Protein Synthesis Inhibitors – Definition, Mechanism, Examples

What are Protein Synthesis Inhibitors?

• Protein synthesis is a complex and sequential process that involves a series of enzymes and structural transformations within organisms. Essential to this process is the ribosome, a biological machine that employs protein dynamics on nanoscales to convert RNA into proteins. Ribosomes are integral to both prokaryotic and eukaryotic cells and serve as the primary site of protein synthesis.
• To understand the role of protein synthesis inhibitors, it’s crucial first to grasp the significance of protein synthesis. The synthesis process comprises multiple stages, including initiation, the formation of the 70s complex, and the elongation phase where polypeptides are produced. Each stage is critical and must proceed without interruption for the successful production of proteins.
• Therefore, when bacteria infect a host, they rely on their protein synthesis machinery to proliferate and spread. As a countermeasure, some antibacterial agents specifically target and disrupt bacterial protein synthesis. They achieve this by interacting with either the 30s or 50s subunit of the bacterial ribosome, thus impeding the synthesis process. Antibiotics such as Aminoglycosides, Macrolides, Tetracycline, Oxazolidinone, and Chloramphenicol are known to inhibit bacterial protein synthesis.
• Besides these antibiotics, a more broad category of compounds called protein synthesis inhibitors exists. By definition, a protein synthesis inhibitor is a substance that halts or decelerates cell growth by directly interfering with the creation of new proteins. Although this description might seem broad, these inhibitors predominantly act at the molecular level on the translational machinery. In most cases, this means they target either the ribosome itself or associated translation factors.
• Then, why are these inhibitors so effective? The answer lies in the structural differences between prokaryotic and eukaryotic ribosomes. Many inhibitors exploit these differences to specifically target bacterial ribosomes, leaving the host’s ribosomes largely unaffected.
• Moreover, protein synthesis inhibitors function at various stages of translation. Some might impede the initiation phase, while others interfere during elongation or termination. By understanding the specific action of each inhibitor, researchers and medical professionals can employ them more effectively against bacterial infections.
• In conclusion, protein synthesis inhibitors play a crucial role in combating bacterial infections. By targeting the essential process of protein synthesis in bacteria, these compounds offer a strategic approach to halt the growth and spread of harmful pathogens.

Types of Protein synthesis inhibitors

On the basis of target organisms inhibitor of protein synthesis is categories as follows-

1. Acting only on prokaryotes
2. Acting on prokaryotes and Eukaryotes
3. Acting only on Eukaryotes

1. Acting only on prokaryotes

• Tetracycline:
  • Origin: Tetracycline is a semisynthetic antibiotic produced through the catalytic hydrogenation of chlorotetracycline, which is derived from the bacterium Streptomyces aurofaciens.
  • Mechanism: It targets the 30S ribosomal subunit of bacteria, specifically obstructing the binding of aminoacyl tRNA to the ribosome’s A site, thereby inhibiting protein elongation.
• Streptomycin:
  • Origin: This antibiotic is extracted from the soil actinomycete, Streptomyces griseus.
  • Mechanism: Classified as an aminoglycoside, streptomycin attaches to the 30S ribosomal subunit. At low concentrations, it prompts misreading of the genetic code, while at elevated concentrations, it obstructs the initiation of protein synthesis.
• Chloramphenicol:
  • Origin: Chloramphenicol is sourced from Streptomyces venezuela.
  • Mechanism: As a bacteriostatic antibiotic, chloramphenicol’s binding is reversible. It functions by associating with the A site of the ribosome’s 50S subunit. This action impedes the correct binding of aminoacyl tRNA to the A site and subsequently hinders the peptidyl transferase activity, crucial for peptide bond formation.
• Erythromycin:
  • Origin: Contrary to the provided information, erythromycin is derived from Saccharopolyspora erythraea (formerly known as Streptomyces erythraeus).
  • Mechanism: This antibiotic functions by binding to the 23S rRNA molecules situated within the 50S ribosomal subunit of bacteria. By attaching to the ribosome’s exit channel, erythromycin restricts the elongation of the peptide chain, stalling protein synthesis.
• Rifamycin:
  • Mechanism: Unlike other antibiotics, rifamycin operates indirectly to inhibit protein synthesis. It hampers the initiation of RNA chains by associating with RNA polymerase, thereby inhibiting RNA synthesis which is a precursor step to protein synthesis.

2. Acting on prokaryotes and Eukaryotes

• Puromycin:
  • Origin: Derived from the bacterium Streptomyces alboniger, puromycin is an antibiotic that has significant implications for protein synthesis.
  • Structural Relevance: Molecularly, puromycin bears a striking resemblance to the 3’ end of an aminoacyl tRNA.
  • Mechanism: This structural similarity allows puromycin to bind to the ribosomal A site and partake in peptide bond formation, resulting in the production of peptidylpuromycin. Crucially, puromycin is unable to participate in translocation. Consequently, it dissociates from the ribosome soon after linking to the peptide’s carboxyl terminus. This action abruptly halts polypeptide synthesis.
  • Applications: Despite its profound impact on protein synthesis, puromycin is not used clinically due to its toxic effects on the host. Nevertheless, its unique mechanism makes it valuable in research contexts, particularly in cell culture studies.
• Actinomycin D:
  • Mechanism: Actinomycin D exhibits a distinct mode of action. Instead of directly targeting the ribosome, it associates with DNA. By doing so, it impedes the movement of RNA polymerase along the DNA strand. This blockage subsequently disrupts both RNA synthesis and, by extension, protein synthesis.

3. Acting only on Eukaryotes

• Cycloheximide:
  • Mechanism: Cycloheximide functions by obstructing the peptidyl transferase activity. This action particularly affects the translocation reaction associated with the 80S eukaryotic ribosomes.
• Anisomycin:
  • Mechanism: Anisomycin targets the ribosomes, where it serves to inhibit the peptidyl transferase reaction, thereby disrupting a crucial step in protein elongation.
• α-Amaniyin:
  • Mechanism: Unique in its action, α-Amaniyin primarily impedes mRNA synthesis. It accomplishes this by preferentially binding to RNA polymerase II, a pivotal enzyme in transcription.
• Diphtheria Toxin:
  • Mechanism: This toxin, derived from the bacterium responsible for diphtheria, catalyzes the ADP-ribosylation of diphthamide – a specialized histidine residue. This modification is found in the eukaryotic elongation factor eEF2. The result of this modification is the inactivation of eEF2, thus hindering its role in protein synthesis.
• Ricin:
  • Source: Ricin is a highly toxic protein extracted from the castor bean plant.
  • Mechanism: Its primary mode of action is on the 60S subunit of eukaryotic ribosomes. It achieves this by depurinating a specific adenosine located in the 23S rRNA, thereby rendering the ribosomal subunit non-functional.

Mechanism of Protein synthesis inhibitors

Protein synthesis, a core biological process, occurs at the ribosomal level, translating bacterial mRNA into proteins through stages like initiation, elongation, and termination. The process demands intricate interactions involving mRNA, tRNA, and ribosomal RNA. However, protein synthesis inhibitors interrupt this delicate process. To grasp the intricacy of these inhibitors’ actions, it is essential to delve into the specific mechanisms each inhibitor employs.

1. Early Stages of Protein Synthesis:

• Rifamycin: Rifamycin acts by inhibiting the transcription of bacterial DNA into mRNA. It achieves this by binding to the beta-subunit of DNA-dependent RNA polymerase.
• alpha-Amanitin: Contrarily, alpha-Amanitin targets eukaryotic DNA transcription machinery, making it a powerful inhibitor of this domain.

2. Initiation:

• Linezolid: This inhibitor acts at the initiation phase, potentially preventing the formation of the initiation complex. However, the exact mechanism remains not fully elucidated.

3. Ribosome Assembly:

• Aminoglycosides: These bind to the bacterial 30S ribosomal subunit, subsequently preventing the assembly of ribosomes.

4. Aminoacyl tRNA Entry:

• Tetracyclines and Tigecycline: These agents block the A site on the ribosome, thus stopping the binding of aminoacyl tRNAs.

5. Proofreading:

• Aminoglycosides: Besides other potential actions, aminoglycosides disrupt the proofreading process. This interference results in an increased error rate in synthesis, leading to premature termination.

6. Peptidyl Transfer:

• Chloramphenicol: This agent impedes the peptidyl transfer step of elongation on the 50S ribosomal subunit found in both bacteria and mitochondria.
• Macrolides: They bind to the 50s ribosomal subunits, inhibiting the peptidyl transfer step.

7. Ribosomal Translocation:

• Macrolides and Clindamycin: Evidence suggests that these inhibitors impede ribosomal translocation. Additionally, Fusidic acid hinders the turnover of elongation factor G (EF-G) from the ribosome.

8. Termination:

• Macrolides and Clindamycin: They cause the premature dissociation of the peptidyl-tRNA from the ribosome.
• Puromycin: Mimicking the structure of tyrosinyl aminoacyl-tRNA, it binds to the ribosomal A site, takes part in peptide bond formation, but soon dissociates, causing premature termination.

To further elucidate, here’s an examination of the mechanisms of specific classes of antibiotics:

• Aminoglycosides: Once inside the cytoplasm, they inhibit bacterial protein synthesis by binding to the A-site of 16s rRNA. This binding leads to mistranslation and results in the integration of incorrect amino acids into polypeptides, damaging the cell.
• Macrolides: Their inhibitory action on bacterial protein synthesis is multifaceted, involving inhibition during the early phase of translation, peptidyl tRNA dissociation, obstruction in peptide bond formation, and interference with the 50s subunit.
• Chloramphenicol: This agent specifically targets the elongation steps, inhibiting the peptidyl transferase activity. By binding to specific sites on ribosomal units, it prevents peptide bond formation.
• Tetracycline: Bacteriostatic in nature, tetracyclines bind to the 30s ribosomal unit, hindering the attachment of transfer RNA and thereby inhibiting protein synthesis.
• Oxazolidinone: They inhibit the formation of the initiation complex. Upon binding to the 50s ribosomal subunit, they prevent the 70s subunit formation and the subsequent translocation of the peptide chain.

Binding site of Protein synthesis inhibitors

1. Inhibitors Binding to the 30S Ribosomal Subunit: The 30S subunit serves as a vital site for the initiation phase of protein synthesis, particularly in bacterial cells. It is responsible for decoding the mRNA into protein. Given its integral role, certain antibiotics target this subunit to hinder protein synthesis.
   • Aminoglycosides: These compounds bind specifically to the 30S ribosomal subunit. Upon binding, they disrupt protein synthesis by causing mRNA misreading and subsequently lead to the integration of incorrect amino acids into the growing peptide chain.
   • Tetracyclines: Another group binding to the 30S subunit, tetracyclines work by preventing the attachment of aminoacyl tRNA to the ribosome. As a result, the elongation phase of protein synthesis is interrupted.
2. Inhibitors Binding to the 50S Ribosomal Subunit: The 50S ribosomal subunit is larger than its 30S counterpart and primarily plays a role in the elongation phase of protein synthesis. Several antibiotics target this subunit, thus obstructing the protein synthesis pathway.
   • Chloramphenicol: This antibiotic binds to the 50S ribosomal subunit and specifically inhibits the peptidyl transferase activity, an essential function during the elongation phase.
   • Clindamycin: By binding to the 50S subunit, clindamycin hinders the elongation phase of protein synthesis, particularly affecting peptide bond formation.
   • Linezolid: An oxazolidinone, Linezolid also targets the 50S ribosomal subunit. It acts by inhibiting the formation of the initiation complex, a crucial step in the beginning of protein synthesis.
   • Macrolides: These agents bind to the 50S subunit, affecting multiple aspects of protein synthesis, including hindering the early phase of translation and causing peptidyl tRNA dissociation.
   • Telithromycin: As a derivative of macrolides, it also binds to the 50S subunit, playing a similar inhibitory role in protein synthesis.
   • Streptogramins: These compounds have an affinity for the 50S ribosomal subunit. Their binding disrupts the elongation phase, causing premature termination of protein synthesis.
   • Retapamulin: Another agent that binds to the 50S subunit, its exact mechanism remains under investigation, but its association with the 50S subunit suggests an inhibitory role in protein synthesis.

Examples of Protein synthesis inhibitors

Protein synthesis inhibitors are molecules that impede the protein synthesis process in cells. They can target various stages of this process, such as initiation, elongation, and termination, and can be specific to prokaryotic or eukaryotic organisms, or sometimes both. Here are some examples of protein synthesis inhibitors:

Prokaryotic-Specific Inhibitors:

1. Tetracycline: Binds to the 30S subunit of the bacterial ribosome, preventing the attachment of aminoacyl-tRNA to the A site of the ribosome.
2. Chloramphenicol: Binds to the 50S subunit and inhibits the peptidyl transferase activity, thereby halting peptide bond formation.
3. Streptomycin: An aminoglycoside that binds to the 30S subunit, causing misreading of mRNA and inhibiting initiation at higher concentrations.
4. Erythromycin: Binds to the 50S subunit and inhibits the translocation step of protein synthesis.
5. Rifamycin: Binds to the DNA-dependent RNA polymerase, preventing the initiation of RNA synthesis.

Eukaryotic-Specific Inhibitors:

1. Cycloheximide: Binds to the 60S subunit of the eukaryotic ribosome, inhibiting the translocation step of protein synthesis.
2. Anisomycin: Inhibits peptidyl transferase activity in eukaryotic ribosomes.
3. α-Amanitin: Binds to RNA polymerase II and III, inhibiting mRNA and snRNA synthesis.

Inhibitors Affecting Both Prokaryotes and Eukaryotes:

1. Puromycin: Structurally similar to an aminoacyl-tRNA and causes premature chain termination by releasing nascent polypeptide chains from the ribosome.
2. Actinomycin D: Interacts with DNA and inhibits RNA polymerase action in both prokaryotes and eukaryotes.

Additional Examples:

1. Linezolid: Binds to the 50S subunit and prevents the formation of the initiation complex in bacteria.
2. Clindamycin: Similar to erythromycin, clindamycin binds to the 50S ribosomal subunit and inhibits the elongation step.
3. Diphtheria Toxin: A bacterial toxin that ADP-ribosylates eEF2, blocking the elongation step in eukaryotic cells.
4. Ricin: Damages the 60S subunit of eukaryotic ribosomes by depurinating rRNA, halting protein synthesis.

Each of these inhibitors has a specific mechanism of action, and while some are used therapeutically to treat infections, others are primarily used as research tools due to their toxicity to human cells.

Mechanism of Resistance to Protein synthesis inhibitors

1. Tetracyclines
   • Efflux Pump Mechanism: One of the primary defense mechanisms against tetracyclines is the efflux pump. Here, active antibiotics are extricated from the cell through transmembrane proteins. Consequently, a substantial reduction in the intracellular concentration of the antibiotics occurs. This is predominantly due to the transportation of the molecule alongside magnesium ions.
   • Cytoplasmic Ribosomal Protection: Besides the efflux pump system, another mode of resistance emerges from cytoplasmic proteins. These specific proteins function to guard the ribosome against the adverse effects of tetracycline.
2. Chloramphenicol
   • Efflux Strategies and Target Mutation: Resistance to chloramphenicol often manifests through efflux mechanisms. Simultaneously, mutations at the antibiotic’s target site further augment the resistance.
   • Permeability Hindrances: Sometimes, the bacterial cell erects barriers, inhibiting the permeation of the antibiotic.
   • Enzymatic Deactivation via Acetylation: There exists an enzyme, phosphotransferase, which inactivates chloramphenicol by acetylation. This enzymatic action stems from genes such as catA and catB, which encode the chloramphenicol acetyltransferases.
   • Outer Membrane Protein Deficiency: A notable decline or absence of specific outer membrane proteins significantly contributes to chloramphenicol resistance.
3. Aminoglycosides
   • Methylation of rRNA: A dominant form of resistance to aminoglycosides originates from the methylation process of the 16S rRNA.
   • Enzymatic Alteration of Structures: Moreover, the resistance is escalated due to certain enzymatic activities that modify the antibiotic structures. Three distinct classes of enzymes involved in this process are aminoglycoside phosphotransferases, aminoglycoside nucleotidyltransferases, and aminoglycoside acetyltransferases. These modifications consequently disrupt the antibiotic’s functioning due to either steric or electrostatic interferences.
4. Macrolides
   • rRNA Alterations: For macrolides, resistance is primarily attributed to modifications in the 23S rRNA or mutations triggered by erm genes. This is primarily due to the demethylation process that occurs at the A2058 residue in 23S rRNA.
   • Horizontal Gene Dissemination: The erm genes, which are instrumental in inducing resistance, can be transferred among pathogens. This horizontal gene transfer further amplifies resistance across a broad spectrum of bacteria.
   • Steric Barriers: A pivotal factor in resistance emerges from steric hindrance, a consequence of the dimethylation of A2058 residues. This alteration significantly compromises the drug’s affinity to its target.
5. Oxazolidonones
   • Target Adjustments: Resistance against oxazolidinones can primarily be attributed to modifications in the target molecules.
   • Role of Cfr Genes: Critical to understanding oxazolidinone resistance is the role played by the cfr genes. These genes, which can be located on plasmids or within the chromosome, contribute significantly to resistance. Furthermore, mutations, especially the Guanine to Uracil transition at the A2058 position of 23S rRNA, play a pivotal role.

References

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