Chloramphenicol Analogues Research Articles

Berberine analog of chloramphenicol exhibits a distinct mode of action and unveils ribosome plasticity

Chloramphenicol (CHL) is an antibiotic targeting the peptidyl transferase center in bacterial ribosomes. We synthesized a new analog, CAM-BER, by substituting the dichloroacetyl moiety of CHL with a positively charged aromatic berberine group. CAM-BER suppresses bacterial cell growth, inhibits protein synthesis invitro, and binds tightly to the 70S ribosome. Crystal structure analysis reveals that the bulky berberine group folds into the P site of the peptidyl transferase center (PTC), where it competes with the formyl-methionine residue of the initiator tRNA. Our toe-printing data confirm that CAM-BER acts as a translation initiation inhibitor in stark contrast to CHL, a translation elongation inhibitor. Moreover, CAM-BER induces a distinct rearrangement of conformationally restrained nucleotide A2059, suggesting that the 23S rRNA plasticity is significantly higher than previously thought. CAM-BER shows potential in avoiding CHL resistance and presents opportunities for developing novel berberine derivatives of CHL through medicinal chemistry exploration.

Detection of florfenicol resistance in opportunistic Acinetobacter spp. infections in rural Thailand.

Florfenicol (Ff) is an antimicrobial agent belonging to the class amphenicol used for the treatment of bacterial infections in livestock, poultry, and aquaculture (animal farming). It inhibits protein synthesis. Ff is an analog of chloramphenicol, an amphenicol compound on the WHO essential medicine list that is used for the treatment of human infections. Due to the extensive usage of Ff in animal farming, zoonotic pathogens have developed resistance to this antimicrobial agent. There are numerous reports of resistance genes from organisms infecting or colonizing animals found in human pathogens, suggesting a possible exchange of genetic materials. One of these genes is floR, a gene that encodes for an efflux pump that removes Ff from bacterial cells, conferring resistance against amphenicol, and is often associated with mobile genetic elements and other resistant determinants. In this study, we analyzed bacterial isolates recovered in rural Thailand from patients and environmental samples collected for disease monitoring. Whole genome sequencing was carried out for all the samples collected. Speciation and genome annotation was performed revealing the presence of the floR gene in the bacterial genome. The minimum inhibitory concentration (MIC) was determined for Ff and chloramphenicol. Chromosomal and phylogenetic analyses were performed to investigate the acquisition pattern of the floR gene. The presence of a conserved floR gene in unrelated Acinetobacter spp. isolated from human bacterial infections and environmental samples was observed, suggesting multiple and independent inter-species genetic exchange of drug-resistant determinants. The floR was found to be in the variable region containing various mobile genetic elements and other antibiotic resistance determinants; however, no evidence of HGT could be found. The floR gene identified in this study is chromosomal for all isolates. The study highlights a plausible impact of antimicrobials used in veterinary settings on human health. Ff shares cross-resistance with chloramphenicol, which is still in use in several countries. Furthermore, by selecting for floR-resistance genes, we may be selecting for and facilitating the zoonotic and reverse zoonotic exchange of other flanking resistance markers between human and animal pathogens or commensals with detrimental public health consequences.

Old Antibiotics Can Learn New Ways: A Systematic Review of Florfenicol Use in Veterinary Medicine and Future Perspectives Using Nanotechnology.

Florfenicol is a broad-spectrum bacteriostatic antibiotic used exclusively in veterinary medicine in order to treat the pathology of farm and aquatic animals. It is a synthetic fluorinated analog of thiamphenicol and chloramphenicol that functions by inhibiting ribosomal activity, which disrupts bacterial protein synthesis and has shown over time a strong activity against Gram-positive and negative bacterial groups. Florfenicol was also reported to have anti-inflammatory activity through a marked reduction in immune cell proliferation and cytokine production. The need for improvement came from (1) the inappropriate use (to an important extent) of this antimicrobial, which led to serious concerns about florfenicol-related resistance genes, and (2) the fact that this antibiotic has a low water solubility making it difficult to formulate an aqueous solution in organic solvents, and applicable for different routes of administration. This review aims to synthesize the various applications of florfenicol in veterinary medicine, explore the potential use of nanotechnology to improve its effectiveness and analyze the advantages and limitations of such approaches. The review is based on data from scientific articles and systematic reviews identified in several databases.

A helper antibody based competitive fluorescence immunochromatographic assay for quantitative detection of florfenicol in poultry eggs.

Florfenicol (FF) is a chloramphenicol analogue used in animals, and florfenicol amine (FFA) is the main metabolite of FF. However, their residues in agricultural products are harmful to human health. A highly specific and sensitive assay for FF/FFA detection needs to be developed since the traditional detection methods are low sensitive. In this study, a new method for rapid quantification of FF/FFA in poultry eggs by fluorescent immunochromatographic assay (HAFIA) was established. The triple antibodies including a primary monoclonal antibody (mAb) specific to the targets FF and FFA, a secondary polyclonal antibody (pAb) labeled with Europium nanoparticles (EuNPs) and a helper monoclonal antibody (hAb) reacting with pAb but not react with mAb or target antigen are designed, which can form structural aggregation complexes in microwells with a single step of reactions. By loading the reaction sample solution, the triple-antibodies (mAb-pAb-hAb)-EuNPs complexes migrate to the test (T) line on nitrocellulose membrane of testing strip, which are competitively captured by the immobilized FF-BSA conjugates on the membrane and the FF/FFA targets in sample solution. Fluorescence on T line is read by a portable fluorescent strip reader in 10 min, and the result is given by the ratio of fluorescent intensity on T line than control (C) line. This new fluorescent testing strip with triple-antibody complexes' amplification signal has 50-fold higher sensitivity than conventional CG-LFIAs, and can detect as low as 0.01 ng/mL florfenicol and 0.1 ng/mL florfenicol amine targets from egg samples. The developed competitive fluorescent immunochromatography method based on auxiliary antibodies has the advantages of high sensitivity and specificity for the rapid and quantitative detection of FF/FFA in poultry eggs.

Bacterial Dehydrogenases Facilitate Oxidative Inactivation and Bioremediation of Chloramphenicol.

Antimicrobial resistance represents a major threat to human health and knowledge of the underlying mechanisms is therefore vital. Here, we report the discovery and characterization of oxidoreductases that inactivate the broad-spectrum antibiotic chloramphenicol via dual oxidation of the C3-hydroxyl group. Accordingly, chloramphenicol oxidation either depends on standalone glucose-methanol-choline (GMC)-type flavoenzymes, or on additional aldehyde dehydrogenases that boost overall turnover. These enzymes also enable the inactivation of the chloramphenicol analogues thiamphenicol and azidamfenicol, but not of the C3-fluorinated florfenicol. Notably, distinct isofunctional enzymes can be found in Gram-positive (e. g., Streptomyces sp.) and Gram-negative (e. g., Sphingobium sp.) bacteria, which presumably evolved their selectivity for chloramphenicol independently based on phylogenetic analyses. Mechanistic and structural studies provide further insights into the catalytic mechanisms of these biotechnologically interesting enzymes, which, in sum, are both a curse and a blessing by contributing to the spread of antibiotic resistance as well as to the bioremediation of chloramphenicol.

Efficient Synthesis of Two Chloramphenicol Derivatives as Antibacterial Agents

Several protocols have been used to prepare some chloramphenicol analogs using different reagents, which can be expensive and difficult to handle. The aim of this investigation was to synthesize six chloramphenicol analogs (compounds 2 to 7) using some chemical strategies to evaluate their biological activity against Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, and Klebsiella pneumoniae with the minimum inhibitory concentration method. The results indicate that protocols used to synthesize chloramphenicol derivatives do not require special conditions such as different pH and higher temperatures to give a good yielding. Besides, only compounds 4 and 6 decreased the bacterial growth of either Gram-positive or Gram-negative bacteria. However, the biological activity of compound 4 was higher compared with both chloramphenicol and compound 6.

MFS1, a Pleiotropic Transporter in Dermatophytes That Plays a Key Role in Their Intrinsic Resistance to Chloramphenicol and Fluconazole.

A recently identified Trichophyton rubrum major facilitator superfamily (MFS)-type transporter (TruMFS1) has been shown to give resistance to azole compounds and cycloheximide (CYH) when overexpressed in Saccharomyces cerevisiae. We investigated the roles of MFS1 in the intrinsic resistance of dermatophytes to CYH and chloramphenicol (CHL), which are commonly used to isolate these fungi, and to what extent MFS1 affects the susceptibility to azole antifungals. Susceptibility to antibiotics and azoles was tested in S. cerevisiae overexpressing MFS1 and ΔMFS1 mutants of Trichophyton benhamiae, a dermatophyte that is closely related to T. rubrum. We found that TruMFS1 functions as an efflux pump for CHL in addition to CYH and azoles in S. cerevisiae. In contrast, the growth of T. benhamiae ΔMFS1 mutants was not reduced in the presence of CYH but was severely impaired in the presence of CHL and thiamphenicol, a CHL analog. The suppression of MFS1 in T. benhamiae also increased the sensitivity of the fungus to fluconazole and miconazole. Our experiments revealed a key role of MFS1 in the resistance of dermatophytes to CHL and their high minimum inhibitory concentration for fluconazole. Suppression of MFS1 did not affect the sensitivity to CYH, suggesting that another mechanism was involved in resistance to CYH in dermatophytes.

Triphenilphosphonium Analogs of Chloramphenicol as Dual-Acting Antimicrobial and Antiproliferating Agents.

In the current work, in continuation of our recent research, we synthesized and studied new chimeric compounds, including the ribosome-targeting antibiotic chloramphenicol (CHL) and the membrane-penetrating cation triphenylphosphonium (TPP), which are linked by alkyl groups of different lengths. Using various biochemical assays, we showed that these CAM-Cn-TPP compounds bind to the bacterial ribosome, inhibit protein synthesis in vitro and in vivo in a way similar to that of the parent CHL, and significantly reduce membrane potential. Similar to CAM-C4-TPP, the mode of action of CAM-C10-TPP and CAM-C14-TPP in bacterial ribosomes differs from that of CHL. By simulating the dynamics of CAM-Cn-TPP complexes with bacterial ribosomes, we proposed a possible explanation for the specificity of the action of these analogs in the translation process. CAM-C10-TPP and CAM-C14-TPP more strongly inhibit the growth of the Gram-positive bacteria, as compared to CHL, and suppress some CHL-resistant bacterial strains. Thus, we have shown that TPP derivatives of CHL are dual-acting compounds targeting both the ribosomes and cellular membranes of bacteria. The TPP fragment of CAM-Cn-TPP compounds has an inhibitory effect on bacteria. Moreover, since the mitochondria of eukaryotic cells possess qualities similar to those of their prokaryotic ancestors, we demonstrate the possibility of targeting chemoresistant cancer cells with these compounds.

Binding and Action of Triphenylphosphonium Analog of Chloramphenicol upon the Bacterial Ribosome.

Chloramphenicol (CHL) is a ribosome-targeting antibiotic that binds to the peptidyl transferase center (PTC) of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving the properties of this inhibitor, we explored ribosome binding and inhibitory properties of a semi-synthetic triphenylphosphonium analog of CHL—CAM-C4-TPP. Our data demonstrate that this compound exhibits a ~5-fold stronger affinity for the bacterial ribosome and higher potency as an in vitro protein synthesis inhibitor compared to CHL. The X-ray crystal structure of the Thermus thermophilus 70S ribosome in complex with CAM-C4-TPP reveals that, while its amphenicol moiety binds at the PTC in a fashion identical to CHL, the C4-TPP tail adopts an extended propeller-like conformation within the ribosome exit tunnel where it establishes multiple hydrophobic Van der Waals interactions with the rRNA. The synthesized compound represents a promising chemical scaffold for further development by medicinal chemists because it simultaneously targets the two key functional centers of the bacterial ribosome—PTC and peptide exit tunnel.

Recent Trends in Synthesis of Chloramphenicol New Derivatives.

Chloramphenicol (CAM), the bacteriostatic broad-spectrum antibiotic, isolated from Streptomyces venezuelae during the “golden era” of antibiotic discovery, nowadays has limited clinical potential due to adverse side effects and frequent antimicrobial resistance. Numerous CAM analogs were synthesized in order to find the derivatives with improved pharmacological properties and activity on resistant bacterial strains. This work aims to summarize the most recent achievements in obtaining new CAM analogs reported during the last five years. Current investigations are mainly focused on elucidating the molecular basis of the mode of CAM action and determining the mechanisms of resistance to this class of antibiotics or on studies of the possible use of the CAM scaffold to search for therapeutic agents with different CAM modes of action—such as selective antiproliferative agents or bacterial cell wall biosynthesis inhibitors. Hopefully, a deeper understanding of the CAM interactions with the target and its specificity will generate research ideas for developing new effective drugs.

Interaction of Chloramphenicol Cationic Peptide Analogues with the Ribosome.

Virtual screening of all possible tripeptide analogues of chloramphenicol was performed using molecular docking to evaluate their affinity to bacterial ribosomes. Chloramphenicol analogues that demonstrated the lowest calculated energy of interaction with ribosomes were synthesized. Chloramphenicol amine (CAM) derivatives, which contained specific peptide fragments from the proline-rich antimicrobial peptides were produced. It was demonstrated using displacement of the fluorescent erythromycin analogue from its complex with ribosomes that the novel peptide analogues of chloramphenicol were able to bind bacterial ribosome; all the designed tripeptide analogues and one of the chloramphenicol amine derivatives containing fragment of the proline-rich antimicrobial peptides exhibited significantly greater affinity to Escherichia coli ribosome than chloramphenicol. Correlation between the calculated and experimentally evaluated levels of the ligand efficiencies was observed. In vitro protein biosynthesis inhibition assay revealed, that the RAW-CAM analogue shows activity at the level of chloramphenicol. These data were confirmed by the chemical probing assay, according to which binding pattern of this analogue in the nascent peptide exit tunnel was similar to chloramphenicol.

Highly Specific Chemiluminescence Immunoassay for the Determination of Chloramphenicol in Cosmetics.

A direct and highly specific chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) method for monitoring chloramphenicol (CAP) in cosmetics has been developed. The anti-chloramphenicol antibody (mAb) adopted in this work for direct immunoassay could bind to CAP specifically, with negligible cross-reactivity (CR) (less than 0.01%) with most CAP analogues, including structurally related thiamphenicol (TAP) and florfenicol (FF). The limit of detection (LOD), measured by IC10, was 0.0021 ng mL−1. The detection range (IC20-IC80) was ranged from 0.00979 to 0.12026 ng mL−1. In spiked cosmetics samples, mean recoveries ranged from 82.7% to 99.6%, with intraday and interday variation less than 9.8 and 8.2%, respectively. Moreover, with the help of HRP-labeled anti-CAP mAb, the method could be processed in fast direct immunoreaction mode. This CL-ELISA method could be applied for specific, rapid, semiquantitative, and quantitative detection of CAP in cosmetics, facilitating the precise quality control of CAP contamination.

Theoretical and experimental research on self-assembly system of molecularly imprinted polymers formed via chloramphenicol and methacrylic acid.

Chloramphenicol was chosen as the imprinting molecule and the methacrylic acid was chosen as the functional monomer to prepare molecularly imprinted polymers. Ethylene glycol dimethacrylate, pentaerythritol triacrylate, and trimethylolpropane trimethylacrylate were used as the cross-linking agents, respectively. The interaction processes between chloramphenicol and methacrylic acid were simulated by using the ωB97XD/6-31G (d,p) method. The self-assembled configuration, bonding sites, binding number, binding energy, and interaction principle of stable complex formed by chloramphenicol and methacrylic acid with different molar ratios have been studied. The selectivity of the most stable complex formed from chloramphenicol and methacrylic acid was discussed with the thiamphenicol and florfenicol as the analogues of chloramphenicol. The results showed that chloramphenicol and methacrylic acid were interacted through the hydrogen bonds. When the molar ratio was 1:10 and pentaerythritol triacrylate as the cross-linking agent, the ordered complex formed by chloramphenicol and methacrylic acid has the largest amount of hydrogen bonds and the lowest binding energy. Scatchard analysis showed that the maximum apparent adsorption capacity was 173.3mg/g (0.536mol/g), and the selection factor of florfenicol was the largest. This study provides a reliable theoretical and experimental basis for the design, preparation, and characterization of chloramphenicol molecularly imprinted polymers.

Detection of floR gene and active efflux mechanism of Escherichia coli in Ningxia, China

Florfenicol is an antibiotic, a fluorinated structural analogue of thiamphenicol and chloramphenicol, approved exclusively for use in Asia for aquaculture since the 1980's. Our study examined the prevalence of florfenicol resistance in bovine mastitis Escherichia coli isolates. A total of 245 E. coli isolates were collected from bovine mastitis in Ningxia Province, China between May 2016 to July 2017 and screened for florfenicol resistance gene, floR gene by PCR analysis. About 7.35% (15/245) of the isolates were positive for floR gene. Minimal inhibitory concentration (MIC) results showed that 9 isolates were susceptible strains and 6 isolates were highly resistant to florfenicol. HPLC (high performance liquid chromatography) results showed that the amounts of florfenicol was significantly improved in the presence of carbonyl cyanide 3-chlorophenylhydrazone (CCCP) as an efflux pump inhibitor. This, therefore, indicates that the employment of florfenicol in conjunction with CCCP in future drug formulations should be considered.

Binding and Action of Amino Acid Analogs of Chloramphenicol upon the Bacterial Ribosome

Antibiotic chloramphenicol (CHL) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino acid analogs of CHL. The L-histidyl analog binds to the ribosome with the affinity exceeding that of CHL by 10 fold. Several of the newly synthesized analogs were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CHL. However, the inhibitory properties of the semi-synthetic CHL analogs did not correlate with their affinity and in general, the amino acid analogs of CHL were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogs showed that CHL derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.

A novel photonic sensor for the detection of chloramphenicol

A novel photonic sensor using a self-cross-linked imprinting close-packed opal (CPO) as a recognition element was developed for the detection of chloramphenicol (CAP). The self-cross-linked imprinting CPO film was consisted of monodisperse molecular imprinted – N-hydroxymethylacrylamide particles (MI-HAM particles) with self-assembling and self-crosslinking properties. A dilute dispersion of these particles was self-assembled to formation of the highly ordered CPO structure, and at the same time the neighboring particles reacted to form covalent bond to stabilize the CPO structure at the air/dispersion. Thus the obtained self-cross-linked imprinting CPO was characterized by a highly stable three-dimensional (3D) CPO structure without the interference of bulk hydrogel matrix, in which numerous CAP recognition sites were dispersed through the molecular imprinted process. The inherent high affinity of the recognition sites allowed the self-cross-linked imprinting CPO to recognize CAP with high specificity, and changes of the periodic structure enabled the self-cross-linked imprinting CPO to transfer the recognition events into readable optical signals. Then the self-cross-linked imprinting CPO film was packed into a reaction cell with an optical spectrometer to establish a novel photonic sensor. A linear relationship was found between the diffraction intensity decrease and CAP concentration in the range from 2ngmL−1 to 512ngmL−1, whereas there was no obvious optical change for CAP analogues, thus indicating that the sensor had selective and sensitive response to CAP molecules. Furthermore, the sensor was applied successfully to detect CAP in drinking water samples. As a result, the developed sensor might offer a potential alternative in routine supervision for residue due to its convenience, low cost, reusability, and high sensitivity as well as selectivity.

Interaction of Chloramphenicol Tripeptide Analogs with Ribosomes.

Chloramphenicol amine peptide derivatives containing tripeptide fragments of regulatory "stop peptides" - MRL, IRA, IWP - were synthesized. The ability of the compounds to form ribosomal complexes was studied by displacement of the fluorescent erythromycin analog from its complex with E. coli ribosomes. It was found that peptide chloramphenicol analogs are able to bind to bacterial ribosomes. The dissociation constants were 4.3-10µM, which is 100-fold lower than the corresponding values for chloramphenicol amine-ribosome complex. Interaction of the chloramphenicol peptide analogs with ribosomes was simulated by molecular docking, and the most probable contacts of "stop peptide" motifs with the elements of nascent peptide exit tunnel were identified.

An Acyl Transfer Reaction Catalyzed by an Epimerase MarH

MarH, a small protein (129 amino acids) belonging to the cupin superfamily, was previously characterized as an epimerase involved in the (2S,3S)-β-methyltryptophan formation in the maremycin biosynthesis. Here, MarH was discovered to act as an acyltransferase that can catalyze the 3-O-acylation of chloramphenicol. Furthermore, MarH can catalyze N-acylation of deacylated chloramphenicol analogue thereby activating them for 3-O-acylation. By systematic site-directed mutagenesis, H64 was revealed as a potential catalytic base that deprotonates the acyl acceptor substrate. Nucleophilic attack at the carbonyl carbon of the acyl donor then gives the acylation product.

Second generation engineering of transketolase for polar aromatic aldehyde substrates

Transketolase has significant industrial potential for the asymmetric synthesis of carboncarbon bonds with new chiral centres. Variants evolved on propanal were found previously with nascent activity on polar aromatic aldehydes 3-formylbenzoic acid (3-FBA), 4-formylbenzoic acid (4-FBA), and 3-hydroxybenzaldehyde (3-HBA), suggesting a potential novel route to analogues of chloramphenicol. Here we evolved improved transketolase activities towards aromatic aldehydes, by saturation mutagenesis of two active-site residues (R358 and S385), predicted to interact with the aromatic substituents. S385 variants selectively controlled the aromatic substrate preference, with up to 13-fold enhanced activities, and KM values comparable to those of natural substrates with wild-type transketolase. S385E even completely removed the substrate inhibition for 3-FBA, observed in all previous variants. The mechanisms of catalytic improvement were both mutation type and substrate dependent. S385E improved 3-FBA activity via kcat, but reduced 4-FBA activity via KM. Conversely, S385Y/T improved 3-FBA activity via KM and 4-FBA activity via kcat. This suggested that both substrate proximity and active-site orientation are very sensitive to mutation. Comparison of all variant activities on each substrate indicated different binding modes for the three aromatic substrates, supported by computational docking. This highlights a potential divergence in the evolution of different substrate specificities, with implications for enzyme engineering.

An ultrasensitive immunochromatographic assay for non-pretreatment monitoring of chloramphenicol in raw milk

A non-pretreatment monitoring method based on immunochromatographic test (ICT) strips was developed to test for chloramphenicol (CAP) in raw milk. This assay was designed to measure competitive binding affinities for CAP molecules in raw milk and CAP antigen immobilized on strips with colloidal gold nanoparticles (GNPs, average diameter of 30 nm) labeled with anti-CAP monoclonal antibody (CAP mAb). Several working conditions, including the CAP solvent, different types and concentrations of CAP mAb, coating antigen, and GNP, were optimized for performance. After optimization, the detection process was carried out in 8 min with a visual limit of detection (LOD) of 0.3 ng/mL for qualitative detection and a LOD of 0.064 ng/mL for semi-quantitative detection. No cross-reactivity was detected toward CAP analogs. Based on these results, this simple and ultrasensitive assay could be thoroughly applied in the on-site testing of CAP in raw milk.