2), 143–152.