Protein synthesis and its selective inhibition

PROTEIN SYNTHESIS AND ITS SELECTIVE INHIBITION

Figure 12.4 outlines the process of protein synthesis involving the ribosome, mRNA, a series of aminoacyl transfer RNA (tRNA) molecules (at least one for each amino acid) and accessory protein factors involved in initiation, elongation and termination. As the process is essentially the same in prokaryotic (bacterial) and eukaryotic cells (i.e. higher organisms and mammalian cells) it is surprising that there are so many selective agents which act in this area (see Figure 12.1).

Figure 12.4 Outline of the process of protein synthesis (translation of messenger RNA) in bacterial cells. The four stages of synthesis are shown: initiation, elongation, translocation and termination with the sites of action of antibiotics. AUG is the start codon on messenger RNA (mRNA) specifying the first amino acid in bacterial proteins, N-formyl-methionine. UAG, UAA and UGA are termination codons specifying no amino acid. 30S and 50S are the subunits of the ribosome. Other protein factors involved in protein synthesis are initiation factors (IF-1, 2, 3), elongation factors (EF-Tu and EF-G) and release factors (RF-1, 2, 3).

Bacterial ribosomes are smaller than their mammalian counterparts. They consist of one 30S and one 50S subunit (the S suffix denotes the size, which is derived from the rate of sedimentation in an ultracentrifuge). The 30S subunit comprises a single strand of 16S rRNA and over 20 different proteins that are bound to it. The larger 50S subunit contains two single strands of rRNA (23S and 5S) together with over 30 different proteins. The subunits pack together to form an intact 70S ribosome. The equivalent subunits for mammalian ribosomes are 40S and 60S, making an 80S ribosome. Some agents exploit subtle differences in structure between the bacterial and mammalian ribosomes. The macrolides, azalides and chloramphenicol act on the 50S subunits in bacteria but not the 60S subunits of mammalian cells. By contrast, the tetracyclines derive their selective action through active uptake and concentration within microbial cells but only limited penetration of mammalian cells.

Aminoglycoside–aminocyclitol antibiotics

Most of the information on the mechanisms of action of aminoglycoside–aminocyclitol (AGAC) antibiotics comes from studies with streptomycin. One effect of AGACs is to interfere with the initiation and assembly of the bacterial ribosome (Figure 12.4). During assembly of the initiation complex, N-formylmethionyl-tRNA (fmet-tRNA) binds initially to the ribosome binding site on the untranslated 5′ end of the mRNA together with the 30S ribosomal subunit. Three protein initiation factors (designated IF-1, 2 and 3) and a molecule of guanosine triphosphate (GTP) are involved in positioning the fmet-tRNA on the AUG start codon of mRNA. IF-1 and IF-3 are then released from the complex, GTP is hydrolysed to guanosine diphosphate (GDP) and released with IF-2 as the 50S subunit joins the 30S subunit and mRNA to form a functional ribosome. The fmet-tRNA occupies the peptidyl site (P site) leaving a vacant acceptor site (A site) to receive the next aminoacyl-tRNA specified by the next codon on the mRNA. Streptomycin binds tightly to one of the protein components of the 30S subunit. Binding of the antibiotic to the protein, which is the receptor for IF-3, prevents initiation and assembly of the ribosome.

Streptomycin binding to the 30S subunit also distorts the shape of the A site on the ribosome and interferes with the positioning of the aminoacyl-tRNA molecules during peptide chain elongation. Streptomycin therefore exerts two effects: inhibition of protein synthesis by freezing the initiation complex, and misreading of the codons through distortion of the 30S subunit. Simple blockage of protein synthesis would be bacteriostatic rather than bactericidal. As streptomycin and the other AGACs exert a potent lethal action, it seems that the formation of toxic, non-functional proteins through misreading of the codons on mRNA is a more likely mechanism of action. This can be demonstrated with cell-free translation systems in which isolated bacterial ribosomes are supplied with artificial mRNA template such as poly(U) or poly(C) and all the other factors, including aminoacyl-tRNAs needed for protein synthesis.

In the absence of an AGAC the ribosomes will produce artificial polypeptides, polyphenylalanine (as specified by the codon UUU) or polyproline (as specified by the codon CCC). However, when streptomycin is added, the ribosomes produce a mixture of polythreonine (codon ACU) and polyserine (codon UCU). The misreading of the codons does not appear to be random: U is read as A or C, and C is read as A or U. If such misreading occurs in whole cells, the accumulation of non-functional or toxic proteins would eventually prove fatal to the cells. There is some evidence that the bacterial cell membrane is damaged when the cells attempt to excrete the faulty proteins.

The effectiveness of the AGACs is enhanced by their active uptake by bacteria, which proceeds in three phases. First, a rapid uptake occurs within a few seconds of contact, which represents binding of the positively charged AGAC molecules to the negatively charged surface of the bacteria. This phase is referred to as the energy-independent phase (EIP) of uptake. In the case of Gram-negative bacteria the AGACs damage the outer membrane causing release of some lipopolysaccharide, phospholipid and proteins but this is not directly lethal to the cells. Second, there follows an energy-dependent phase of uptake (EDP I) lasting about 10 minutes, in which the AGAC is actively transported across the cytoplasmic membrane. A second energy-dependent phase (EDP II) which leads to further intracellular accumulation follows after some AGAC has bound to the ribosomes in the cytoplasm. Although the precise details of uptake by EDP I and EDP II are not clear, both require organisms to be growing aerobically. Anaerobes do not take up AGACs by EDP I or EDP II and are consequently resistant to their action.

Tetracyclines

This group of antibiotics is actively transported into bacterial cells, possibly as the magnesium complex, achieving a 50-fold concentration inside the cells. Mammalian cells do not actively take up the tetracyclines (small amounts enter by diffusion alone) and it is this difference in uptake that determines the selective toxicity. Resistance to the tetracyclines occurs through failure of the active uptake system or the action of active efflux pumps, which remove the drug from the cells before it can interfere with ribosome function. Other resistance mechanisms involve ribosomal protection and modification. Protein synthesis by both bacterial and mammalian ribosomes is inhibited by the tetracyclines in cell-free systems. The action is on the smaller subunit. Binding of just one molecule of tetracycline to the bacterial 30S subunit occurs at a site involving the 3′ end of the 16S rRNA, a number of associated ribosomal proteins and magnesium ions. The effect is to block the binding of aminoacyl-tRNA to the A site of the ribosome and halt protein synthesis. Tetracyclines are bacteriostatic rather than bactericidal, consequently they should not be used in combination with β-lactams, which require cells to be growing and dividing to exert their lethal action.

Chloramphenicol

Of the four possible optical isomers of chloramphenicol, only the d-threo form is active. This antibiotic selectively inhibits  protein  synthesis  in  bacterial  ribosomes  by binding to the 50S subunit in the region of the A site involving  the  23S  rRNA.  The  normal  binding  of  the aminocyl-tRNA in the A site is affected by chloramphenicol in such a way that the peptidyl transferase cannot form a new peptide bond with the growing peptide chain on the tRNA in the P site. Studies with aminocyl-tRNA fragments containing truncated tRNA chains suggest that the shape of the region of tRNA closest to the amino acid is distorted by chloramphenicol. The altered orientation of this region of the aminoacyl-tRNA in the A site is sufficient to prevent peptide bond formation. Chloramphenicol has a broad spectrum of activity, which covers Gram-positive and Gram-negative bacteria, mycoplasmas, rickettsia and chlamydia. It has the valuable property of penetrating into mammalian cells and is therefore the drug of choice for treatment of intracellular pathogens, including Salmonella enterica serovar Typhi, the causative organism of typhoid. Although it does not inhibit 80S ribosomes, the 70S ribosomes of mammalian mitochondria are sensitive and therefore some inhibition occurs in rapidly growing mammalian cells with high mitochondrial activity.

Macrolides and azalides

Erythromycin is a member of the macrolide group of antibiotics; it selectively inhibits protein synthesis in a broad range of bacteria by binding to the 50S subunit. The site at which it binds is close to that of chloramphenicol and involves the 23S rRNA. Resistance to chloramphenicol and erythromycin can occur by methylation of different bases within the same region of the 23S rRNA. The sites are therefore not identical, but binding of one antibiotic prevents binding of the other. Unlike chloramphenicol, erythromycin blocks translocation. This is the process by which the ribosome moves along the mRNA by one codon after the peptidyl transferase reaction has joined the peptide chain to the aminoacyl-tRNA in the A site. The peptidyl-tRNA is moved (translocated) to the P site, vacating the A site for the next aminocyl-tRNA. Energy is derived by hydrolysis of GTP to GDP by an associated protein elongation factor, EF-G. By blocking the translocation process, erythromycin causes release of incomplete polypeptides from the ribosome. It is assumed that the azalides, such as azithromycin, have a similar action to the macrolides. The azalides have improved intracellular penetration over the macrolides and are resistant to the metabolic conversion which reduces the serum half-life of erythromycin.

Clindamycin

This agent binds selectively to a region of the 50S ribosomal subunit close to that of chloramphenicol and erythromycin. It blocks elongation of the peptide chain by inhibition of peptidyl transferase.

Streptogramins Quinupristin And Dalfopristin

The two unrelated streptogramins, quinupristin and dalfopristin, have been used in combination (in a 30 : 70 ratio) to treat infections caused by staphylococci and enterococci, particularly methicillin-resistant Staph. aureus (MRSA) and VRE. Their action is synergistic, and is generally bactericidal compared with either agent used alone or compared with antibiotics in the macrolide group. The main target is the bacterial 50S ribosome, with the formulation acting to inhibit protein synthesis. The agents bind sequentially to the 50S subunit; dalfopristin alters the shape of the subunit so that more quinupristin can bind. Dalfopristin blocks an early step in protein synthesis by forming a bond with the ribosome, preventing elongation of the peptide chain by the peptidyl transferase. Quinupristin blocks a later step by preventing the extension of peptide chains and causing incomplete chains to be released. The overall effect is to block elongation. Use of streptogramins is limited by vasculitis, causing pain on intravenous administration.

Oxazolidinones—linezolid

Oxazolidinones such as linezolid act at the early stage of protein synthesis, preventing the formation of the initiation complex between the 30S subunit, mRNA and fmet-tRNA.

Mupirocin

The target of mupirocin is one of a group of enzymes which couple amino acids to their respective tRNAs for delivery to the ribosome and incorporation into protein. The particular enzyme inhibited by mupirocin is involved in producing isoleucyl-tRNA. The basis for the inhibition is a structural similarity between one end of the mupirocin molecule and isoleucine. Protein synthesis is halted when the ribosome encounters the isoleucine codon through depletion of the pool of isoleucyl-tRNA.

Fusidic acid

This steroidal antibiotic does not act on the ribosome itself, but on one of the associated elongation factors, EF-G. This factor supplies energy for translocation by hydrolysis of GTP and GDP. Another elongation factor, EF-Tu, promotes binding of aminoacyl-tRNA molecules to the A site through binding and hydrolysis of GTP. Both EF-G and EF-Tu have overlapping binding sites on the ribosome. Fusidic acid binds the EF-G : GDP complex to the ribosome after one round of translocation has taken place. This prevents further incorporation of aminoacyl-tRNA by blocking the binding of EF-Tu : GTP. Fusidic acid owes its selective antimicrobial action to active uptake by bacteria and exclusion from mammalian cells. The equivalent elongation factor in mammalian cells, EF-2, is susceptible to fusidic acid in cell-free systems.

Pleuromutilins - Retapumilins

These agents bind to the 23S rRNA component of the 50S bacterial ribosome and block peptide bond formation by interfering with the binding of the peptidyl transferase region with the aminoacyl-tRNA substrates in the A and P sites on the ribosome. This mechanism is different to that of other peptidyl transferase inhibitors (chloramphenicol and clindamycin) so cross-resistance to these agents does not occur.