Most drugs that inhibit protein synthesis or translation block the action of the

Inhibitor Molecules

Sordarins

Protein synthesis inhibitors include sordarins which selectively inhibit fungal protein synthesis by blocking the function of elongation factor 2 (EF-2) and ribosomes. They are absent in human cells.

Sphingolipid biosynthesis inhibitors are also being currently investigated to identify new antifungal targets. Recent studies show that DNA topoisomerases are apparently suitable targets for drugs, e.g., eupolauridine, a potential inhibitor of topoisomerase has an advantage of being nontoxic to mammalian cells.

Tacrolimus

It was formerly known as FK506; the 23-membered natural product (NP) macrolide lactone is involved in blocking T-cell activation. Its mode of action is similar to cyclosporins but they are structurally unrelated. It blocks calcineurin, a Ca2+-calmodulin-dependent serine-threonine protein phosphatase subsequently blocks Calcium-dependent events, such as IL-2 gene transcription, nitric oxide synthase activation, cell degranulation, and apoptosis. Calcium signaling is responsible for this pathogen in responding to several stresses.

Ascomycin

Also called immunomycin/FR-900520/FK520, it is an analog of tarcolimus isolated from fermentation broths of Streptomyces species. It inhibits the production of Th2 (INF- and IL-2) and Th2 (IL-4 and IL-10) cytokines. Additionally, ascomycin preferentially inhibits the activation of mast cells.

8.2.3 Protein Synthesis Inhibitors

Protein synthesis inhibitors represent another major group of clinically useful antibacterials, such as erythromycin, tetracycline, chloramphenicol, and aminoglycosides. They selectively interact with the 70S bacterial ribosome and spare the 80S eukaryotic ribosome particle. Macrolide, lincosamide (Figure 8-4D), and streptogramins (Figure 8-4E) (MLS) antibiotics represent three classes of structurally diverse protein biosynthesis inhibitors used clinically against Gram-positive bacteria, chlamydia, mycoplasmas, anaerobes, Hemophilus, Legionella, and Neisseria, while other macrolides such as tylosin and virginimycin are in veterinary use. Despite continuous interest, such as their effectiveness in the treatment of Mycobacterium avium in immune compromised patients and the relatively low level of general clinical resistance, there have been few reports of screening for macrolide protein synthesis inhibitors.

A general assay adopted for protein synthesis inhibition is the incorporation of a radiolabeled amino acid into trichloroacetic acid (TCA) precipitable protein. This assay has been developed using natural mRNA or poly (A) as a template. A major drawback of this assay is the simultaneous detection of nucleic acid inhibitors due to their effect on protein synthesis; hence, the need for a specific secondary assay. Naveh et al. (1984) and Ulitzur (1986) described a sensitive assay for DNA interacting agents as well as protein synthesis inhibitors based on the induction of the luciferase system by acridine dyes in dark variants of Photobacterium leiognathi. Yao and Mahoney (1989)developed a direct competitive enzyme-linked immunosorbent assay (ELISA) using a rabbit polyclonal antibody to 23-amino-O-mycaminosyl-tolonolide conjugated to alkaline phosphatase. They detected several known compounds in fermentation broths but no new compounds resulted from this assay. However, the antibody approach could be readily applied to detect other antibiotics.

The induction of simultaneous resistance to MLS was exploited to develop a plate assay by Ganguli (1989) in which discs of sublethal concentration of erythromycin were placed adjacent to fermentation broths to detect distorted zones from an MLS compound on an overlay of inducible Staphylococcus aureus. Grividomycins and swalpamycin; a neutral 16-membered macrolide with a novel aglycone was discovered by using this assay (Franco et al. 1987; Chatterjee et al. 1987). The plate assay was further modified when induction was described to be caused by translational attenuation in the leader region of the Erm gene (Dubnau 1984). The product of the Erm gene (methylase) methylates adenine 2058 in the 23S rRNA, conferring simultaneous resistance to MLS antibiotics (Weisblum 1983). A LacZ fusion in the ErmC gene produced a construct capable of β-galactosidase induction with sublethal concentrations of erythromycin (Gryczan et al. 1984). The modified assay was used to characterize a new series of erythromycin derivatives (Figures 8-4B and Figures 8-4C) that were active against both inducible and constitutively resistant S. aureus (Goldman and Kadam 1987; Fernandes et al. 1989). These compounds were also shown to bind to methylated ribosomes (Goldman and Kadam 1987). A similar test was developed to use the thiostrepton resistance determinant of Streptomyces azureus, which also confers resistance by methylating an adenine residue in the 23S RNA (Thompson et al. 1982). Neoberninomycin produced by Micrococcus luteus was detected in this assay (Biskupiak et al. 1988). The nature of the leader sequence in ErmC allowed a way to confirm translational attenuation by a different mechanism; namely, the depletion of charged isoleucine residues caused by pseudomonic acid, a potent inhibitor of isoleucyl-tRNA synthetase (Kadam 1989). As shown in Figure 8-5, the presence of pseudomonic acid adjacent to erythromycin allows growth of erythromycin-sensitive bacteria between the two antibiotic discs. This was demonstrated to be due to the ability of pseudomonic acid, a non-macrolide antibiotic used to induce ErmC methylase (Figure 8-6), which in turn generates methylated ribosomes, unable to bind erythromycin and inhibit protein synthesis and cell growth. The leader peptide in ErmC has three isoleucines that possibly cause ribosome stalling in several places, while the leader in ErmD contains no isoleucine residues. In principle, the assay could be used to detect other tRNA synthetase inhibitors by simply adjusting the amino acid residues in the leader region. Another novel class of protein synthesis inhibitors, the oxazolidinones, was described by Barry (1988). These compounds are thought to bind to ribosomes in a way that permits elongation and termination, but prevents subsequent initiation from natural mRNA.

The aminoglycoside antibiotics include a group of structurally related polycationic compounds composed of amino sugars connected by glycosidic linkages. They act on the 30S ribosomal subunit causing inhibition of protein synthesis and subsequent cell lysis. Although their mode of action is not clear, translational misreading appears to be the prevailing consensus opinion. Over the last 30 years, very few new techniques have appeared for the discovery of aminoglycosides. Numata et al. (1986) has described the use of a hypersensitive klebsiella to test 20,000 soil isolates. Seven of the ten positives were known aminoglycosides and one produced a novel amino sugar antibiotic (Tsuno et al. 1986). Yao and Mahoney (1984) again used a gentamicin-derived antibody to develop an ELISA to screen fermentation broths for the presence of compounds that could compete with gentamicin. A novel screening procedure was described by Saitoh et al. (1988) in which compounds were selected for the ability to induce the growth of a streptomycin-dependent strain of Escherichia coli. They discovered a new nontoxic, broad-spectrum antibiotic, boholmycin, using this assay. Hotta et al. (1983) devised an interesting method in which actinomyces isolated on an aminoglycoside-containing medium were characterized by their pattern of resistance to several aminoglycosides because aminoglycoside producers were previously found to possess individual resistance patterns. This step led to an enrichment for aminoglycoside producers. Researchers at Kyowa and Abbott Laboratories used a specific panel of organisms with a known mechanism of aminoglycoside inactivation to dereplicate for known compounds (Jackson 1988). This procedure led to the isolation of three new compounds; sagamicin, seldomycin, and fortimicin (see Figure 8-4G) (Nara et al. 1977). A similar screen also produced lysinomycin (Kurath et al. 1984).

3 INFLUENCE OF CYCLOHEXIMIDE ON MRNA DECAY

The protein synthesis inhibitor cycloheximide has repeatedly been shown to stabilize a variety of mRNAs, including those with AUUUA motifs (60). The mechanism for this effect remains elusive and has been ascribed to the inhibition of synthesis of a labile protein necessary for rapid mRNA decay (39) or the need for continuous ribosome movement (38, 41). We used this well-known effect of cycloheximide to assess the validity of our mRNA transfection system. Transfected lymphocytes were treated with cycloheximide (15 μg/ml) prior to measuring decay rates. Based on Northern blot analysis, both wild-type hGM-AUUUA and mutant hGM-AUGUA mRNAs were stabilized tot12>90min . Under these conditions, no detectable intracellular or extracellular GM-CSF protein was detectable over a 6-hr period. These data further demonstrate that protein synthesis must be functional for labile mRNAs, such as GM-CSF, to decay.

Abstract

Aminoglycosides are protein synthesis inhibitors. Gentamicin is the commonest agent in this class in therapeutics. Aminoglycosides have a broad spectrum that covers both Gram-positive (e.g., staphylococci) and Gram-negative pathogens (e.g., Enterobacterales, Pseudomonas aeruginosa) with notable exceptions. They are poorly active against streptococci and enterococci (though they are synergistic when combined with cell wall active agents). They also have no activity on anaerobes. Aminoglycosides are widely used in the treatment of Gram-negative infections such as urinary tract and intra-abdominal infections. They are indicated in infective endocarditis caused by Gram-positive cocci. Some agents in this class have useful activity against mycobacteria. Inhalational aminoglycosides are commonly used in patients with cystic fibrosis. Aminoglycosides are toxic to the kidneys and possess significant ototoxicity. Research into pharmacokinetics of aminoglycosides has contributed to changes in dosing: once daily dosing is now considered as standard. Once a day dosing has led to significant reduction in toxicity while retaining efficacy. Aminoglycosides demonstrate a post-antibiotic effect. Resistance to these agents is mainly caused by aminoglycoside modifying enzymes belonging to three major groups namely acetyltransferases, adenyltransferases, and phosphotransferases, RNA methyltransferase enzymes, target site mutation, and efflux. Plazomicin is a recently licensed aminoglycoside that shows resilience to several aminoglycoside modifying enzymes.

Axonal mRNA Transport in Developing Axons

Developing axons are characterized by their ability to grow, navigate, and innervate their target region. Each of these processes requires cytoskeletal rearrangements. Significantly, mRNAs encoding cytoskeletal proteins and cytoskeletal-associated proteins have been isolated from axons of developing neurons from a wide range of organisms. Given the importance of cytoskeletal dynamics in developing neurons, this suggests multiple roles for local translation in regulating axon and growth cone behavior, such as axon growth and pathfinding.

Axon guidance

As axons grow, their growth cones encounter a wide variety of signals that influence their navigation. Growth cones react to local attractive and repulsive cues in their environment by extending and retracting filopodia (fingerlike protrusions) and lamellipodia (flattened, veillike extensions), which determines the net direction of migration. Turning is caused by extension of filopodia on one side and withdrawal or collapse of filopodia on the other. Using two different chemotropic assays, the behavior of Xenopus retinal ganglion cell (RGC) axons in vitro has provided an insight into the role of local mRNA translation in growth cone guidance. In turning assays, growth cones grow toward a gradient of an attractant and turn away from a gradient of repellent over the course of 30–60 min. In collapse assays, bath application of a repellent causes complete withdrawal of growth cone filopodia and lamellipodia within 10 min. Embryonic Xenopus RGC growth cones are attracted to a source of netrin-1, but this attractive response is blocked by protein synthesis inhibitors. Moreover, protein synthesis inhibitors block the repulsive turning as well as the collapse response of RGCs toward the repellents Slit-2 and Semaphorin 3A (Sema3A). It is important to note that this inhibition of chemotropic responses by protein synthesis inhibitors also takes place in growth cones that have been severed from their cell bodies, showing that the translation occurs locally, in the growing axon. Indeed, netrin-1, Slit-2, and Sema3A are able to induce rapid (within 5–10 min) protein translation in the growth cone as measured by phosphorylation of eIF4E and eIF4EBP and the incorporation of labeled amino acids. It is striking that netrin-1, Slit-2, and Sema3A elicit local translation by activating different intracellular pathways involving mitogen-activated protein kinases (MAPKs). For example, netrin-1 and Slit-2, but not Sema3A, induce translation that depends on p38 MAPK. The netrin-1, Slit, and Sema3A pathways are dependent on mammalian TOR, indicating that cue-induced translation is cap-dependent.

These findings have provided evidence that growth cone turning and collapse depend on local protein synthesis, but which mRNAs are translated in these processes? Since growth cone turning involves remodeling of the microtubule and actin cytoskeleton, translation of mRNAs encoding cytoskeletal regulators are good candidates. Consistent with this hypothesis, Slit-2 has been shown to induce a protein synthesis-dependent increase in ADF/cofilin in Xenopus RGC growth cones. Moreover, Sema3A-mediated growth cone collapse in DRG neurons is dependent on the local translation of the small GTPase RhoA. RhoA is a key regulator of the actin cytoskeleton, and its mRNA is targeted to the growth cone by a specific sequence located in its 3′UTR. The local translation of RhoA is both sufficient and necessary for Sema3A-mediated growth cone collapse, suggesting that basal levels of RhoA are not sufficient to mediate the Sema3A responses. Thus, the local translation of cytoskeletal proteins provides the developing axon and growth cone with a mechanism to rapidly modulate the cytoskeleton in response to extracellular cues (Figure 3).

Figure 3. Diagram showing the role of axonal messenger RNA (mRNA) transport and local translation in growth cone behavior in developing axons. (a) Growth cones exposed to Sema3A induce the translation of RhoA, a small guanosine triphosphatase. The newly synthesized RhoA is required and sufficient to mediate Sema3A-induced growth cone collapse. (b) Local translation at the midline intermediate target. Translation of a reporter construct encompassing green fluorescent protein (GFP) and the 3′ untranslated region (3′UTR) of the EphA2 receptor is upregulated as growth cones enter the midline and reaches high levels as they emerge from the midline. Translation of the reporter construct is dependent on the cytoplasmic polyadenylation element sequence in the 3′UTR, suggesting that cytoplasmic polyadenylation element binding protein (CPEB) may be involved in mRNA localization or translational control.

Local mRNA translation in growth cones also plays a role in growth cone adaptation, a process whereby the growth cone desensitizes and resensitizes to a guidance cue. Adaptation is thought to be an integral part of the steering mechanism, enabling growth cones to adjust their sensitivity in a way that allows them to navigate in gradients of axon guidance cues. In Xenopus spinal neurons, attractive turning responses toward netrin-1 or brain-derived neurotrophic factor are attenuated if either of the chemoattractants is present in the bath. After 30 min, the desensitization disappears, and the growth cones regain their normal turning behavior. Protein synthesis inhibitors block this resensitization process, indicating that locally synthesized proteins play a role in regaining responsiveness. Collapse assays in Xenopus retinal growth cones show that desensitization and resensitization can even occur at a much shorter timescale (2–5 min) in response to repellents. Resensitization in response to Sema3A and netrin-1 under repulsive conditions is ligand-specific and requires new protein synthesis. Growth cone resensitization to Sema3A and netrin-1 correlates with a protein synthesis-dependent increase of their respective receptors, neuropilin and deleted in colorectal cancer, at the plasma membrane, indicating that resensitization might involve, at least partly, the translation of guidance cue receptors. In this way, growth cones can adjust their sensitivity as a function of exposure to a specific ligand.

Although functional studies on axonal mRNA transport are mostly conducted in vitro, local protein synthesis has also been implicated in axon navigation in vivo. When growing over long distances, axons use intermediate targets to which they are directed by local attractive and repulsive cues. After reaching the intermediate target, the axons must continue their journey and change responsiveness to the cues that guide them to the next target. One way to change responsiveness at an intermediate target is by changing the repertoire of guidance receptors at the plasma membrane. Studies in chick embryonic commissural neurons have suggested that this can be achieved by local translation of a receptor. Spinal commissural axons are initially attracted toward the midline floor plate, but after crossing the midline, axons lose responsiveness to the midline attractants and gain responsiveness to a new set of guidance cues. When these commissural axons are transfected with a construct encompassing the 3′UTR of the EphA2 receptor and GFP, protein expression is detected in the cell bodies but not in the growth cones. However, the protein is expressed in the growth cones of axons that have crossed the midline, suggesting that translation of the reporter construct is selectively activated after midline crossing. Translation is dependent on a cytoplasmic polyadenylation element binding element in the 3′UTR, indicating that CPEB might target the EphA2 mRNA to the growth cone.

Thus, local protein synthesis may play a crucial role in axon guidance. It has been known for decades that axons that are separated from their cell body retain their ability to grow and even make accurate guidance decisions. The ability of axons to locally translate proteins may explain this observation. By locally synthesizing proteins, the growth cone can autonomously respond to its environment. Different guidance cues along the pathway might stimulate the transport and the translation of a specific subset of mRNA needed for immediate growth cone turning, adaptation, and responses at intermediate targets. Moreover, protein synthesis may play a role presynaptically in the establishment of arbors and synapses, and studies in invertebrates indeed suggest a role for local translation in regulating synaptic connections in response to activation. It should be noted that not all cues elicit protein synthesis in the growth cones, indicating that local translation is not a ubiquitous mechanism in cue-directed growth.

1 Introduction

Macrolides are protein synthesis inhibitors that have been in clinical use for several decades. The first member of this class, erythromycin, was derived from Streptomyces erythreus (now named Saccharopolyspora erythraea) in 1949. This work was carried out by Abelardo Aguilar, a physician working for Eli Lilly and Company in the Philippines. Heilman and colleagues published the first clinical paper on the antibiotic shortly after, in 1952, when the medicine was marketed under the name ilotycin (Heilman et al., 1952). Later, azithromycin and clarithromycin were developed as agents with enhanced spectrum and improved pharmacological profiles. Ketolides were also obtained from erythromycin and are recognized as a related but separate class of antibiotics. Macrolides in clinical use are chemically made up of lactone rings with one or more sugar moieties (such as desosamine and cladinose) linked to the macrocyclic core. They contain a 14 (e.g., erythromycin, clarithromycin, and roxithromycin), 15 (e.g., azithromycin), or 16-membered lactone ring. Two subfamilies are recognized within the 16-membered macrolide family. These are the tylosin subfamily (desmomycin, tilmicosin, and tylosin A) and the leucomycin-spiramycin subfamily (e.g., carbomycin, josamycin, leucomycin, and spiramycin) (Arsic et al., 2018). Azithromycin is derived from the 14-membered ring by addition of a nitrogen atom. Ketolides are also derived from the 14-membered compounds. They have a ketone group in position 3 instead of l-cladinose sugar. They are active against macrolide resistant bacteria as they interact with the ribosome at two separate sites. Addition of fluorine atom led to the development of solithromycin, the first fluoroketolide, which is able to bind to the ribosome on as many as three sites (Fernandes et al., 2017; Hardy et al., 1988). Nafithromycin, a lactone ketolide, is in early stages of development (Flamm et al., 2017).

Erythromycin has a spectrum similar to that of penicillin and hence widely used for similar indications in patients allergic to the latter group of agents. Erythromycin is unstable at low pH where it is metabolized into inert molecules namely 8,9-anhydroerythromycin-6,9-hemiketal and anhydrorterythromycin-6,9:9,12-spiroketal. New chemicals were designed to avoid the inactivation of the parent compound into ketal metabolites. These include roxithromycin which has an N-oxime side chain attached to the macrolactone ring, dirithromycin with an amino group replacing a 9-keto group, and clarithromycin with a methyl group in the 6-O position. All these modifications contribute towards resilience in an acidic environment. The advancements in macrolides and the related compounds received formal recognition from the American Chemical Society (Jelic and Antolovic, 2016). Unfortunately, the contribution of the pioneer investigator, Abelardo Aguilar, was not celebrated in his lifetime.

Erythromycin, clarithromycin, and azithromycin are the most widely used macrolides in the treatment of bacterial infections. Azithromycin has enhanced Gram-negative spectrum when compared to erythromycin (this is thought to be related to the nitrogen content). Another macrolide, spiramycin, is used in the setting of toxoplasmosis. The macrolide antibiotics are safe and are well tolerated by patients. Some of them, in particular the 16-membered compounds, are used in veterinary medicine as antibiotics and feed supplements (Arsic et al., 2018).

Macrolides have an enterprising past, a formidable present, and an exciting future. However, their fate depends on how bacterial resistance emerges in the years and decades to follow and the extent to which the advances in structural chemistry can keep pace with growing resistance by generating novel molecules with improved efficacy and reduced toxicity.

7.4 Resistance to Aminoglycosides

Aminoglycosides are protein synthesis inhibitors that act primarily by impairing bacterial protein synthesis through binding to prokaryotic ribosomes. Several mechanisms of aminoglycoside resistance have been described in Gram-positive and Gram-negative bacteria. Enzymatic modification and inactivation of antibiotics are the most prevalent mechanisms of aminoglycoside resistance (Ramirez & Tolmasky, 2010; Vakulenko & Mobashery, 2003). Based upon reactions they catalyze, aminoglycoside-modifying enzymes are divided into three classes: N-acetyltransferases (ACC), O-adenyltransferases (ANT), and O-phosphotransferases (APH). Each class of the aminoglycoside-modifying enzymes has a unique resistance profile based on the type of enzymatic modification and the site of modification (Zhao, Mukherjee, et al., 2015). To date, a large number of aminoglycoside resistance genes have been identified, but only a couple dozen have been detected in Campylobacter. These include aad9, aadE, sat4, aphA-1, 3, 4, and 7, aac(6′), ant(6′), aacA4, aph(3′)-Ic, aph(3′)-IIIa, aph(2″)-If, f1, f2, and f3 and aph(2″)-Ib, Ic, Ih, and Ig and two bifunctional aac(6′)-Ie/aph(2″)-Ia (also named aacA/aphD), and aac(6′)-Ie/aph(2″)-Ia, which confer resistance to various aminoglycosides (Chen et al., 2013; Qin et al., 2012; Yao et al., 2017; Zhao, Mukherjee, et al., 2015; Zhao, Tyson, et al., 2015). The unique resistance gene cluster, aadE-sat4-aphA-3, which confers resistance to streptomycin, streptothricin, and kanamycin, respectively, has been found in C. jejuni and C. coli isolated from food animals, retail meats, and humans (Qin et al., 2012; Zhao, Tyson, et al., 2015). Additionally, this resistance cluster has been detected in the chromosome, as well as on a plasmid (Chen et al., 2013; Qin et al., 2012; Zhao, Tyson, et al., 2015).

WGS analysis of gentamicin-resistant (GenR) Campylobacter strains from the NARMS program showed that variants of aminoglycoside 2″-phosphotransferase genes [aph(2″)] were the major contributors to the GenR phenotype. Nine variants of aph(2″), including six [aph(2″)-Ib, Ic, If1, If3, Ih, and aac(6′)-Ie/aph(2″)-If2] that were identified for the first time in Campylobacter, were described (Zhao, Mukherjee, et al., 2015). Almost all GenR Campylobacter (98.7%) were coresistant to tetracycline, and 65.8% of human isolates were resistant to three or more antimicrobials (Zhao, Mukherjee, et al., 2015). Several studies conducted in China showed that the prevalence of GenR Campylobacter isolated from broiler chicken and swine was much higher than in the United States, and the aph(2″)-If gene was the dominant gentamicin resistance gene (Chen et al., 2010; Qin et al., 2011; Yao et al., 2017). APH(2″) family is genetically diverse, and the percentage of amino acid identity between subfamilies can be as low as 25.9% (Zhao, Mukherjee, et al., 2015). The variants of aph(2″) genes have been detected in both the chromosome and on a plasmid. Based on the G + C content of the plasmid backbone and on the structure of resistance gene cluster, it has been suggested that most aph(2″) genes are from an exogenous source, likely from Gram-positive bacteria since APH(2″) is widely distributed in enterococci and staphylococci (Toth, Frase, Antunes, & Vakulenko, 2013; Zhao, Mukherjee, et al., 2015). A study by Toth et al. showed that aph(2″)-If not only confers resistance to gentamicin but also confers resistance to other aminoglycosides, such as kanamycin, tobramycin, dibekacin, and sisomicin (Toth et al., 2013).

Macrolide and Azalide Antibiotics

This group of protein synthesis inhibitors, the most notable of which is erythromycin, acts on protein elongation, rather than initiation. They have a broad spectrum of action, and affect a wide range of Gram-positive and Gram-negative bacteria. As with chloramphenicol, these drugs bind to the 23S ribosomal RNA associated with the 50S ribosomal subunit. Changes in methylation of 23S ribosomal RNA can confer resistance to both macrolide and azalide antibiotics but this is associated with slightly different regions of the ribosomal RNA molecule. Binding of chloramphenicol to the 50S ribosome can also inhibit binding of macrolides, and vice versa. This implies a common but different target for each agent. Unlike chloramphenicol, the macrolides block translocation of the ribosomal complex along the mRNA. After the peptidyl transferase has formed a peptide bond between the nascent peptide bound to the P site and the aminoacyl-tRNA at the A site, the whole ribosome must relocate to the next codon for translation to proceed. Energy for this process is derived by hydrolysis of GTP to GDP mediated through the accessory elongation factor (Ef-G). By blocking the translocation incomplete peptides are lost from the ribosome–mRNA complex, the ribosomal subunits dissociate and initiation must recommence. It is assumed that the azalides have a mode of action similar to erythromycin, but they have better intracellular penetration and are less readily metabolized by the host with a resulting improved serum half-life.

3 Mechanism of action

Clindamycin is a protein synthesis inhibitor which is generally bacteriostatic but can have bactericidal effects at higher concentrations. Clindamycin binds to 50S subunit of the ribosome and interferes with peptide chain initiation and also stimulates the dissociation of peptidyl-tRNA from ribosomes, a property that it shares with erythromycin, spiramycin, and josamycin. Clindamycin, spiramycin and josamycin act on the peptidyl transferase center and hence cause dissociation of the transferase enzyme with only 2–4 amino acid residues (in contrast, erythromycin leads to dissociation with 6–8 amino acid residues). The distance between drug and peptidyl transferase center is least for josamycin (4.2 A°), approximately 4.6 A° for clindamycin, 7 A° for spiramycin, 10.6 A° for erythromycin, and 12.9 A° for telithromycin. Thus, they all block peptide synthesis allowing variable space for the exit of the nascent peptides. The build-up of dissociated peptidyl-tRNA leads to a paucity of free tRNA pools which causes cellular toxicity. The chemistry of clindamycin contributes to its mechanism of action. Clindamycin molecule has structural resemblance to l-Pro-Met and the d-ribosyl ring of adenosine which allows it to interfere with peptide elongation. The structure of erythromycin is different and it is not a direct inhibitor of peptidyltransferase (Spizek and Rezanka, 2017; Tenson et al., 2003). Additionally, clindamycin treatment can make bacterial cells more susceptible to the action of phagocytes (Dhawan and Thadepalli, 1982).

4.14.5.2 Neurotransmitter Effects of Protein Synthesis Inhibition

The actions of anisomycin and other protein synthesis inhibitors on cell responses such as activation of MAPK and superinduction of protein and gene expression are general across cell types and organ systems. However, there are also dramatic effects of protein synthesis inhibitors on neuronal functions, effects that seem likely to represent an additional mechanism by which multiple protein synthesis inhibitors may impair memory formation for reasons that can be attributed to abnormal cellular responses to protein synthesis inhibition. As noted with regard to superinduction of genes, these are mechanisms that do not lead to the conclusion that de novo protein synthesis triggered by an experience is a mechanism of memory formation. One of these actions is that memory-impairing injections of anisomycin into the amygdala or hippocampus result in an enormous dumping of norepinephrine, dopamine, serotonin, and acetylcholine into extracellular space (Canal et al., 2007; Qi and Gold, 2009). Infusions of several neurotransmitters and their receptor agonists can themselves impair memory at high doses, i.e., at the high end of an inverted-U dose–response function. After anisomycin injections into the amygdala, the increases in biogenic amine levels ranged from 1000% to 20,000% of baseline. For comparison, inhibitory avoidance training results in increases in hippocampal and amygdala norepinephrine levels of 20%–100% above baseline (Morris et al., 2010; Canal et al., 2007; Williams et al., 1998; Galvez et al., 1996), suggesting that anisomycin results in extraordinary supraphysiological release of neurotransmitters. Moreover, administration of the β-adrenergic receptor blocker, propranolol, blocked amnesia induced by anisomycin, likely due to the blockade of norepinephrine-induced amnesia (Canal et al., 2007).

As with protein synthesis inhibition, inhibition of CREB also impairs memory and neurotransmitter functions. CREB antisense, infused into the amygdala, impaired 48-h memory for inhibitory avoidance training and also impaired release of norepinephrine in the amygdala during training (Canal et al., 2008). In that experiment, the impairment of memory in antisense-treated rats was reversed by injection of the β-adrenergic agonist, clenbuterol, into the amygdala immediately after training, i.e., rescuing the neurochemical and memory impairments induced by CREB antisense. Thus, one response to inhibition of protein synthesis, by anisomycin and by CREB antisense, is to generate abnormal neurotransmitter release profiles, i.e., perhaps another response common to protein synthesis inhibition by multiple inhibitors. There is a wealth of information showing that pharmacological actions targeting many neurotransmitters can impair memory. Protein synthesis inhibitors offer another route, albeit an indirect one, to interfering with normal neurotransmitter release patterns with subsequent failures of cell–cell communication needed for memory formation.