What is Protein Synthesis?

Protein synthesis is the process whereby amino acids are linked together to form proteins using mRNA as a template.

Overview

The production of proteins from an mRNA template is a multistep process that includes:

  • Activation of amino acids to allow them to be joined together
  • Initiation of translation by binding of translation machinery to the mRNA template
  • Elongation of the protein by successive addition of amino acids
  • Termination and release of the mRNA template
  • Folding and posttranslational processing of the final protein

As each step in the process relies on numerous essential components, protein synthesis is susceptible to inhibition at multiple stages. Because the translation machinery differs slightly between prokaryotic and eukaryotic organisms, bacterial translation machinery can be selectively targeted for inhibition. As a result, protein synthesis is the most common mechanism of action for naturally occurring antibiotics. Interestingly, because the mitochondria of eukaryotic cells often have bacteria-like translation machinery, off target effects in human cells can occur.

Existing Products

Existing protein synthesis inhibitors are primarily used as antibiotics, although some of these antibiotics also have anti-parasitic effects.

Tetracycline (natural product)Broad spectrum antibioticPrevents addition of new amino acids to a protein by occupying an important transfer RNA binding site on the 30S bacterial ribosome.
Product Indication Notes
Chloramphenicol Broad spectrum antibiotic Prevents formation of bonds between amino acids through interactions with the 50S bacterial ribosome. Also inhibits protein synthesis in mitochondria of mammalian cells.
Streptomycin Tuberculosis First antibiotic ever used to successfully treat tuberculosis. Binds the 30S component of the bacterial ribosome.
Doxycycline (synthetic derivative of tetracycline) Broad spectrum antibiotic; used for malaria prophylaxis in travelers; used off label for the treatment of filarial worms (lymphatic filariasis and onchocerciasis) Doxycycline has the same mechanism of action as tetracycline in bacteria.
Paromomycin Amoebiasis and leishmaniasis Paromomycin inhibits protein synthesis by binding to 16S rRNA, part of the 30S bacterial ribosome. In eukaryotic pathogens such as amoeba and leishmania, the drug appears to bind to the equivalent site on the eukaryotic ribosome, although the mechanism of action has not been fully elucidated.
Azithromycin Broad spectrum antibiotic, also used in treatment of trachoma Macrolide antibiotic, binds to the 50S ribosomal subunit inhibiting peptidyl transfer and also results in premature dissociation of peptidyl tRNA from ribosomes.
Erythromycin Broad spectrum antibiotic, often used in those allergic to penicillin Macrolide antibiotic that potentially causes premature dissociation of peptidyl tRNA from ribosome as well as inhibiting peptidyl transfer
Cycloserine Second line drug against tuberculosis Oxazolidone that targets an early step in protein synthesis involving the binding of N-formyl methionyl tRNA to the ribosome
Linezolid (Zyvox) Used to treat serious infections caused by gram positive bacteria, Oxazolidone that works by inhibiting initiation of protein synthesis

Protein Synthesis Inhibitors as Broad Spectrum Antibiotics

Differences between eukaryotic (i.e., human, animal, protozoan parasite, helminth) and prokaryotic (i.e., bacterial) translation machinery facilitate selective or semi-selective inhibition of the prokaryotic machinery with antibiotics. The bacterial 70S ribosome consists of two subunits, the larger 50S subunit and the smaller 30S subunit. Eukaryotes, on the other hand, have an orthologous, although more complex, 80S ribosome consisting of the larger 60S subunit and the smaller 40S subunit. The majority of inhibitors of protein synthesis in bacteria target the 30S or 50S subunits of the bacterial 70S ribosome with minimal cross reactivity with the eukaryotic ribosome. However, as eukaryotic mitochondria have bacteria-like ribosomes some cross reactivity can occur.

Protein Synthesis Inhibitors as Neglected Tropical Disease Therapeutics

Streptomycin, an antibiotic derived from the actinomycete Streptomyces griseus, was the first antibiotic successfully used to treat tuberculosis. The compound, like many antibiotic protein synthesis inhibitors, binds to the 30S subunit of the bacterial ribosome. Although drug resistance has ultimately limited the usefulness of streptomycin for the treatment of tuberculosis, streptomycin provides proof of concept that protein synthesis is a viable therapeutic target for the treatment of tuberculosis.

The mechanism of action of protein synthesis inhibitors in protozoan parasites, such as the parasites that cause malaria and leishmaniasis, is not as well understood. The parasites that cause malaria and leishmaniasis have eukaryotic 80S ribosomes in their cytosol. Additionally, the malaria parasite has a unique organelle called an apicoplast that shares many properties with both bacteria and the plant organelle known as a chloroplast. The apicoplast has its own translation machinery including a 70S-like ribosome. The efficacy of doxycycline for the prophylactic treatment of malaria is thought to involve inhibition of this 70S-like apicoplast ribosome thus blocking synthesis of important apicoplast proteins.1

The efficacy of paromomycin for the treatment of leishmaniasis appears to be less straight forward. Although paromomycin is known to inhibit a subcomponent of the 30S ribosome of bacteria, biochemical studies in leishmania parasites suggest that paromomycin inhibits both cytosolic (eukaryotic) and mitochondrial (prokaryotic-like) ribosomes.2 Biochemical studies of paromomycin on leishmania parasite ribosomes suggest that the drug preferentially binds the leishmania ribosome over the human ribosome.3 However, paromomycin may have additional effects on membrane permeability that also contribute to its efficacy.2

The final group of neglected tropical diseases for which protein synthesis inhibitors have been used is in the treatment of filarial worms, a subset of parasitic worms or helminths that includes the parasites that cause lymphatic filariasis and onchocerciasis. Filarial worms are colonized by commensal bacteria of the genus Wolbachia. These commensal bacteria are essential for the production of microfilaria, the circulating form of the parasite that is transmitted by mosquitoes, and for the survival of the adult worms (macrofilaria) that produce the microfilaria. Clinical trials have demonstrated the efficacy of doxycycline against both the microfilaria and macrofilaria worms.4,5 Doxycycline is not in widespread use for lymphatic filariasis or other filarial worm infections, but these studies served as an important proof of concept that the commensal Wolbachia bacteria are a good target for future therapeutic development.

References

  1. Dahl EL and Rosenthal PJ (2008) “Apicomplast translation, transcription and genome replication: targets for antimalarial antibiotics.”TRENDS in Parasitology 24: 279-284.
  2. Jhingran A et al. (2009) “Paromomycin: Uptake and resistance in Leishmania donovani.” Molecular and Biochemical Parasitology 164: 111-117.
  3. Fernandez MM et al. (2011) “Differential effects of paromomycin on ribosomes of Leishmania mexicana and mammalian cells.” Antimicrobial Agents and Chemotherapies 55: 86-92.
  4. Hoerauf A et al. (2003) “Doxycycline as a novel strategy against bancroftian filariasis-depletion of Wolbachia endosymbionts fromWuchereria bancrofti and stop of microfilaria production.” Med Microbiol Immunol (Berl) 192: 211–216.
  5. Taylor MJ (2005) “Macrofilaricidal activity after doxycycline treatment of Wuchereria bancrofti: a double-blind, randomised placebo-controlled trial." The Lancet 365: 2116–2121.

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Pipline

Analysis

Protein synthesis inhibitors are in development for tuberculosis and leishmaniasis. As protein synthesis is a biological process that occurs in all organisms, further exploration of protein biosynthesis machinery as therapeutic targets for neglected tropical diseases is warranted. However, inhibitor selectivity for the machinery of the neglected tropical disease over the machinery of the human host will be essential.

The relative strengths, weaknesses, opportunities, and risks for natural products that are currently in use or in development for neglected tropical diseases are summarized here.

  Strengths Weaknesses Opportunities Risks
Nitroimidazoles
Relevant neglected tropical diseases:

Tuberculosis (multiple on market products; PNU-100480, phase II; AZD5847, phase I)

Leishmaniasis (paromomycin, on market but currently in clinical trials as part of combination therapies)
Well proven drug target across multiple diseases

Multiple on market products already in use, especially for bacterial infections
Potential off target effects in human cells Identification of selective inhibitors of protein synthesis machinery for additional neglected tropical diseases

New combination therapies
Products with the same mechanism of action are already on market 

Drug resistance is common for on market antibiotics using this mechanism of action

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Database Resources

There are several online resources providing information on protein synthesis inhibitors including:

Assays

Monitoring protein synthesis activity generally involves monitoring incorporation of C14-labeled radioactive amino acids.1 There are numerous cell-free systems that can be used to monitor protein synthesis, some of which have been adapted for high throughput screening.2-4

References

  1. Martin R (ed.) Protein Synthesis: Methods and Protocols, 1998. Book information is available here
  2. Boddeker N et al. (2002) “Characterization of a novel antibacterial agent that inhibits bacterial translation.” RNA 8: 1120–1128.
  3. Novac O et al. (2004) “Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen.” Nucleic Acids Research 32: 902-915.
  4. Promega Protocol and Applications: Protein expression, available here.

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To learn how you can get involved in neglected disease drug, vaccine or diagnostic research and development, or to provide updates, changes, or corrections to the Global Health Primer website, please view our FAQs.